ClBBX genes were identified. ClBBXs were grouped into five clades  based on their conserved domains and phylogenetic relationships. Most of the ClBBXs (84%) might be localized in the nuclei or cytoplasm. The classification of ClBBXs was consistent with their gene structures. They were unevenly distributed in nine chromosomes except for Chr4 and Chr10, with the largest number of six members in Chr2. Segmental duplications were the major factor in ClBBX family expansion. Some BBXs of watermelon and Arabidopsis evolved from a common ancestor. In total, 254 hormonal and stress-responsive cis elements were discovered in ClBBX promoters. ClBBXs were differentially expressed in tissues, and the expression levels of ClBBX15 and 16 were higher in aboveground tissues than in roots, while the patterns of ClBBX21a, 21b, 21c, 28 and 30b were the opposite. With salicylic acid, methyl jasmonate and salt stress conditions, 17, 18 and 18 ClBBXs exhibited significant expression changes, respectively. In addition, many ClBBXs, including ClBBX29b, 30a and 30b, were also responsive to cold and osmotic stress. In summary, the simultaneous response of multiple ClBBXs to hormonal or abiotic stress suggests that they may have functional interactions in the stress hormone network. Clarifying the roles of key ClBBXs in transcriptional regulation and mediating protein interactions will be an important task. Our comprehensive characterization of the watermelon ClBBX family provides vital clues for the in-depth analysis of their biological functions in stress and hormone signaling pathways.Plant B-BOX (BBX) zinc finger transcription factors play crucial roles in growth and development and the stress response. Although the BBX family has been characterized in various plants, systematic analysis in watermelon is still lacking. In this study, 25 watermelon  Arabidopsis and grapevine BBXs were grouped into five structure groups (I–V) based on the number of B-box and CCT domains, and significantly clustered with dicotyledonous plants rather than rice, as is consistent with their position in plant classification [Plant transcription factors (TFs) function mainly through transcriptional regulation ,2. With fication ,8.Arabidopsis BBX members functioned in a HY5-dependent manner [CmBBX13 delayed flowering time [CmBBX22 negatively regulated drought resistance [GbBBX25 enhanced the salt adaptability of transgenic poplar [BBX proteins widely participate in physiological and biochemical processes in plants ,9,10. Fot manner . They fot manner . Both pet manner ,15. Addit manner . Sweet ct manner . Chrysaning time , while Csistance . Ginkgo c poplar . These rBBX family members is the prerequisite for a comprehensive analysis of the interaction among different BBXs. The characterization of the BBX family in many plants has been reported. The number of cotton BBXs ranged from 17 (Gossypium arboreum) to 37 (G. hirsutum), and the expansion of this family in cotton occurred mainly through segmental replication [VviBBXs in grapevine might participate in powdery mildew infestation, hormone signaling, and seed abortion [SlBBXs were induced by drought or heat stress, and some were strongly induced by ABA, gibberellic acid (GA), or ethephon [StBBX family (consisting of 30 members) might be made up of the key players in the circadian rhythm network [PbBBXs were specifically expressed in pollen tubes [CaBBXs might regulate anthocyanin accumulation [BBX (ClBBX) family to date.The identification of lication . The 25 abortion . Most ofethephon . The pot network . Brieflyen tubes . Most ofCitrullus lanatus) is a cucurbitacea crop and one of the top five most commonly consumed fresh fruits [ClBBX family. In this study, 25 genes encoding watermelon ClBBXs were identified. The gene structure, domains, phylogeny, and synteny were determined. The cis elements involved in stress and hormone responsive were analyzed in ClBBX promoters. In addition, the expression of ClBBXs in tissues and their response to salicylic acid (SA), methyl jasmonate (MeJA) and abiotic stresses  were analyzed. Our work will be helpful for the further functional analysis of watermelon ClBBXs.Watermelon  was used as a BLAST query against the Cucurbit Genomics Database (CuGenDB), and the Arabidopsis BBXs were also used as a BLAST query against CuGenDB. Briefly, 31 candidate BBX sequences were obtained, of which 6 sequences were removed due to their e-value of >0.01. The remaining protein sequences were assessed for the presence of B-box zinc finger domains through Pfam and SMART. Finally, 25 watermelon BBX candidate members were identified and designated as ClBBXs. The structure type of ClBBXs  was based on the classification rules of BBX family members in Arabidopsis (ClBBXs ranged from 378 (ClBBX29b) to 1,503 bp (ClBBX27), the length of deduced proteins ranged from 125 to 500 amino acids, and the molecular weights ranged from 13.42 to 56.18 kDa. The number of introns in ClBBXs ranged from 0 to 4. The isoelectric points of ClBBXs ranged from 4.38 (ClBBX29a) to 7.49 (ClBBX15), and only three proteins  belonging to Structure V were stable. Additionally, most ClBBXs were predicted to be localized in the nucleus or cytoplasm, while ClBBX30b might function primarily in chloroplasts  in zinc fingers and arginine residues (R) in CCT domains were highly conserved.Among the 25 ClBBXs, 4 members each belonged to Structure I and II groups, 3 members belonged to Structure III groups, 8 members belonged to Structure IV group, and 6 members belonged to Structure V group. Except for ClBBX3 from the Structure I group which had no B-box2 domain, all other ClBBXs from Structure I and II groups contained B-box1, B-box2, and CCT domains. All members of the Structure III group contained B-box1 and CCT domains, those of the Structure IV group contained two B-box domains, while those of the Structure V group had only B-box1 . As showArabidopsis, 25 from grapevine, 25 from pear, 23 from pepper, 30 from potato, 30 from rice and 29 from tomato, were used to construct a phylogenetic tree  had one intron, 11 genes (44%) had two or more introns, and only ClBBX27 from Clade V had four introns  and three members from Clade III  had only one intron, while all four genes of Clade II  contained three introns. The genes without introns belonged mainly to Clade V  and only ClBBX21b belonged to Clade IV. The structures of Clade IV genes were relatively complex, with seven out of eight genes containing at least one intron. The structures of ClBBXs may be related to their phylogenetic classification.Briefly, 5 (20%) of the 25  introns . All fouClBBXs were widely distributed in 9 out of 11 chromosomes except for Chr4 and Chr10 (ClBBXs (6), while Chr3 and Chr9 both possessed only one gene. Two ClBBXs were distributed on Chr1 and Chr11, respectively. Chr5, Chr6, and Chr8 each contained three genes, and four members were distributed on Chr7.Watermelon nd Chr10 . Chr2 coClBBXs was analyzed. Nine segmental duplication events were detected, but no tandem duplication events were identified . G-box elements were not found in ClBBX7a and 7b promoters, while the GT1-motif was absent in the promoters of ClBBX6a, 12 and 28. The largest number of G-box and GT1-motifs existed in the promoter of ClBBX29a (10) and ClBBX21c (6), respectively  was the highest among that of the 19 ClBBX promoters, while the number of W-box, WRE3 and TC-rich repeat elements in each promoter was less than three  was the highest. The largest number of AAGAA-motif, ABRE, ERE and TCA-elements was found in promoters of ClBBX7b (5), ClBBX29a (10), ClBBX29b (7) and ClBBX24 (5), respectively. Furthermore, among the 16 ClBBX promoters containing ABRE3a elements, only ClBBX15 and ClBBX24 promoters contained two elements, and the other 14 promoters contained only one  (28%) were higher in shoots than in roots. Except for ClBBX10 (the only gene with the highest transcription level in stems), the levels of six other genes in stems were lower than those in cotyledons and true leaves. Conversely, five genes  (20%) were preferentially expressed in roots rather than in shoots. The genes preferentially expressed in cotyledons (9) and true leaves (10) accounted for 76% of the total 25 genes. It was noteworthy that the transcription levels of ClBBX14, ClBBX15 and ClBBX16 in cotyledons were the highest (40~50-fold that in roots) and their levels in true leaves were much higher than those in roots (30~44-fold).To investigate the potential functions of tyledons A. Among ClBBXs in fruit development, an expression atlas was created for the genes against the RNA-seq data of four developmental stages of watermelon fruits, including white fruits , white-pink fruits (F-DAP18), pink fruits (F-DAP28), and red ripe fruits (F-DAP34) (ClBBXs (40%) were relatively low at different stages. For instance, ClBBX28 was almost not expressed in fruits, while ClBBX10, 16 and 21c were not detected in F-DAP28, and their levels were also very low at the other three fruit stages. Differently from the stable low expression pattern of ClBBX6b in different stages, ClBBX3, 19b and 30a all had the highest levels in F-DAP18. The levels of the other 15 members in fruits were high. Among them, seven genes  maintained high levels at different stages. The transcription levels of ClBBX7a, 14 and 20 were higher in early stages (F-DAP10 and F-DAP18) than in late periods (F-DAP28 and F-DAP34), whereas the reverse was true for ClBBX15. The levels of ClBBX24 and 30b were lowest in F-DAP10, while those of ClBBX29b were the lowest in F-DAP28  B. The tr F-DAP28 B.ClBBXs contained many phytohormone response elements. To explore the response of ClBBXs to plant hormones, the effects of SA and MeJA treatment on their transcription levels were analyzed  and four  genes were up-regulated and down-regulated, respectively, while the expression of other genes was not significantly changed (more than two-fold). With the extension of time after treatment, the levels of many genes showed obvious fluctuations. For example, the level of ClBBX30b was lowest at 12 h and highest after 24 h, while that of ClBBX14 and ClBBX16 was the exact opposite. The levels of ClBBX10, 21c and 27 were the highest after 3 h, while that of ClBBX6b was the lowest. Briefly, 6 h after SA treatment, only one (ClBBX30b) and seven  genes and were significantly up- and down-regulated, respectively. The promoters of analyzed . As showClBBXs significantly regulated at different times after MeJA treatment  and three down-regulated genes  were identified 1 h after MeJA treatment. Briefly, 3 h after treatment, ClBBX7a, 21c and 30b were up-regulated, and their levels reached the peak, but ClBBX14 and 16 were down-regulated and showed the lowest levels. At 6 h, ClBBX6b and 28 had the lowest levels; however, only ClBBX5 was up-regulated and peaked, with a nearly 9-fold increase. The levels of ClBBX14, 16 and 29b reached the highest at 12 h, while those of ClBBX29a and 30b reached the lowest. At 24 h, the number of significantly down-regulated genes (7) was the highest and their levels were also the lowest, whereas only ClBBX30b was significantly up-regulated.However, the effect of MeJA on the expression of this family genes was relatively weak, which was reflected in the number of reatment B. Four uClBBXs , and it did not respond to melatonin (L-MT) and/or cold stress (L-Cold). Similarly, ClBBX21b was not detected in L-Cold and L-Cold + MT tissues. Compared to L-CK, eight and three genes were significantly induced and inhibited by cold stress, respectively. Among the 11 genes, the up-regulated genes included ClBBX3, 7a, 19a, 19b, 29a, 29b, 30a and 30b, while the down-regulated genes were ClBBX7b, 14 and 21a. Except for the induced expression of ClBBX30b, almost all ClBBXs did not respond to melatonin. In contrast to cold treatment, the combined melatonin and cold treatment only induced the expression of ClBBX10 and 21c, and inhibited ClBBX6b and 30b. These data indicate that the role of ClBBX family genes in the melatonin-mediated cold stress response pathway may be limited or indirect. Moreover, the RNA-seq data of watermelon roots treated with PEG6000 (20% polyethylene glycol 6000 for 6 h) were used to analyze the effects of osmotic stress on ClBBX expression. As shown in ClBBX5, 6a, 6b, 7b, 15, 19b, 20, 21a, 22, 24, 27, 29b, 30a, and 30b. Only ClBBX16 and 28 were inhibited by PEG6000 treatment, and the other nine genes were not sensitive to osmotic stress.The RNA-seq data of leaves treated with melatonin (150 μM for 3 d) and cold stress (4 °C for 36 h) were used to analyze the effects of combined melatonin and cold treatment (MT + Cold) on the expression of ClBBXs A. As a sClBBXs under salt conditions, the levels of each ClBBX in watermelon (XN-8) leaves treated with salt (100 mM NaCl for 24 h) were monitored via qRT-PCR  did not change significantly within 24 h for salt stress treatment. The levels of seven genes were up-regulated, among which four genes  and two genes (ClBBX6a and 7a) peaked at 1 h and 3 h, respectively, while ClBBX30b reached the highest level at 24 h with a 6.5-fold increase. Eight genes were down-regulated under salt conditions, and ClBBX29b and 30a decreased sharply at 3 h and 12 h, respectively. Two (ClBBX6b and 19b) showed the lowest levels at 6 h, while the lowest levels of four other genes genes  were delayed until 24 h. Additionally, ClBBX27 peaked at 3 h and decreased significantly at 24 h, and both ClBBX21c and 28 decreased significantly at 1 h and peaked at 6 h, but the lowest levels of ClBBX28 appeared at 24 h. Therefore, ClBBXs may play a synergistic role in the watermelon salt stress response.Many plant BBXs were involved in salt stress . To inve qRT-PCR . The levBBX members in different crops is particularly important. With whole genome sequencing, analysis of BBX family has been performed in many species. However, identification of watermelon ClBBX family is still lacking.Plant BBXs play key roles in regulatory networks controlling biological processes. BBX proteins belong to zinc finger TFs, and a thorough understanding of the function of each protein in physiological processes will facilitate their applications in crop genetic improvement . TherefoClBBXs were systematically identified. The number was same as that in grapevine [Arabidopsis (32) [Ipomoea trifida (34) [Melilotus albus (20) [Arabidopsis had more BBXs than watermelon did in all clades except for Clade V. Tomato mainly had an advantage in Clade I and II, with two more members in each clade than watermelon. Although the protein number in Clade I, II, III and IV of rice was higher than that in potato, there were five members less than that in potato in Clade V. The remarkable variation in the protein number of the same clades may reflect species-specific duplication or deletions during evolution [In this study, 25 watermelon rapevine  and pearrapevine , while isis (32) , tomato sis (32) , potato sis (32) , Ipomoeaida (34)  and riceida (34) . Nonetheida (34) , and Melbus (20) . Althougvolution . Arabidopsis [Arabidopsis AtBBXs and five potato StBBXs of Clade I also contained a valine–proline (VP) motif of 6-amino acid (G-I/V-V-P-S/T-F) at the C-terminal, which may be important for protein interaction [Ipomoea trifida [BBXs contained one or two B-box motifs (B-box1 and B-box2), and sometimes also had a CCT domain . Five Cybidopsis , pear [2bidopsis , and ricbidopsis . It was bidopsis . These veraction ,23. Amoneraction  and Ipom trifida . ClBBXs, five genes were intronless, nine genes had one intron, four members contained two introns, six members had three introns, and only one gene contained four introns. Genes belonging to Clade I and III had only one intron, genes belonging to Clade 2 had three introns, and genes without introns were mainly distributed in Clade V. The genes in Clade IV had the greatest variation in introns, ranging from zero to three, which is consistent with the expansion and diversification of Clade IV members (ClBBXs without introns (20%) was much higher than that of grapevine (12%) [ClBBXs containing similar gene structures clustered together in phylogenetic trees, such as the genes with no introns (ClBBX30a and 30b), genes with one intron  and genes with three introns   [Gene structure was related to the phylogenetic relationship of a gene family . Among t members . The perne (12%) , pear (1ne (12%) , pepper ne (12%) , and tomne (12%) . ClBBXs and 19b) , indicatn tandem , indicatn tomato  and pearn tomato . Twenty-bidopsis , which wsis (26) , suggestClBBXs in various tissues was analyzed. The levels of most ClBBXs varied greatly in roots, stems, true leaves and cotyledons , accounting for 56%  and MYC  existed in the promoter of each gene, implying their potential functions in the stress response [ClBBX19b, 29b, 30a, and 30b) were markedly regulated by salt, cold and osmotic stress . It was suggested that gene expression may depend on the number, type and location of cis-acting elements in promoters. Other ClBBXs not regulated by stress may also indirectly participate in the abiotic stress response. Arabidopsis BBX24/STO was not induced by salt, but the overexpression of BBX24/STO enhanced the salt tolerance of transgenic plants, suggesting that this gene participated in salt stress responses [In addition to growth development and hormonal signaling, BBXs were also involved in abiotic stress responses ,36. All response . Four mec stress . Their pelements . It was esponses . TherefoClBBX21b, 28 and 30b in roots were much higher than those in aboveground tissues  and used as a query to search watermelon (97103) genome v2 in CuGenDB . The amino acid sequences of the Arabidopsis BBXs [http://smart.embl-heidelberg.de/, accessed on 12 June 2023) [Citrullus lanatus BBX (ClBBX) genes, and annotated based on their homology with Arabidopsis AtBBXs. Sequences of BBXs from Vitis vinifera (VviBBXs) [Pyrus bretschneideri (PbBBXs) [Solanum lycopersicum (SlBBXs) [Capsicum annuum (CaBBXs) [Solanum tuberosum (StBBXs) [Oryza sativa (OsBBXs) [To identify potential watermelon BBX family members, the hidden Markov model (HMM) profile for the B-box-type zinc finger domain (PF00643) was downloaded from Pfam (ermelon 9103 genomne 2023) . The preVviBBXs) , Pyrus b(PbBBXs) , Solanum(SlBBXs) , Capsicu(CaBBXs) , Solanum(OsBBXs)  were obthttps://www.expasy.org/, accessed on 12 June 2023). The chromosomal positions and intron numbers of ClBBXs were retrieved from CuGenDB. The subcellular locations were predicted via WoLF PSORT .The number of AAs, MW, pI and instability index   of ClBBXhttp://www.ebi.ac.uk/interpro/search/sequence/, accessed on 12 June 2023) programs were used to identify the conserved domains. Weblogo 3  was used to create the sequence logos [The CLUSTALW program was used to align the full sequences of BBXs. The NJ phylogenetic tree was constructed using MEGA-X . The parce logos .ClBBXs were identified based on their chromosomal position and the relative distance derived from CuGenDB. The diagrams of exon–intron structures of watermelon ClBBXs was generated using Gene Structure Display Server 2.0  [−9 substitutions per site per year. The divergence time (T) was calculated based on the following formula: T = Ks/(2 × 6.56 × 10−9) × 10−6 million years ago [The chromosomal locations of ne 2023) . Syntenyne 2023) . Nonsynoears ago .ClBBX promoters were submitted to PlantCARE online  for cis element prediction. With the intention to perform functional annotation identification, 25 ClBBX protein sequences were uploaded to AmiGO 2  and were utilized for gene ontology (GO) analysis. The GO analysis of ClBBXs contains biological process, molecular function, and a cellular component.The regions (2000 bp upstream of the transcription start site ATG) of ClBBXs were retrieved from CuGenDB. Specifically, the data for fruits of cultivar Dumara were obtained 10, 18, 28, 34 days after pollination  [ClBBXs are listed in ClBBX were used for drawing a heatmap.FPKM values for watermelon  and 34) . The osm and 34) . The lea and 34) . The FPKClBBXs. Seedlings were grown under the condition of 28 °C (day for 16 h)/22 °C (night for 8 h) for 30 d. The roots, stems, true leaves and cotyledons from seedlings were collected for tissue expression analysis. To study the effect of phytohormones on gene expression, seedlings were treated with 100 μM SA and MeJA for 0, 1, 3, 6, 12, and 24 h, respectively. SA and MeJA were dissolved in 0.004% ethanol, and control seedlings were treated with 0.004% ethanol. To investigate the effects of salinity on ClBBX gene expression, seedlings were treated with Hoagland solution containing 100 mM NaCl for 0.5, 1, 3, 6, 12, and 24 h, and control samples were mock-treated with Hoagland solution only. The leaves were sampled from the treated and untreated plants. The control plants were used as the corresponding controls to avoid the effects of the circadian clock on expression analysis [Watermelon cultivar XN-8 was used for the expression analysis of ClBBX .Total RNA was extracted using a total RNA kit . RNA quality and concentration were determined using NanoDrop 2000 Spectrophotometer . First-strand cDNAs were synthesized via the reverse transcription of 1400 ng of total RNA using the One-Step gDNA Removal and cDNA Synthesis Super Mix kits . The reverse-transcribed cDNA (20 μL) was diluted to a final volume of 200 μL and used for quantitative RT-PCR (qRT-PCR) experiments. Primer Premier 5.0  was used to design the primer pairs for each ClBBX . The spe2O. The reaction system was as follows: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. The fluorescent signal was measured at the end of each cycle, and the melting curve analysis was performed by heating the PCR product from 60 °C to 95 °C. Watermelon yellow-leaf-specific proein8 (ClYLS8) and β-actin (ClACT) were used as the reference genes [ClBBXs were calculated using the 2−△△CT method [ChamQ Universal SYBR qPCR Master Mix  was used to perform qRT-PCR reactions on qTOWER3 Series-Real-Time Thermal Cyclers . The total reaction system was 20 μL, including 10 μL of SYBR qPCR Master Mix (2×), 0.4 μL of each primer (10 μM), 2 μL of the cDNA samples, and 7.2 μL of ddHce genes . Three tce genes . The levT method , and heaT method .ClBBXs were identified. The comprehensive analysis of the ClBBX family revealed the conserved domains, classification, subcellular localization, gene structures, phylogenetic relationships, chromosomal distributions, genomic synteny, cis elements in promoters, and expression patterns. Most of ClBBXs were differentially expressed in tissues . The transcription of some ClBBXs was regulated by hormones  and abiotic stresses . These data indicate that watermelon ClBBXs may be involved in growth and development, hormone signaling pathways, and the abiotic stress response. Detecting the subcellular localization and transcriptional activity of key ClBBXs  regulated by abiotic stress and hormones, obtaining materials with different ClBBX expression levels through genetic transformation and genome editing techniques, and clarifying their functions in hormone and stress-induced signaling pathways will be an important task. Our research not only enriches the BBX family of horticultural crops, but also provides a foundation for the functional characterization of watermelon ClBBX family genes.In this study, 25 watermelon


AX-10887638 and AX-94875830, which control shoot length and root–shoot ratio on chromosomes 6A and 6D, respectively. Additionally, 39 SNPs were pleiotropic for multiple traits. Further functional analysis of the genes near the 39 SNPs shows that some candidate genes play key roles in encoding proteins/enzymes, such as transporters, hydrolases, peroxidases, glycosyltransferases, oxidoreductases, acyltransferases, disease-resistant proteins, ubiquitin ligases, and sucrose synthetases. Our results can potentially be used to develop low-nitrogen-tolerant species using marker-assisted selection and provide a theoretical basis for breeding efficient nitrogen-using wheat species.Nitrogen fertilizer input is the main determinant of wheat yield, and heavy nitrogen fertilizer application causes serious environmental pollution. It is important to understand the genetic response mechanism of wheat to nitrogen and select wheat germplasm with high nitrogen efficiency. In this study, 204 wheat species were used to conduct genome-wide association analysis. Nine phenotypic characteristics were obtained at the seedling stage in hydroponic cultures under low-, normal, and high-nitrogen conditions. A total of 765 significant loci were detected, including 438, 261, and 408 single nucleotide polymorphisms (SNPs) associated with high-, normal, and low-nitrogen conditions, respectively. Among these, 14 SNPs were identified under three conditions, for example,  Triticum aestivum L.) is a pivotal crop in global agriculture. It is widely cultivated and plays an indispensable role in human food production [Wheat  and NRT1.1 [Zm00001d004123 (controlling crown number), Zm00001d051083 (controlling root length and area), Zm00001d025554 (controlling plant height), and Zm00001d050798 . These genes were related to lactoylglutathione lyase, leucine-rich repeat receptor-like serine/threonine protein kinase, aspartic protease synthesis, and E3 ubiquitin protein ligase EL5 synthesis [OsTCP19, OsNAC42, OsNPF61, OsNLP4, OsNiR, etc.) were identified based on GWASs in rice. Therefore, GWASs can help to improve our understanding of the molecular basis of traits related to N-efficient species and lay a foundation for breeding such species.Wang et al. used a hydroponic system to analyze the natural variation in 22 root traits and 6 stem traits in 213 maize inbred lines under NN and LN conditions. Through GWAS, 297 significant single nucleotide polymorphisms (SNPs) were detected at different N levels and 51 candidate genes with amino acid variations in coding regions or differentially expressed were identified under LN conditions. Subsequently, researchers re-sequenced the candidate gene nditions . Schmidtd NRT1.1 . Fu et aAt present, the overapplication of N fertilizer in agricultural production has become a common phenomenon, but most of the current studies have been conducted under LN and normal N (NN) conditions ,36,37,38The variation in the phenotypic traits of the 204 wheat accessions under the three conditions was extensive and showed a normal distribution. Nine phenotypic traits were measured and best linear unbiased prediction (BLUP) values were calculated in HN, LN, and NN conditions . Compareh2) ranges of the nine phenotypic traits were from 0.135 (SDW) to 0.92 (RL), from 0.327 (TDW) to 0.79 (RL), and from 0.381 (SDW) to 0.92 (RL) in the LN, NN, and HN conditions, respectively. However, the h2 of RL in the NN condition was lower (0.79) than that of the other two conditions, indicating a significant environmental effect on RL  compared to that of the A (4.6 Mb) and B (3.5 Mb) genomes. The LD attenuation distance for the whole genome was 3.5 Mb  was detected in both the HN and LN conditions. AX-108847203 (1A) and AX-108871687 (6B) were detected in both the NN and LN conditions. AX-111028996 (1B) was detected in all three conditions.In the HN condition, three important RDW-related loci were distributed on chromosomes 1B, 3B, and 6D . The relA total of 46, 11, and 92 SNPs were significantly correlated with SL, SFW, and SDW, respectively .AX-111124688 (6A), AX-111170306 (1A), AX-111724995 (1A), AX-108847203 (1A), AX-109988415 (2A), AX-109913880 (4B), AX-110832233 (4D), AX-89398511 (4D), AX-108774819 (7B), AX-109052370 (7B), AX-109327847 (7B), AX-110483224 (7B), AX-110490694 (7B), AX-111506964 (7B), and AX-111609105 (7B) could be detected in both the NN and LN conditions. Two SNPs, AX-94550983 (1B) and AX-109627540 (7D), were detected in both the HN and NN conditions. AX-108876381 (6A) was detected in all three conditions.The SNPs significantly correlated with SL were mainly located on chromosomes 1A, 1B, and 7B . NotablySNPs significantly related to SFW were mainly found in the LN condition , and theAX-109326114 (1D), AX-108962141 (1B), AX-110832233 (4D), AX-108847203 (1A), and AX-111170306 (1A) were detected in both the NN and LN conditions.For SDW, most of the loci detected in the LN condition were distributed on 2D, 4D, 5D, and 6B . Among tA total of 104, 193, and 195 SNPs significantly correlated with TFW, TDW, and RSR were detected under different conditions .AX-109815802 (4B) was detected in both the NN and HN conditions.SNPs that were significantly related to TFW were mainly distributed on chromosomes 5A, 3B, and 7D . In the AX-108962141 (1B) and AX-110832233 (4D) were found in all three conditions.In terms of TDW, significant SNPs were concentrated on chromosomes 2D and 5D . In the AX-109374174 (2A), AX-110042217 (2A), and AX-94762593 (6D) appeared in two conditions, and AX-94875830 (6D) appeared in all three conditions.SNPs were significantly associated with RSR . Two impIn total, 765 significantly related SNPs were detected, including 438, 261, and 408 single nucleotide polymorphisms (SNPs) associated with HN, NN, and LN conditions, respectively . It is wAX-108962141 (1B) was associated with RDWN, RFWN, SLL, SDWL, SDWN, TDWH, TDWL, and TDWN; AX-108847203 (1A) and AX-111170306 (1A) were associated with RDWL, RDWN, SLL, SLN, SDWL, SDWN, TDWL, and TDWN; AX-109815802 (4B) and AX-109327593 (4B) exerted pleiotropic effects on RFWN, SFWH, SFWN, TFWH, and TFWN  were found near the three SNP loci of AX-1089962141, AX-110832233, and AX-111051748, and TraesCS2A02G161000 near AX-11004221 encodes trehalose 6-phosphate, TraesCS2A02G447200 encodes transcription factor MYB93, and TraesCS2A02G577000 encodes serine/threonine protein kinase. Two candidate genes on chromosome 2D were related to AX-111405488, including TraesCS2D02G107500 (encoding peroxidase) and TraesCS2D02G109400 (aspartate protease family); TraesCS6D02G360800, a candidate gene related to AX-94875830 on chromosome 6D, encodes phosphoenolpyruvate carboxylase kinase. The functions of these genes were mainly concentrated on stress resistance, oxidative stress responses, and protein modification pathways. Therefore, we speculate that these genes are involved in nitrogen metabolism and may play a role in nitrogen accumulation ; this is in the same region as the SNP locus QPh-4D (16–19 Mb) that controlled plant height in previous studies and may, therefore, be the same QTL site. AX-110026721, identified at 34.32 Mb on chromosome 2A, is 2 Mb apart from the locus at 30.9–32.0 Mb on chromosome 2A [AX-109621732, AX-111672733, and AX-94559451); these are 0.38, 0.000035, and 1.34 Mb away from Affx-109177363, Affx-109254520, and Affx-88661145, which were associated with TDW, SDW, and TFW in a previous study [AX-109517098) and the previous SNP for RFW under normal moisture conditions (AX-109055035) was approximately 0.73 Mb. In the HN condition, AX-109741930 and AX-108966945 on chromosome 6D for RDW were 0.97 and 0.019 Mb away from AX-110986216 and AX-108905447 for RDW, respectively [In this study, certain SNP loci were located close to known loci associated with traits. For example, in the LN and NN conditions, a polymorphic SNP locus on chromosome 4D controlled the length of the aboveground portion  were detected in all conditions when controlling for the same trait. These SNPS are more adaptable to N conditions, and further studies on these important QTLs and SNPs will be conducted in the future.Meanwhile, we identified some possible new QTLs within our known range; some important QTLs contained more than nine consecutive SNPs were only associated under a single condition, such as QTLs related to RSR, RFW, and RDW on 1B (322.4–325.2 Mb), 3B (521.8–524.5 Mb), and 6D (467.2–471.9 Mb) under HN condition, QTL sited on 1B (392.7–394 Mb) under NN condition, and QTLs correlated with both SFW and TDW traits on 2D (59.2–60 Mb) and 5A (513.9–518.3 Mb) under LN condition. These results indicate that these QTLs are affected by nitrogen levels and belong to nitrogen-sensitive QTL sites. Therefore, these QTLs have certain reference value for the subsequent marker-assisted selection of wheat varieties with high efficiency. Five SNPs , Hebei (33), Sichuan (36), Yunnan (27), Xinjiang (25), and other important wheat growing areas. A total of 36 were imported varieties, from the United States, France, Italy, Mexico, and other countries. The panel possesses high genetic diversity of wheat breeding .2O2 for 20 min, washed with distilled water, soaked in saturated CaSO4 for 6 h to disinfect the seeds, and washed under flowing distilled water until the seeds germinated. Germination paper from Anchor Company, USA was sterilized at 121 °C for 20 min to prevent fungal growth during seedling development. Each soaked wheat seed was placed upside down approximately 1.5 cm from the edge of the blotting paper, spaced approximately 1.5 cm from the next wheat seed. In total, 12 seeds of each variety were tested for each treatment. The seeds were covered with another sheet of blotting paper and rolled up from one side to the other. The rolled wheat seeds were placed in different hydroponic nutrient solutions for each treatment in an incubator with the following conditions: photoperiod, 14/10 h (light/dark); relative humidity, 45%; temperature, 25 °C; and light intensity, 8600 Lux. The nutrient solution was replaced every two days to maintain cleanliness and stability. After culturing for 15 days, all traits were determined. The experiment was repeated four times.The paper roll culture method  was usedThe root length  and shoot length  of the 12 seedlings were measured manually. Plants were divided into shoots and roots. Excess water was removed with absorbent paper before weighing with a 1/10,000 balance to obtain the root fresh weight  and shoot fresh weight . Each plant was killed at 105 °C for 30 min and dried to a constant weight at 80 °C. The root dry weight  and shoot dry weight  were determined. The root–shoot ratio (RSR), total dry weight , and total fresh weight  were calculated.h2) of each trait.Variance analysis was performed on the phenotypic data of the nine traits under treatment with different nitrogen concentrations using SPSS software . Using the R software  package “lme4”, optimal linear unbiased prediction (BLUP) values were obtained for each trait from the four repetitions, and these predictors were later used for GWAS analysis. The formula below was used to calculate the generalized heritability  method . After q2 dropped to half its maximum value [2 values and SNP pair distances and fitting these points to a smooth curve using Origin software . p = 0.001 was set as the threshold for all conditions. A neighbor-joining tree was constructed using the TASSEL 5.0  [The population structure of the panel was inferred using the STRUCTURE 2.3.4 software  for eachum value . The LD versity) .p-value of 0.001 was used to estimate significant SNPs [In order to avoid too many false positives in the results of association analysis, the affinities of the tested natural population were taken as covariates in the subsequent association analysis . The calant SNPs ,77. The t-test was used to detect and compare the significance of the differences in the target traits between the two haplotypes. Origin 2021 was used to generate a boxplot for haplotype analysis. The R package “CMPlot” was used to produce a Manhattan map and to mine candidate genes for wheat traits within the LD attenuation range of these SNP sites. The SNP sequence was used to search the Chinese Spring Wheat Genome Database and obtain the interval sequence. The BLAST sequence was compared in the NCBI database. Gene function was annotated using Gene Ontology  and the Kyoto Encyclopedia of Genes and Genomes pathway database . To investigate potential candidate genes, the Ensembl Plants database  was used to download and function query the nearby genes of significant SNPs under the three conditions [Stable SNPs in the four experiments were selected for favorable allele analysis. In the analysis of allele effects on each trait, alleles with positive effects leading to higher trait values were described as “favorable alleles,” whereas those leading to lower values were “unfavorable alleles” . Based onditions .In this study, 204 wheat accessions were evaluated related traits under low-, normal, and high-nitrogen conditions and were genotyped using Wheat55K SNP array. GWAS analysis shows that a total of 765 SNPs was significantly associated with nine traits in wheat under three different nitrogen conditions. Additionally, there are three, one, and two important QTL loci found to be associated only under HN, NN, and LN conditions, respectively. The resulting data were further analyzed to identify relevant SNP loci and candidate genes, which can be used to breed new wheat species with relatively low nitrogen fertilizer requirements and high efficiency by using marker-assisted selection in the future to achieve efficient conservation and utilization of nitrogen and nitrogen resources.


The development of female or male sex characteristics occurs during fetal life, when the genetic, gonadal, and internal and external genital sex is determined . Any discordance among sex determination and differentiation stages results in differences/disorders of sex development (DSD), which are classified based on the sex chromosomes found on the karyotype.This chapter addresses the physiological mechanisms that determine the development of female or male sex characteristics during fetal life, provides a general classification of DSD, and offers guidance for clinical, biochemical, and genetic diagnosis, which must be established by a multidisciplinary team. Biochemical studies should include general biochemistry, steroid and peptide hormone testing either at baseline or by stimulation testing. The genetic study should start with the determination of the karyotype, followed by a molecular study of the 46,XX or 46,XY karyotypes for the identification of candidate genes.46,XX DSD include an abnormal gonadal development , an androgen excess (the most frequent) of fetal, fetoplacental, or maternal origin and an abnormal development of the internal genitalia. Biochemical and genetic markers are specific for each group.Diagnosis of DSD requires the involvement of a multidisciplinary team coordinated by a clinician, including a service of biochemistry, clinical, and molecular genetic testing, radiology and imaging, and a service of pathological anatomy. I1)EMX2, CBX2, NR5A1, GATA4, and WT1) , , . A. A12]. AA variety of diagnostic algorithms have been designed , 14], , , 16], w, w16], w3)a)Biochemical tests and, especially, hormone determinations, play a crucial role in initial diagnosis of DSD, follow-up, and monitoring of response to treatment. There are two major groups of hormones: steroid and peptide hormones.Steroid hormones  are syntPeptide hormones are determined by noncompetitive immunoassays, which have a high sensitivity but which specificity is not always known. The lack of gold-standard methods and inconsistent standardization hinder inter-assay comparison. Thus, age- and sex-specific reference values are required for each assay , 28]. P. P28]. Pb)In some cases, basal hormone testing is not informative enough and stimulation tests are required to identify secretion deficiencies.There are three types of functional tests:b-1)®) at a dose of 0.25 mg . Adrenal enzyme deficiencies are investigated through determination of a range of hormones and precursors at baseline and at 60 min from stimulation , , , , 40]. C. C40]. Cc-2)in vitro . A. Ain viterformed .Structural analysis of a specific candidate gene is performed by automated Sanger DNA sequencing, which involves PCR amplification of coding and flanking regions and, eventually, of the promoter region. However, the introduction of high-throughput DNA sequencing allows whole exome testing (coding regions) or screening a panel of candidate genes. In addition, broad expressivity in some DSD phenotypes could be explained by an oligogenic origin, in which interaction of multiple genes could give rise to a phenotype unique to each individual . The avaII1)Gonadal development disorders with 46,XX karyotype include PGD and CGD, ovotesticular gonads (ovotesticular DSD) and testis development (testicular DSD) .BMP15, ESR2, FOXL2, MYRF, NR5A1, NUP107, and SOX8 genes. In most cases, the effect is dominant (except for NUP107) and, in some cases, they are associated with other phenotypic characteristics  concentrations. Other precursors such as androstenedione, 17-α-hydroxyprogesterone (17OH-P), and T also are at prepubertal concentrations, whereas dehydroepiandrosterone (DHEA) and its sulphate increase during normal adrenarche. A milder and relatively frequent clinical form is early menopause or early ovarian failure, in which biochemistry shows an early increase in LH and FSH concentrations, reduced levels of AMH (a good marker of ovarian reserve), and low levels of E2 and progesterone (P). Evidence is progressively published on monogenic causes , among teristics .SRY gene to an autosome. Some monogenic causes are associated with complex phenotypes such as mutations in NR2F2, RSPO1, SOX10, WNT4 and, more recently, WT1; in the case of the NR5A1 gene, only the p.Arg92Trp mutation causes ovotesticular or testicular development; duplications in the FGF9, SOX3, and SOX9 genes have also been described.Ovotesticular or testicular development shows genital ambiguity  from birth, due to fetal exposure to elevated levels of T. The biochemical profile in newborn or infant is similar to that observed in males with 46,XY karyotype. During childhood, the ability of the gonads to produce T can be assessed by HCG testing. During puberty, there is an increase in T concentrations that does not reach normal male concentrations, resulting in elevated levels of LH and FSH. Most monogenic causes give rise either to an ovotesticular or testicular DSD . The fir2)When the gonads differentiate into ovaries and the internal genitalia are female, fetal exposure to elevated levels of androgens causes the virilization of the external genitalia. The origin of these androgens may be fetal, fetoplacental, or maternal .a)CYP21A2 gene). Its “simple virilizing” form is associated with cortisol deficiency, ACTH elevation, and the accumulation of 17OH-P, androstendione, and T . The most frequent cause is 21-hydroxylase deficiency , 50] .CYP21A2 gene in homozygosity or in compound heterozygosity, the effect of which is the total or almost-total inactivation of enzyme activity in the most severe forms with concomitant salt loss, as well as in the simple virilizing forms. There is an association between the genotype and the degree of virilization of patients with typical forms of the enzyme deficiency  (T (TCYP21AOther less frequent causes of CAH are 3β-hydroxy-steroid dehydrogenase type 2 (3βHSD2), 11β-hydroxylase, and cytochrome P-450 oxidoreductase (POR) deficiencies .HSD3B2 gene) catalyzes two sequential reactions converting pregnenolone into P, 17OH-pregnenolone into 17OH-P, and DHEA into androstendione (HSD3B2 inactivating mutations in homozygosis or compound heterozygosis (The enzyme 3βHSD2 (tendione . Patientozygosis .The enzyme 11β-hydroxylase converts 11-deoxicortisol into cortisol and 11-deoxycorticosterone into corticosterone . 11β-hydCYP11B1 gene in homozygosis or compound heterozygosis (There are mild forms of the enzyme deficiency . Moleculozygosis .GRα or NR3C1)   causes cESR1 gene)   is a verb)c that plays a crucial role in electron transfer from NADPH to microsomal enzymes P450 . POR deficiency is characterized by a partial, varying impairment of different enzyme activities (POR-inactivating mutations in homozygosis or compound heterozygosis (P450-oxidoreductase (POR) is a flavoprotein bound to the membrane of cytochrome tivities : 17α-hydtivities . The steozygosis .CYP19A1 gene in homozygosis or compound heterozygosis (The enzyme aromatase (CYP19) catalyzes T conversion into E2 and DHEA conversion into estrone (E1). In the absence of aromatase activity, the placenta cannot convert DHEA sulphate, produced in large amounts by the fetal adrenal gland, into estrogens  and transforms it into T, which causes virilization of the 46,XX fetus and the mother . Patientozygosis .Androgen-producing fetal or placental tumors: cases have been reported of congenital adrenal tumors causing the virilization of a 46,XX fetus.c)Excess androgens may be transferred from the mother to the 46,XX fetus, either by secretion from virilizing tumors during pregnancy (including pregnancy luteoma and Krukenberg tumor), by pharmacological therapies or by environmental pollutants with androgenic effects, or by poor therapeutic monitoring during pregnancy of a mother with CAH .3)Some individuals may exhibit isolated malformations of female internal genital ducts . These malformations may be caused by incomplete development or the presence of abnormal structures . They arHOXA13 gene); MURCS syndrome ; and the MRKH (Mayer–Rokitansky–Kuster–Hauser) syndrome types I and II, where several genetic disorders have been described; and finally WNT4-inactivating mutations, which have been associated with ovotesticular or testicular development with potential Mullerian duct aplasia (There are no specific biochemical markers. Only amenorrhea, dysmenorrhea, and infertility may give some diagnostic guidance. Malformations include aplasia or hypoplasia of the uterus and the fallopian tubes, a bicornuate or bipartite uterus that may be associated with malformations in other systems or tissues, such as the hand–foot–genital syndrome (associated with the  aplasia .


Flammulina filiformis, a typical agaric fungus, is a widely cultivated and consumed edible mushroom. Elongation of its stipe (as the main edible part) is closely related to its yield and commercial traits; however, the endogenous hormones during stipe elongation and their regulatory mechanisms are not well understood. Gibberellin (GA) plays an important role in the regulation of plant growth, but little has been reported in macro fungi. In this study, we first treated F. filiformis stipes in the young stage with PBZ (an inhibitor of GA) and found that PBZ significantly inhibited elongation of the stipe. Then, we performed GA-targeted metabolome and transcriptome analyses of the stipe at both the young and elongation stages. A total of 13 types of GAs were detected in F. filiformis; the contents of ten of them, namely, GA3, GA4, GA8, GA14, GA19, GA20, GA24, GA34, GA44, and GA53, were significantly decreased, and the contents of three  were significantly increased during stipe elongation. Transcriptome analysis showed that the genes in the terpenoid backbone biosynthesis pathway showed varying expression patterns: HMGS, HMGR, GPS, and FPPS were significantly upregulated, while CPS/KS had no significant difference in transcript level during stipe elongation. In total, 37 P450 genes were annotated to be involved in GA biosynthesis; eight of them were upregulated, twelve were downregulated, and the rest were not differentially expressed. In addition, four types of differentially expressed genes involved in stipe elongation were identified, including six signal transduction genes, five cell cycle-controlling genes, twelve cell wall-related enzymes and six transcription factors. The results identified the types and content of GAs and the expression patterns of their synthesis pathways during elongation in F. filiformis and revealed the molecular mechanisms by which GAs may affect the synthesis of cell wall components and the cell cycle of the stipe through the downstream action of cell wall-related enzymes, transcription factors, signal transduction and cell cycle control, thus regulating stipe elongation. This study is helpful for understanding the roles of GAs in stipe development in mushrooms and lays the foundation for the rational regulation of stipe length in agaric mushrooms during production. Flammulina filiformis is a large-scale edible mushroom produced industrially worldwide for its excellent culinary and medicinal value, including antitumour, antihypercholesterolemic, antihypertension, and other therapeutic benefits , and it can promote cell division because it shortens the time between the G1 and S phases of the cell cycle . Their main physiological function in plants is to promote plant growth and development, mainly through an increase in cell number and cell elongation. GA can increase cell division in some plants  medium and incubated at 25°C at constant temperature and protected from light; substrate cultivation experiments were conducted according to the method described by Tao et al.  was sprayed on young fruiting bodies as an inhibitor of GA synthesis  and pass the supernatant through a 0.22 μm filter membrane, then carry out HPLC-MS/MS detection (the extraction process should be kept on the ice box at 4°C).Hormone extraction: (1) take out the ultra-low temperature preserved sample, grind it in liquid nitrogen to dry powder, weigh the appropriate amount of fresh sample in a glass test tube; (2) add isopropyl alcohol-water-hydrochloric acid extraction solution to the glass test tube; (3) add 8 μL of 1 μg/ml internal standard solution and shake at low temperature for 30 min; (4) add dichloromethane and shake at low temperature for 30 min; (5) centrifuge at low temperature for 5 min at 13,000 r/min and extract the lower organic phase; (6) avoid light, blow dry the organic phase with nitrogen, and re-solvate with methanol (0.1% formic acid); (7) centrifuge at 4°C for 10 min  column: Poroshell 120 SB-C18 reversed-phase column ; (2) column temperature: 30°C; (3) mobile phase: A:B=(methanol/0.1% formic acid):(water/0.1% formic acid); (4) elution gradient: 0–1 min, A = 20%; 1–9 min, A increasing to 80%; 9–10 min, A = 80%; 10–10.1 min, A decreasing to 20%; 10.1–15 min, A = 20%; (5) injection volume: 2 μl, A = 80%; 9–10 min, A = 80%; 10–10.1 min, A decreases to 20%; 10.1–15 min, A = 20%; (5) Injection volume: 2 μl.Mass spectrometry conditions: electrospray ionization (ESI) source atomization temperature: 400°C, curtain gas (CUR): 15 psi, spray voltage, IonSpray voltage (IS): 4,500 V, atomization gas pressure (Gas1): 65 psi, auxiliary gas pressure (Gas2): 70 psi, monitoring mode: multiple reaction monitoring mode (MRM). Gas2: 70 psi, monitoring mode: MRM (multiple reaction monitoring mode). In the Q-Trap 6500, each ion pair is scanned for detection based on optimized declustering potential (DP) and collision energy (CE). The amount of GA is quantified in ng/g.Samples used for RNA extraction weighed 0.5 g. Total RNA was extracted from samples using the OMEGA E.Z.N.A. A plant RNA kit  was used for transcriptome sequencing when the A260/A280 ratio of RNA was between 2.0 and 2.2 .F. filiformis (6-3). Subsequently, after converting the SAM files to BAM files and reordering them using SAMtools software, the raw read counts of each gene in each sample were obtained using FeatureCounts software, and after filtering out the non-expressed genes using awk command, the count values were then converted to FPKM values using TBtools .The extracted RNA was entrusted to high-throughput transcriptome sequencing by Beijing Novogene Technology Co., Ltd. Transcriptome data preprocessing is as follows: First, quality assessment of the raw transcriptome data was performed using FastQC, followed by quality control using fastap software to obtain clean data. The SAM file was obtained by comparing the clean data using Hisat2 software using the reference genome and annotation information of the laboratory-assembled t-test. P-values of < 0.05 were considered to be statistically significant.The values in the statistical plots are the mean ± standard deviation of the three biological replicates; the significance analysis was performed using a F. filiformis strain FY006 is shown in After growing out of the primordium, the growth of the fruiting body of F. filiformis was investigated, and the phenotypes of the fruiting bodies before and after PBZ treatment are shown in F. filiformis.The effect of PBZ (an inhibitor of GA biosynthesis) on the growth of F. filiformis, targeted metabolomic analysis was performed on stipe samples at different developmental stages, and the contents of 19 GAs in the stipe of F. filiformis at the young fruiting body stage and elongation stage were determined by LC-MS/MS, namely GA1, GA3, GA4, GA5, GA6, GA7, GA8, GA9, GA13, GA14, GA15, GA19, GA20, GA24, G29, GA34, GA44, GA51, and GA53; the heatmap of GA content  is shown in To investigate the synthesis and accumulation of GAs in the stipe of Among the 13 GA species with detectable levels, the highest GA29 content was found in the elongation stage stipe. Ten species of GAs were downregulated from the young fruiting body to the elongation stipe, namely, GA3, GA4, GA8, GA14, GA19, GA20, GA24, GA34, GA44, and GA53, among which nine species were downregulated by more than 50% except for GA34, which was downregulated by 33.6%. Among GAs with increased content were GA5, GA9 and GA29, which were present at significantly higher levels during the elongation stage than during the young fruiting body stage, with GA5 increasing by 15.5% and GA9 and GA29 increasing by more than 2-fold.F. filiformis stipe  compared with the young fruiting body stage (YF), for a total of 635 DEGs.To investigate the molecular regulation mechanism of GA during the development of F. filiformis, which includes the GA biosynthesis pathway, were not annotated in the KEGG background database.The results of the KEGG enrichment analysis of differentially expressed genes showed that the DEGs upregulated in the elongation stage were mainly enriched in lipid metabolism, carbohydrate metabolism, glycan biosynthesis and metabolism, biosynthesis of secondary metabolites, and terpenoid backbone biosynthesis . The dowA. chinensis from the juvenile stage to the mature stage showed that cellular components (CC) were mainly enriched in membrane (GO:0016020), endoplasmic reticulum (GO:0005783), transporter complex (GO:1990351), and oxidoreductase complex (GO:1990204); molecular function (MF) was mainly enriched in oxidoreductase activity (GO:0016491), transmembrane transporter activity (GO: 0022857), gluconokinase activity (GO:0046316), and transcription regulatory region sequence-specific DNA binding (GO:0000976); biological process (BP) is mainly enriched in response to stimulus (GO:0050896), oxidation–reduction process (GO:0055114), secondary alcohol biosynthetic process (GO:1902653), and oligopeptide transport , J , M , and Z (cytoskeleton). The specific groups of downregulated genes include A (RNA processing and modification), C (energy production and conversion), F (nucleotide transport and metabolism), and Y (nuclear structure). Among the specific groups, the C group enriched with downregulated genes had the largest difference.F. filiformis , and all four enzyme-encoding genes were upregulated in expression, which would increase the formation of the diterpenoid synthesis precursor GGPP and thereby affecting the synthesis of GA.Using genes related to terpenoid backbone biosynthesis and diterpenoid biosynthesis (contains GA biosynthesis), the KEGG pathway related to terpenoid biosynthesis in plants and fungi, as a reference, we used local BLASTp with 6-3 as the reference genome, setting an e-value<1e-5, and combined the results with the annotation results of the three major databases SwissProt, TrEMBL, and Nr to obtain homologues that might be involved in GA biosynthesis in liformis . Eleven ynthesis ; HMGS, HF. filiformis in response to endogenous hormones. Six upregulated DEGs involved in the signal transduction pathway were screened using the KEGG pathway and KOG enrichment results combined with transcriptome data  was significantly upregulated and Ghfp2 and Ghfp3 were significantly downregulated; Elf2 (elongation factor 2) gene was significantly upregulated; Psph (phosphopyruvate hydratase) gene was significantly downregulated.According to the results by Li et al., the rapid elongation of the stipe is also closely related to the remodeling of the cell wall structure. We investigated the transcription level of cell wall-related enzyme-encoding genes in F. filiformis and all agaric fungi. In this study, we applied targeted metabolomic and transcriptomic analyses to reveal the molecular mechanism by which GA regulates stipe elongation. First, we determined the contents of 19 GAs in the stipe of F. filiformis using LC-MS. Among the 13 detectable GAs, 10 GAs  were downregulated during the elongation stage, and 3 GAs  were upregulated during the elongation stage. The changes in GA content may be closely related to the elongation of the stipe, and the accumulation of various GAs in the young fruiting body stage may lay the foundation for the rapid elongation of the stipe in the following developmental period. Among more than 130 GAs identified so far, GA1, GA3, GA4, and GA7 had the highest biological activity in plant  was downregulated with the elongation of the stipe, and similarly, the GA4 content showed a downregulated pattern from the primordium stage to the fruiting body stage in Sanghuangporus baumii, with a similar trend of change in F. filiformis , and protein kinases may be second messengers in the GA signaling pathway  and v5-210801 (FfZCP68) are both Zn2Cys6-type transcription factors, and v5-207229 and v5-212490 are both Cys2His2 (C2H2) type transcription factors. It is hypothesized that Cys2His2 (C2H2) and Cys6 (C6) zinc-finger type transcription factors have important roles in the process of GA affecting stipe elongation.The signal transduction pathway is an important pathway involved in growth and development of FfGs6 was significantly upregulated in yellow F. filiformis, which is consistent with the previous findings  can be found at: NCBI—PRJNA999472.HL, BX, and YT conceived and designed the research. HL, XM, WX, LS, and SY conducted the experiments. HL, SY, XM, HJ, ZL, YZ, BX, and YT analyzed the data and revised the manuscript. HL, SY, XM, and YT wrote the manuscript. All authors have read and approved the manuscript.


As a key nonviralgene therapy vector, poly(β-amino ester)(PAE) has demonstrated great potential for clinical application aftertwo decades of development. However, even after extensive effortsin structural optimizations, including screening chemical composition,molecular weight (MW), terminal groups, and topology, their DNA deliveryefficiency still lags behind that of viral vectors. To break throughthis bottleneck, in this work, a thorough investigation of highlybranched PAEs (HPAEs) was conducted to correlate their fundamentalinternal structure with their gene transfection performance. We showthat an essential structural factor, branch unit distribution (BUD),plays an important role for HPAE transfection capability and thatHPAEs with a more uniform distribution of branch units display bettertransfection efficacy. By optimizing BUD, a high-efficiency HPAE thatsurpasses well-known commercial reagents , jetPEI, and Xfect) can be generated. This work opens anavenue for the structural control and molecular design of high-performancePAE gene delivery vectors. During the recent Covid-19 outbreak,nucleic acid vaccines from Pfizer, Moderna, Johnson & Johnson,AstraZeneca, etc., were mass-produced and supplied worldwide, savinghundreds of millions of lives.11 Duringthe past two decades it has emerged rapidly in gene delivery withthe advantages of biodegradability, easy synthesis and modification,and high gene delivery efficiency.12 In 2016, highlybranched PAEs were synthesized by Wang et al. (HPAEs) via a one-pot“A2 + B3 + C2”-type Michael addition approach.13 It was found that the branched structure significantlyenhanced the transfection efficiency compared to the correspondinglinear PAE (LPAE) and commercial transfection reagents. Additionally,different optimization strategies, for example, molecular designs and a precise synthesis , were applied to efficientPAE development.16 So far, thousands of PAE vectors have been synthesizedand developed for clinical applications.18 However, their transfection performance is stillnot comparable to that of viral vectors.19 The gene delivery behavior of polymer vectors isclosely related to their chemical structure, thus, to pursue the aimof improving the gene delivery performance of PAEs, the structuralproperties of PAEs have been continuously studied and optimized. While,through the studies during the past few decades, all the common structuralfactors of PAEs and topology etc.) have been explored so far,22 it seems that the optimization of the structure ofPAEs has reached its limit. Does this mean that the optimization ofPAEs has come to an end?As one of the notablepolymer vectors, poly(β-amino ester)(PAE) was first developed and applied as a transfection reagent byLanger’s group in 2000.To answer this question, it is necessaryto go back and carefullyconsider the fundamental synthesis and structure of PAEs to identifywhether there are any critical structural characteristics that affectthe polymer transfection efficiency but have not been realized. Inthis work, on the basis of the current most promising HPAE, a newstructural factor, branch unit distribution (BUD), was for the firsttime found to impact the gene transfection performance. The correlationbetween the BUDs of HPAE and their gene transfection performance wasinvestigated by comparatively analyzing the behavior of differentHPAEs with different BUDs during the key steps of gene transfection.The results from this work provide a new principal for the futuredevelopment of highly efficient transfection vectors from the perspectiveof polymer structural control.13  and proton nuclear magnetic resonance spectroscopy (1H NMR). Mw,GPC values of 15.3, 12.2, and 13.2 kDa with the same dispersity(Đ) of 2.6, respectively. The Mark–Houwinkexponent alpha (α) values of HPAE-A1, HPAE-B1, and HPAE-C1 are0.26, 0.27, and 0.28, respectively , pentaerythritol tetraacrylate, and 1,4-butanediol diacrylate  via a facile one-pot “A2 + B4 + C2”-type Michaeladdition approach13 1A at var13 1.1-.Moreover, as shown by flow cytometry (S3), thepercentage of GFP-positive RDEBK cells achieved by HPAE-A1 and HPAE-B1polyplexes is only 37% and 64% at the polymer/DNA weight ratio (w/w)of 20:1, respectively. In contrast, HPAE-C1 polyplexes achieved amuch higher level of GFP-positive cells, 77% and 89% at w/w ratiosof 20:1 and 30:1 respectively, which is also greater than that ofcommercial reagents .In addition, the median fluorescence intensity (MFI) of RDEBK cellstransfected by HPAE-C1 polyplexes at a w/w ratio of 30:1 is higherthan that transfected by HPAE-A1, HPAE-B1, and the commercial reagentcontrols.Subsequently,HPAE-A1, HPAE-B1, and HPAE-C1 were applied to thein vitro analysis to assess their transfection capability. The efficiencyof these three HPAE vectors in gene transfection was evaluated byexamining the cytoplasmic green fluorescent protein (GFP) expression.Well studied commercial transfection reagents, Lipo3000, jetPEI, andXfect, were used as positive controls, providing a good benchmarkingfor comparison. The human-derived embryonic kidney cells (HEK-293)and a disease model, recessive dystrophic epidermolysis bullosa keratinocytes(RDEBK), were used for in vitro assessment. 25 The first step for successfulgene delivery is that vectors can effectively package DNA to formnanosized polyplexes. The interaction of HPAE-A1, HPAE-B2, and HPAE-C3with DNA was analyzed by a PicoGreen assay, dynamic light scattering(DLS), and transmission electron microscopy (TEM). As shown in Figure S4), indicating the strong electrostatic interaction of threeHPAE vectors with DNA. Then, the serum stability of the polyplexesof HPAE-A1, HPAE-B1, and HPAE-C1 was tested. Given that DNA uptakeis a continuous process and there is interference of serum proteinsin the physiological environment, polyplex stability is an essentialfactor that needs to be considered for efficient gene delivery. To further confirm and analyze the mechanism behind the abovevariationsin HPAE gene transfection performance, HPAE-A1, B1, and C1 were carefullystudied in terms of several key steps in gene transfection: DNA bindingand condensation, serum stability, polyplex cellular uptake, and DNAprotection.Đ, and BD exhibitsimilar transfection performance. Therefore, we considered whetherthere were other critical structure factors, beyond those that arecommonly evaluated, including topology, composition, MWs, Đ, BD, etc., that could cause the different transfectionperformances of HPAE-A1, HPAE-B1, and HPAE-C1. To clarify this, furtherstudy is required to explore more subtle structural differences betweenHPAE-A1, HPAE-B1, and HPAE-C1, the findings from which may open anew avenue for the PAE vector design.The above results demonstrated that compared to HPAE-A1 andHPAE-B1,HPAE-C1 contributed more to the polyplex stability and to DNA protection,which are favorable for cellular uptake, thus ultimately promotinghigher gene transfection efficiency. However, these results contradictour conventional understanding that polymer vectors that have similartopology, composition, MWs, 28 The introductionof “Bn” not only brings more terminal groups, but alsochanges the configuration of the macromolecule from a 2D linear toa 3D branched structure. The more “Bn” present, themore pronounced this transition. Since HPAE is composed of a groupof macromolecules of different sizes (Đ >2),13 where the structure of the multifunctionalmonomerand bifunctional monomer are randomly distributed, the distributionof the branching unit “Bn” on different macromolecularchains may vary depending on the synthesis method. This inspired usto hypothesize that, besides the common properties  of the entire HPAE polymer, the branchunit distribution in HPAE could play a role for gene delivery efficiency.As mentioned before, minimalchanges in the polymer structure couldhave an important impact on transfection efficiency. Therefore, itis conceivable that the variations in the HPAE structure due to thedifferent synthesis concentrations could be responsible for theirdifferent transfection properties. The synthesis strategy of HPAEis to introduce a multifunctional monomer “Bn” to thestep growth polymerization (SGP) process.With this question in mind, HPAE-A1, HPAE-B1, and HPAE-C1 werefractionated to obtain a series of polymer components within differentranges of MWs, HPAE-AS1–AS5, HPAE-BS1–BS5, and HPAE-CS1–CS5,respectively, via elution fractionation. As depicted in Table S1 show the GPC and 1H NMR characterization results for each individual component HPAE-AS1–AS5,HPAE-BS1–BS5, and HPAE-CS1–CS5 fractionated from HPAE-A1,HPAE-B1, and HPAE-C1 respectively. These data clearly illustrate theparallel movements of the molecular weights from low to high, Mw,GPC = 4542–30525 Da for HPAE-AS1–AS5; Mw,GPC = 2444–26535 Da for HPAE-BS1–BS5; Mw,GPC = 6321–27130 Da for HPAE-CS1–CS5.1H NMR), the ratiosof PTTA units to BDA units on each polymer component fractionatedfrom HPAE-A1, HPAE-B1, and HPAE-C1 were calculated here to evaluatethe BUD, i.e., PTTA distribution in HPAE-A1, HPAE-B1, and HPAE-C1(Table S1 and S5–S7). Then, using a method similar to Based on the above BUD characterization, it is understandable whyHPAE-C1 exhibited better transfection performance compared to HPAE-A1and HPAE-B1, though all general structural information was similar.On the one hand, the higher ratio of PTTA units in the low MW componentsof HPAE-C1 brought more terminal groups, which contributed to theshielding of the DNA charges, regular coil folding, and DNA compression.On the other hand, the lower content of PTTA in the high MW componentsof HPAE-C1 enabled it to form a more flexible high-MW structure, whichcould wrap more tightly around the polyplex, resulting in the superiorstability of the nanoparticle structure. Therefore, the HPAE-C1/DNApolyplex with its better capacity for DNA binding, protection, andserum stability is more favorable for cellular uptake, thus improvingthe gene transfection efficiency. According to the above studies,the BUD of HPAE, which has not been evaluated in previous HPAE vectordesigns, has an important effect on gene delivery performance.Đ, and BD. Inspired by this,a thorough investigation of the subtle structural characteristicsof the HPAEs was conducted and correlated with their behavior duringthe key steps of gene transfection. We show here for the first timethat BUD acts as an essential structural factor for HPAE transfectioncapability. It is demonstrated that HPAE, with a more uniform distributionof branch units , maintainsbetter transfection efficacy. This provides valuable insights intothe structural control and future development of high-performancepolymer gene delivery vectors.In this work it is reported that the HPAE vector synthesized ata lower reaction concentration achieved better gene delivery performancecompared to the HPAE vectors synthesized at higher reaction conditionswith similar MWs,


Joubert syndrome (JS) is a group of rare ciliopathies, mainly characterized by cerebellar dysplasia representing the “molar tooth sign (MTS)” on neuroimaging, hypotonia, and developmental delay. Having a complicated genotype-phenotype correlation due to its rich genetic heterogeneity, JS is usually combined with other organic defects affecting the retina, kidney, and liver. This report aimed to present new cases and novel variants of JS.TCTN2, CPLANE1, INPP5E, NPHP1, and CC2D2A genes.Five unrelated patients who were diagnosed with JS, with or without typical clinical characteristics, received integrated examinations, including whole-exome sequencing (WES) and Sanger sequencing. We identified nine pathogenic variants in the TCTN2, CPLANE1, and INPP5E genes were reported. The findings broadened the genotypic spectrum of JS and contributed to a better understanding of genotype-phenotype correlation.Four novel pathogenic mutations in the The online version contains supplementary material available at 10.1186/s12920-023-01669-7. NPHP1 and AHI1 associated with JS came to light [OFD1 gene, and recently an autosomal dominant pattern is unveiled by causative variants in the SUFU gene [Joubert syndrome (JS) is a rare developmental malformation, also known as a ciliopathy, which is mainly inherited in an autosomal recessive manner . Accordito light , 5. So fto light . JS-causto light , which nto light . The abeUFU gene , 9.The classical features of JS include the “molar tooth sign (MTS)” on neuroimaging, hypotonia with subsequent ataxia, and developmental delay (DD) or intellectual disability . The canPreviously, a group of diseases with MTS and typical clinical features of JS combined with systemic involvements were named “JS-related disorders”. Such diseases were divided, based on associated comorbidities, into clinical subtypes as follows: classic or pure JS, JS with retinal disease (JS-Ret), JS with renal disease (JS-Ren), JS with oculorenal disease (JS-OR), JS with hepatic disease (JS-H), JS with oral-facial-digital features (JS-OFD), JS with acro-callosal features (JS-AC), and JS with Jeune asphyxiating thoracic dystrophy (JS-JATD). “Joubert syndrome” is now used as a general term for this group of diseases .TCTN2, CPLANE1, and INPP5E genes that enriched the genotypic spectrum of JS.In this study, we described five patients with or without the typical clinical symptoms of JS, and using whole-exome sequencing (WES), we pointed out four novel pathogenic mutations in the Patient 1 was a 3-month-old boy admitted to our department due to hypotonia. The patient, who was the third child in the family, was delivered vaginally at term and weighed 3280 g at birth. His parents insisted on the delivery, despite of the fetal brain MRI performed at 28 weeks of gestation suggesting the cerebellar vermis was absent. The patient also had a hearing impairment and failed to control his head with a soft neck. Gesell scores for the patient indicated a moderate to severe DD: 40 in gross motor, 54 in fine motor, 40 in adaptability, 40 in language ability, and 34 in social ability. His cranial MRI records revealed a typical MTS, an enlarged fourth ventricle, and cerebellar vermis dysplasia. However, the images could not be recovered due to a data disaster. Other routine physical exams and biochemical tests were all normal. His parents and the 6-year-old sister were healthy. The 12-year-old sister, showing claw-like hands with slender, curving fingers and intellectual disability, was unable to walk until she was five years old and fell frequently. We failed to follow up on this patient and his family members due to the COVID-19 outbreak in 2019.Patient 2, female, the only child, was admitted to our department at 9 months due to delayed developmental milestones. She had hypotonia, sitting unsteadily and making incomprehensible sounds. Her mother was treated with levothyroxine because of hypothyroidism during pregnancy, though, the patient was born at term with a birth weight of 3000 g and with a normal thyroid function. However, she had poor visual fixation and esotropia after birth. At the age of 10 months, her Gesell assessment revealed a moderate to severe DD: 35 in gross motor, 35 in fine motor, 35 in adaptability, 40 in language ability, and 40 in social ability. Cranial MRI showed a typical MTS and “batwing” appearance of fourth ventricle  and c.1147G > T (p.E383*) (NM_024809.5)  (NM_023073.4), the other is a haploid replication of exon 34–41 (NM_023073.4), detected by fluorogenic quantitative PCR  and c.1393G > A (p.V465I) (NM_019892.6)  (NM_000272.5), while both his parents sheltered a heterozygous deletion in the NPHP1 gene (exon 1–20) respectively  and c.4238G > A (p.C1413Y) (NM_001080522.2)  Translational Medical Research Center Co. Ltd.  for trio-based WES. Sanger sequencing and the American College of Medical Genetics and Genomics (ACMG) clinical practice guidelines  were appTCTN2, CPLANE1, INPP5E, NPHP1, and CC2D2A genes, confirming the JS diagnosis.The diagnosis of JS takes MTS as a necessary criterion, classically characterized by thickened and elongated superior cerebellar peduncles, with the major manifestations including hypotonia and DD. As one of the ciliopathies, JS also has overlapping symptoms with Meckel syndrome, nephronophthisis or other diseases, especially the extra-neuro symptoms such as polydactyly, retinal dystrophy, polycystic kidney, and renal/liver fibrosis . In thisTCTN2 gene comprising of previously unreported mutations c.916 C > T (p.Q306*) and c.1147G > T (p.E383*). Compared to other JS-associated genes, TCTN2 gene mutations are more likely to result in intellectual disability and less likely to result in renal, hepatic, or retinal involvement [TCTN2 localizes to the outer compartments of TZ  and is colvement . SimilarCPLANE1 lead to dysfunction of TZ, abnormalities of ciliogenesis and Shh signal transduction, causing defects in cerebellar developmental [CPLANE1 gene is the latest discovery. The other genomic alteration c.1819_1820insT in the CPLANE1 gene is novel, but the amino acid change p.Y607Lfs*12 (rs777686211) shares the same result with a previously reported case  [A component of TZ, CPLANE1 is commonly associated with JS cases, of which over 125 mutations were identified, accounting for about 8–14% of all JS patients , 23. The07Lfs*6) .NPHP1 gene (exon 1–20) results in loss of function of the protein, which has been observed in several other JS patients [CC2D2A and AHI1. The relationship between JS genotype and seizure is ambiguous, but seizure management is similar for JS and non-JS individuals [CC2D2A gene was previously reported in JS [NPHP1 and CC2D2A similarly function as essential components of TZ, playing roles in ciliary development. In terms of Patient 4, the homozygous deletion in the patients , 25. Notpatients , 25, sugividuals . As for ed in JS , and c.2ed in JS .INPP5E gene was previously detected in a patient with inherited retinal degenerations [Unlike the previously mentioned proteins, INPP5E localizes to various subcompartments of primary cilium in a TZ-dependent manner and regulates ciliogenesis through phosphoinositide 3-kinase signaling pathway . The varerations , and c.6Since there is no curative therapy for patients with JS, only regular follow-up and symptomatic treatment are practicable. Although a gene-targeted therapy for JS has entered the early stage of research, the extreme genetic heterogeneity of JS and the differences between human and animal models make the research exceedingly difficult . PrenataIndeed, our research had certain limitations. First, we cannot regain the MRI images of Patient 1 and lost the follow-ups, which makes it an imperfect case. Second, patients from other cities cannot receive timely rehabilitation and developmental assessments because of the COVID-19 epidemic. Third, the siblings of Patient 1 and 3 also had related symptoms but they did not receive proper diagnosis and treatment due to family reasons.TCTN2, CPLANE1, and INPP5E genes. Our findings enrich the spectrum of pathogenic variants in JS and provide more practical experience in genetic diagnosis and counseling. Hoping that our reports could shed light on the clinical diagnosis of JS and trigger further studies on therapies.In conclusion, we diagnosed JS in five patients, and the WES data revealed four novel variants in the Below is the link to the electronic supplementary material.Supplementary Material 1


Maize serves as a crucial nutrient reservoir for a significant portion of the global population. However, to effectively address the growing world population’s hidden hunger, it is essential to focus on two key aspects: biofortification of maize and improving its yield potential through advanced breeding techniques. Moreover, the coordination of multiple targets within a single breeding program poses a complex challenge. This study compiled mapping studies conducted over the past decade, identifying quantitative trait loci associated with grain quality and yield related traits in maize. Meta-QTL analysis of 2,974 QTLs for 169 component traits  revealed 68 MQTLs across different genetic backgrounds and environments. Most of these MQTLs were further validated using the data from genome-wide association studies (GWAS). Further, ten MQTLs, referred to as breeding-friendly MQTLs (BF-MQTLs), with a significant phenotypic variation explained over 10% and confidence interval less than 2 Mb, were shortlisted. BF-MQTLs were further used to identify potential candidate genes, including 59 genes encoding important proteins/products involved in essential metabolic pathways. Five BF-MQTLs associated with both quality and yield traits were also recommended to be utilized in future breeding programs. Synteny analysis with wheat and rice genomes revealed conserved regions across the genomes, indicating these hotspot regions as validated targets for developing biofortified, high-yielding maize varieties in future breeding programs. After validation, the identified candidate genes can also be utilized to effectively model the plant architecture and enhance desirable quality traits through various approaches such as marker-assisted breeding, genetic engineering, and genome editing. Zea mays L.), the third most prevalent cereal crop after wheat and rice, is consumed as a staple food in many developed and developing countries worldwide  constructing a high-density consensus genetic map, ii) identifying robust and consistent MQTLs with narrower confidence intervals compared to initial QTLs, iii) determining precise flanking markers for MQTLs to facilitate MAB, iv) validating MQTLs through genome-wide association studies (GWAS), v) identifying CGs within promising MQTL regions, and vi) conducting expression analysis of the identified CGs. It is anticipated that these efforts will greatly enhance the selection efficiency for various yield-related and quality traits in maize.in silico expression analysis, v) synteny analysis among the maize, wheat, and rice for the key genomic regions. By employing these steps, the study aimed to gain insights into the genetic basis underlying both yield and quality-related traits in maize and related cereals.In the present analysis, a MQTL analysis was conducted to identify the genomic regions associated with yield and quality-related traits in maize. The analysis involved following five key steps: i) bibliographic search and compilation of QTL mapping studies related to quality and yield-associated traits, ii) integration of high quality linkage maps and markers from the individual studies to create a consensus map, iii) prediction of MQTLs and their validation using GWAS, iv) mining genes within potential MQTL regions and https://scholar.google.com/) and PubMed Central (https://www.ncbi.nlm.nih.gov/pmc/), using appropriate keywords. The collected studies provided essential data for the meta-QTL analysis, including the size and type of the mapping population, the specific map used in each study, the marker positions associated with QTLs, the traits linked to the QTLs, the confidence interval of the markers, the LOD score, and the phenotypic variation explained by individual QTLs (represented as R2 or PVE). However, certain studies were not included in the meta-analysis due to the lack of important data. Further, to facilitate clarity and uniqueness, the QTLs used in the present study were assigned distinctive names, which included the trait name, followed by the chromosome number, and finally, the number of the QTL on the chromosome.The various traits that impact both maize quality and yield are illustrated in https://www.maizegdb.org/data_center/map) These maps were carefully compiled and enriched to ensure accuracy and reliability. Furthermore, all the markers flanking the QTLs extracted from different studies were integrated into this consensus map, facilitating the projection of a greater number of QTLs. The construction of the consensus map was accomplished using the LPMerge package within the R environment as described by To construct a highly dense consensus map, the present study incorporated two high-quality, high resolution, mixed marker  linkage maps, i.e., “ISU Integrated IBM 2009” and IBM2 (intermated B73/Mo17) . Additionally, QTLs without available LOD or PVE values were excluded, as well as those lacking information on flanking markers and genetic positions. Subsequently, the selected QTLs were projected onto the consensus map using BioMercator v4.2.3 software, which served as the projection tool . For QTLIn this study, maize quality and yield-related traits were considered as meta-traits. A meta-QTL refers to a genomic region that contains two or more QTLs for a trait, obtained from at least two different mapping studies. The prediction of meta-QTLs was carried out using a two-step process described by https://www.maizegdb.org/), GrainGenes (https://wheat.pw.usda.gov/GG3/), and Gramene (https://www.gramene.org/) were utilized to obtain information on the nucleotide sequences of the flanking markers. The obtained sequence information was then subjected to BLAST analysis against the reference genome of maize available on the Ensembl Plants database (https://plants.ensembl.org/index.html).To calculate the LOD and PVE values of the predicted MQTLs, the average of the LOD and PVE values of the initial QTLs comprising the meta-QTL was taken. In cases where multiple MQTLs were predicted on a single chromosome; a standardized naming procedure was followed. Each meta-QTL was assigned a name starting with “MQTL,” followed by the numeric representation of the chromosome number, a period, and another numeric value indicating the sequence of the MQTL on that specific chromosome . To obtain the physical position of each meta-QTL, the flanking markers associated with them were used. Published studies or online databases such as MaizeGDB  and rice (Oryza sativa) were conducted using the MaizeMine tool on MaizeGDB. The wheat and rice homologous collected through MaizeMine were assessed through Ensembl Plants to retrieve their chromosomal location on respective genomes. MQTLs containing similar gene models located on conserved genomic regions across wheat, rice, and maize were considered as ortho-MQTLs.Each gene identified within the MQTL region was analysed for expression patterns using the qTeller tool available on MaizeGDB. qTeller provides information on gene expression across different tissues throughout maize development. To gain insights into the functional characteristics of the identified genes, as well as to explore potential orthologous relationships, functional descriptions and orthologous studies in wheat  ranging from 3.86% to 21.41%. Among the reported MQTLs, 19 had high LOD scores (≥5) and PVE values (≥10%), namely,: MQTL1.4, MQTL2.4, MQTL2.8, MQTL3.5, MQTL5.3, MQTL6.4, MQTL6.8, MQTL7.2, MQTL7.3, MQTL7.4, MQTL7.5, MQTL7.7, MQTL7.8, MQTL8.1, MQTL8.3, MQTL8.4, MQTL8.6, MQTL10.7, MQTL10.8 . The aveAll the MQTLs were physically mapped onto the maize reference genome Zm-B73-REFERENCE-NAM-5.0. The physical CI of these MQTLs ranged from 2,049 bp (MQTL7.3) to 39.95 Mb (MQTL 4.4), with a mean physical CI of 3.30 Mb. Collectively, these MQTLs occupied a physical length of 224.52 Mb across the genome refer to . Among tOut of the 68 MQTLs reported, a subset of 10 MQTLs was selected and designated as “breeder-friendly MQTLs” (BF-MQTLs). The selection criteria for identifying BF-MQTLs were as follows: a PVE value of more than 10%, a genetic CI of less than 2 cM, a physical CI of less than 1 Mb, and involvement of at least 10 initial QTLs from different studies. Detailed information on these BF-MQTLs can be found in The physical positions of MQTLs identified through meta-analysis were compared with the physical positions of significant markers (MTAs) identified for the same traits using the GWAS approach. Data obtained from 10 different GWAS studies provided information on 250 MTAs, that overlapped with 47 MQTLs out of 68 reported MQTLs. The count of overlapping MTAs or adjacent MTAs (within a 1 Mb region surrounding the MQTL regions) varied for each MQTL, with MQTL9.3 exhibiting the highest number of overlaps with 36 MTAs. Notably, among the overlapped MTAs, 35 were associated with oil-related traits, 26 were associated with yield-related traits, 17 were associated with carotenoid-related traits, 2 were associated with sugar-related traits, and 1 was associated with protein-related traits see . It is iZm00001d002000), two genes associated with kernel length , three genes associated with kernel number per row , one gene associated with kernel thickness , two genes associated with kernel weight , one gene associated with moisture content , and four genes associated with grain yield . Interestingly, three breeder-friendly MQTLs  were found to be associated with these reported genes that influence grain yield.GWAS studies provided valuable insights into the genes associated with the traits under investigation see . Specifiviz., GRMZM2G059138, GRMZM5G828253, GRMZM2G118423, GRMZM2G176542, GRMZM2G125268, GRMZM2G122767, GRMZM2G417435, GRMZM2G169089, GRMZM2G077789, GRMZM2G136072), two genes were associated with starch content , and one gene was validated for grain protein content . Two breeder-friendly MQTLs  were found to be associated with these reported genes affecting grain quality. Notably, these genes encode essential enzymes  involved in significant metabolic pathways and transcription factors , as well as proteins  that are believed to play crucial roles in influencing grain yield and quality , four genes were expressed in shoot axis internodes, 41 genes were expressed in stem and shoot apical meristems, 49 genes were expressed in reproductive tissues, 52 genes were expressed in roots and internodes, 53 genes were expressed in leaves, and 54 genes were expressed in seeds.Additionally, the expression analysis of these genes was performed using the expression analysis tool within the MaizeMine, revealing tissue-specific expression patterns . Among tSynteny analysis was conducted to identify conserved regions of the reported maize MQTLs in the genomes of wheat and rice. In total, 59 genes identified in the maize genome within the 10 BF-MQTL regions corresponded to 42 genes in the rice genome  and 150 etc.), and phytic acid, which are essential for the maize-consuming population  exhibited QTLs associated with both grain quality and yield. Among these promising BF-MQTLs, MQTL 6.8 had the highest number of associated genes (12), followed by MQTL1.4 with 10 genes, and 7 genes each for MQTL2.4, MQTL2.8, and MQTL6.4. Based on these findings, BF-MQTL1.4, 2.4, 2.8, 6.4, and 6.8 appear to be the most promising candidates for improving both grain yield and quality. However, further investigations are required to identify functional variants of these genes within the MQTL regions.The MQTLs validated through GWAS were further characterized as breeder-friendly based on their high PVE and low CI values. Among the 10 validated BF- MQTLs in the present study, a total of 59 CGs were reported. etc.Several genes identified within the validated hotspot regions encode enzymes involved in various metabolic processes, such as terpenoid biosynthesis (1-deoxy-D-xylulose-5-phosphate reductoisomerase) , auxin dZmNAC128 and ZmNAC130, have been identified to play crucial roles in the accumulation of starch and protein in maize, as highlighted in a study by In addition to enzymes, some genes also encode for potent transcription factors. An example of such transcription factors is the C2H2-type domain-containing proteins, known as zinc finger proteins, which play a pivotal role in regulating plant growth and development . EukaryoAZG1 plays a crucial role in facilitating adenine and guanine transport across membranes.A few genes reported within the MQTL regions were categorized as peroxisome biogenesis protein 22 that participates in peroxisome assembly , multipl2+ and chelate the cytosolic Ca to regulate Ca homeostasis (Two genes were associated with apoptosis involving senescence-associated gene 20, and ubiquitin carboxyl-terminal hydrolase 19 . Severaleostasis . Tr-typeeostasis . The aboDuring the synteny analysis, for MQTL1.4, the maximum number of orthologs were present on chromosome 9 in rice and on chromosomes 5 and 7 in wheat. These conserved genes encode major transcription factors and enzymes that play important roles in major metabolic pathways. For MQTL2.4 and 2.8, rice orthologs were found on chromosome 4 and on chromosome 2 in wheat, respectively. MQTL6.4 showed conserved sequences on chromosome 6 in rice and chromosome 7 in wheat. In MQTL6.8, orthologs were present on chromosome 5 in rice and on chromosomes 1 and 7 in wheat. In MQTL8.1, orthologs accumulated on chromosomes 3, 4, 5, and 7 in wheat, and on chromosomes 6, 3, and 1 in rice. In MQTL10.8, conserved sequences were reported on chromosome 4 in rice and chromosome 2 in wheat. Overall, chromosomes 1, 2, 3, 4, 5, and 7 exhibited conserved regions in wheat for quality and yield-associated traits, with the maximum number of genes on chromosome 7 . In riceThe present study successfully compiled the results of various mapping studies on yield and quality-related traits. A total of 68 MQTLs and 7 singletons were identified in the current study. More than half of the MQTLs were validated through GWAS, and a total of 10 MQTLs were defined as BF-MQTLs based on their CI and PVE values. A comprehensive analysis revealed nearly 60 different genes within the genomic regions of these BF-MQTLs. The putative genes analysed in this study encode essential enzymes involved in multiple metabolic pathways and transcription factors that impact the regulation of key genes. Additionally, some genes were found to be expressed in chloroplasts and peroxisomes, while others function as transporters and signal proteins. Four potential BF-MQTLs were identified to significantly influence both maize yield and grain nutrient quality, making them highly recommended for further utilization in marker-assisted breeding. The overall findings from this study provide valuable insights into robust genomic regions that can be targeted in future investigations to enhance maize yield and nutrient quality.


The cation channel of sperm (CatSper) is a sperm-specific, pH-sensitive, and Ca2+-permeable ion channel. It is responsible for the predominant Ca2+ entry in mammalian sperm and is involved in nearly every event of sperm to acquire fertilizing capability. In addition, CatSper also serves as a pivotal polymodal chemosensor in mammalian sperm by responding to multiple chemical cues. Physiological chemicals  provoke Ca2+ entry into sperm by activating CatSper and thus triggering sperm functions. Additionally, synthetic and natural chemicals  affect sperm functions by regulating CatSper-dependent Ca2+ signaling. Therefore, understanding the interactions between CatSper and extracellular ligands sheds light on the mechanisms underlying male infertility and offers innovative diagnostic and treatment approaches. This underscores the importance of CatSper as a crucial regulatory target in male reproduction, linking sperm function with the extracellular environment. In conclusion, this review comprehensively summarizes the relevant studies describing the environmental factors that affect CatSper in humans and rodents.After sperm enter the female reproductive tract, the physicochemical and biochemical microenvironment undergoes significant changes. In particular, the large changes in various ions encountered by sperm may alter the physiology of sperm, ultimately compromising capacitation and fertilization. Thus, the rapid response to environmental variations is vital for sperm functions. For example, Calcium, the most crucial ion for sperm functions, enters into sperm via Ca From sperm motility to acrosome reaction, [Ca2+]i orchestrates these key events of sperm function in the correct time and order i i . Althoug [Ca2+]i . Herein,CATSPER1, CATSPER2, CATSPER3, and CATSPERE have been found in infertile men i i . In thes2+]i . In addiincrease  , an endogenous CatSper inhibitor i . Therefonhibitor . P4 bind+ influx . P4-indulization ,32.2+ influx induced by P4 i. In addition, seminal fluids contain high concentrations of PGs I in a biphasic manner with similar amplitudes, and potentiates Ca2+ currents similarly to P4 in human sperm i [2+]i in mouse sperm i  and moduse sperm . In addise sperm ,43,44.2+]i I elevation. Indeed, the Ca2+ mobilized by ZP glycoproteins requires CatSper to enter sperm, implying that ZP-glycoprotein-induced Ca2+ influx is dependent on CatSper i . Xia and2+]i  found thh et al.  showed t CatSper ,47. The 2+. BSA can induce sperm capacitation and increase [Ca2+]i in several mammals, but these effects are absent in the sperm of Catsper1-knockout mice and could be restored by an EGFP-CATSPER1 fusion protein i via a G-protein-coupled receptor/olfactory G-protein/cAMP/PKA pathway i, and downregulated the expression of Catsper1, Catsper2, Catsper3, and Catsper4 through Ten-eleven translocation 1 [2+]i, and inhibits P4-induced Ca2+ influx and sperm functions in humans [2+]i in human sperm i i . A recentinoxate . Of thes CatSper ,99. ThesCatsper1, Catsper2, Catsper3, and Catsper4, as well as the sperm motility associated gene Mvh, were decreased significantly i [2+]i i . In addi2+]i . Specifi]i [2+]i . Therefo6-ethenoadenosine and 1,N2-ethenoguanosine, resulting in DNA structure instability and leading to single-strand breaks [Oxidative stress occurs when the generation of reactive oxygen species (ROS) exceeds the natural antioxidant defenses of bodies. Thus, the precise balance of ROS and antioxidants within sperm are necessary for capacitation and fertilization. The major effect of oxidative stress compromising sperm function is caused by two principal mechanisms, DNA damage and lipid peroxidation . In humad breaks . Once thd breaks . Hence, Catsper in the testes of young adult and aged male mice, which are the genes responsible for sperm motility [To counteract ROS damage, the human body has developed a variety of antioxidant strategies. For instance, non-enzymatic antioxidants contained within the seminal fluid, like vitamin E and selenium . Interesmotility ,110. Meamotility ,110. Con2+ influx into sperm and it is also a polymodal chemosensor that harbors structurally diverse binding sites for extracellular factors. CatSper regulates key sperm physiological processes, including capacitation, hyperactivation, the acrosome reaction, and chemotaxis, by responding to physiological ligands. These processes are necessary to produce sperm capable of fertilization. In addition, the interaction between CatSper and extracellular factors, such as EDCs and drugs, may potentially disrupt sperm function and induce male infertility [CatSper plays a key role in male fertility by controlling extracellular Caertility . Consequ