Pectinodesmus pectinatus is a green alga of commercial interest in sewage purification. Clarification of its organelle genomes is helpful for genetic manipulation, taxonomic revisions and evolutionary research. Here, de novo sequencing was used to determine chloroplast genome and mitochondrial genome of P. pectinatus strain F34. The chloroplast genome was composed of a large single-copy (LSC) region of 99,156 bp, a small single-copy (SSC) region of 70,665 bp, and a pair of inverted repeats (IRs) with a length of 13,494 bp each separated by LSC and SSC. The chloroplast genome contained 69 protein-coding genes, 25 transfer-RNA (tRNA) genes, 3 ribosomal RNA (rRNA) genes. The mitochondrial genome was 32,195 bp in length and consisted of 46 unique genes, including 16 protein-coding genes, 27 tRNA genes and 3 rRNA genes. The predominant mutations in organelle genomes were T/A to G/C transitions. Phylogenic analysis indicated P. pectinatus was a sister species to Tetradesmus obliquus and Hariotina sp. within the Pectinodesmus genus. In analysis with CGView Comparison Tool, P. pectinatus organelle genomes displayed the highest sequence similarity with that of T. obliquus. These findings advanced research on the taxonomy and phylogeny of Chlorophyceae algae and particularly revealed the role of P. pectinatus in microalgae evolution. Phylogenetic analysis with organelle genomes leads to clarification of the evolutionary relationships . ComparaChlamydomonas sp., uncovering the existence of genetic materials outside the nucleus of photosynthetic eukaryotes [Chlamydomonas reinhardtii was successfully sequenced [Photosynthetic eukaryotes convert light energy into the chemical energy in chloroplasts, which are metabolically active and semi-autonomous organelles existed in cyanobacteria, algae and plants . In 1962karyotes . In 1978equenced . The chlequenced . Determiequenced ,12,13. Aequenced . UnderstMitochondria is another two-layer membrane-coated semi-autonomous organelle, which is the place for cells to carry out aerobic respiration and produce energy. As same as chloroplasts, mitochondria have their own genetic material and genetic system. In addition to energy supplement, mitochondria are also involved in processes such as cell differentiation, information transmission, apoptosis, cell growth and cell cycle . An everHariotina, Pectinodesmus, Scenedesmus and Tetradesmus. Scenedesmus obtusus XJ-15 and Tetradesmus obliquus IPPAS S-2023 have showed their potential in lipid bioproduction [Coelastrella rubescens and Coelastrella astaxanthina are rich in astaxanthin and canthaxanthin [Coelastrella sp. KGU-Y002 is another promising Scenedesmaceae algae for biosynthesis of astaxanthin, adonixanthin, and zeaxanthin [Scenedesmaceae is a family belonging to the class Chlorophyceae and includes 43 genera, such as oduction . Coelastaxanthin ,19. Anotaxanthin . Despiteaxanthin , only a Pectinodesmus pectinatus (NCBI Taxonomy ID: 91197), formerly named as Scenedesmus pectinatus, is a species of chlorophycean algae in the family Scenedesmaceae and a common and important planktonic microalgae distributed globally in ponds, lakes, ditches, small puddles, various cultivation tanks and other water bodies all over the world [P. pectinatus is rarely single-celled and usually composed of four to eight cells, sometimes dozens of cells [P. pectinatus makes a high impact on environments and is economically important [P. pectinatu can directly uses organic matter as a carbon and nitrogen source when it is attached to other aquatic plants to form a colloidal layer to absorb organic matter. In this way, P. pectinatus rapidly degrades organic matter in the water and purifies the water. More importantly, P. pectinatus is highly resistance to organic pollutants, and produces oxygen during photosynthesis, facilitating bacteria in the water to decompose organic matter as well. Moreover, P. pectinatus is easy to be cultivated in large quantities. Thus, P. pectinatus has great potential for use in research on water pollution control [P. pectinatus cells can also be used as a feed for poultry since they are rich in protein and vitamins [The he world ,25. P. pof cells . P. pectmportant ,28,29,30 control . BesidesPectinodesmus species based upon the morphological characteristics, the canonical taxonomic classification is always incomplete for species identification of the genus Pectinodesmus. Clarification of phylogenetic relationships is urgent for taxonomic revision and evolutionary study of P. pectinatus. Recently, phylogenomic studies of several strains have provided valuable insights into the phylogeny of the Chlorophyceae class, but only limited information on relationships within the Scenedesmaceae family, especially the Pectinodesmus genus, is available [P. pectinatus. As for the family Scenedesmaceae, only Tetradesmus obliquus strain UTEX 393 chloroplast genomes , Tetradesmus obliquus strain KS3-2 mitochondrial genome (NC_002254) and Tetradesmus obliquus strain UTEX 78 mitochondrial genomes  are available. Tetradesmus obliquus strain DOE152z mitochondrial genome (CM007919), Scenedesmus sp. PABB004 mitochondrial genome (CM026549) and Coelastrella sp. MACC-549 chloroplast genome (CM031383) is assembled only but not yet annotated.Because of differences opinion on taxonomic classification of various vailable ,31,32,33vailable . HoweverP. pectinatus organelle genomes, we examined the complete sequence and genetic characterization of the chloroplast and mitochondrial genomes of P. pectinatus F34. Moreover, we investigated the phylogeny of P. pectinatus among the related genera and resolved the phylogenetic position through in-depth analysis. These results will be helpful for the organelle genetic engineering of P. pectinatus, which is the promising candidate for sewage purification.Here, to provide a better understanding of the complexity of Pectinodesmus pectinatus strain F34 was obtained from the Third Institute of Oceanography (TIO) of the Ministry of Natural Resources in Xiamen, Fujian, China. P. pectinatus F34 was cultured in f/2 liquid medium at 22 ± 1 °C in a 12 h:12 h light-dark cycle. Total genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method and Illumina sequencing was performed by using the NEBNext UltraTM DNA Library Prep Kit for 500-bp paired-end library. Sequencing was carried out on the Illumina HiSeq2500 platform . About 3.77 Gb raw data was generated with reads whose length was 250 bp. A number of potential reads were extracted from the pool of Illumina reads using BLAST searches against corresponding genomes of related species and the NOVOPlasty results [P. pectinatus F34 are NC_036668 and NC_036659, respectively. results . The Ill−5 against several protein databases: NCBI non-redundant (Nr) protein database, Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) terms. All tRNA genes were further confirmed through online tRNAscan-SE [http://snapgene.com, accessed on 16 November 2021). Codon usage, GC content, and relative synonymous codon usage (RSCU) were analyzed by CUSP program in EMBOSS  [Preliminarily gene annotation was carried out through the online program Dual Organellar GenoMe Annotator (DOGMA) and CPGAVAS with plastid/bacterial genetic code and default conditions ,37. FuncAscan-SE ,39. The Ascan-SE . The graAscan-SE ,42. The er 2021) . The GC −3), and sequence identity greater than 90%  [P. pectinatus F34 by using MISA software . In this study, only perfect repeats were considered with basic motifs ranging from 1 to 6 nucleotides and a minimum repeat length of 10 nucleotides (for mono-), 6 nucleotides (for di-), 5 nucleotides  by default. To analyse the nucleotide composition of the motif, motifs repeated on a complementary strand were considered equivalents and grouped into one motif. For instance, the motif AG is equivalent to GA, TC, CT, and so forth.Using online available REPuter program, the sizes and locations of forward, palindrome, reverse and complement sequences were determinedaccording to the following criteria: minimum repeat size of 30 bp, maximum computed repeats of 5000  .The detehttp://www.drive5.com/muscle/, accessed on 9 December 2021) [http://darwin.uvigo.es/software/prottest_server.html, accessed on 10 December 2021) [www.evolgenius.info/evolview/, accessed on 21 December 2021) [The complete sequences and GenBank files of all other chloroplast genomes were downloaded from Genbank. Phylogenetic analysis was carried out based on protein-coding genes from GenBank. This study made use of 25 complete chloroplast genomes and 15 complete mitochondrial genomes of chlorophycean algae, which are listed in er 2021) . The beser 2021) . Phylogeer 2021) . MrBayeser 2021) . The phyer 2021) . The CGVer 2021) .P. pectinatus in terms of the evolution of the chlorophycean algae and to characterize the genetic content and structure of chloroplast and mitochondrial genomes, its organelle genomes were sequenced. A total of 3.76 Gb data composed of 15,061,892 Illumina Hiseq 2500 Pair-End reads were extracted by aligning against the NOVOPlasty and Abyss assembled chloroplast genomes. Finally, reads were finally employed to assemble the P. pectinatus F34 chloroplast genome, whose final complete sequence with gene annotation has been deposited in the NCBI GenBank database with the accession number NC_036668. This complete chloroplast genome was finally assembled into a circular double-stranded DNA molecule of 196,809 bp in length with a typical quadripartite structure found in most terrestrial species, containing an LSC of 99,156 bp and an SSC of 70,665 bp separated by two inverted repeated rDNA sequences (IRA and IRB), each of which spanning 13,494 bp, respectively (Aiming to determine the phylogenetic position of ectively .P. pectinatus chloroplast genome is 70.06% and is more than the that of Arabidopsis (63.7%), tobacco (62.2%), rice (61.1%) or maize (61.5%) . The GC content of LSC and SSC regions was 28.75% and 28.02%, respectively, which were lower than that of the two IR regions (39.36% and 39.36%). It can be inferred that the GC richness of the ribosomal RNA (rRNA) genes (54.88%) contribute to the high GC contents in the IR regions, which is similar to that of most green algae and land plants.The composition of nucleotides of chloroplast genome was biased toward adenine and thymine (A + T) with a guanosine and cytosine (G + C) content of 29.94% . The totP. pectinatus F34 mitochondrial genome have been obtained and assembled well. As the completeness of the genome assembly is affected by conditions such as the mitochondrial DNA content and sequence complexity in the total DNA of the sample genome, for areas with complex structures or low sequencing coverage, PCR and Sanger sequencing were used for verification to ensure the completeness and accuracy of the mitochondrial genome sequence. Its final complete sequence with gene annotation has been deposited in the NCBI GenBank database with the accession number NC_036659. The complete mitochondrial genome of P. pectinatus was composed of 32,195 bp with a GC content of 40.78% and was more than 6 times smaller than its chloroplast genome  accounted for 46.70% of the genome, and the non-coding regions, including intergenic spaces, accounted for the remaining 53.30% of the genome. In total, there were 102 genes (excluding open reading frames (ORFs)), which were scattered singly or in groups throughout the entire genome, including 69 protein-coding genes, 25 transfer-RNA (tRNA) genes and 3 ribosomal-RNA (rRNA) genes  genes . The genen algae ,54. MajoP. pectinatus chloroplast genome were important genes involved in the photosynthesis process. Among them, there were 5 genes coding for protein subunits of the cytochrome complex, 7 genes coding for proteins being a part of the photosystem I (PS I), 16 genes coding for proteins being a part of the photosystem II (PS II), 6 genes encoding the ATPase chloroplast subunits, 12 genes encoding the NADH dehydrogenase subunits and one gene encoding the RuBisCo large subunit. In addition, 29 P. pectinatus chloroplast genes were related to the gene expression processes, including 5 genes encoding the chloroplast RNA polymerase subunits and 22 genes encoding protein components of the ribosomes. The transcription of chloroplast genes in angiosperm plants is carried out by two types of RNA polymerases, the bacterial-type multi-subunit RNA polymerase (PEP) encoded by the chloroplast genome and the T3/T7 phage-type RNA polymerases (NEP) encoded by the nucleus genome [P. pectinatus rpoA gene that encoding α subunit of the PEP core, together with the genes of the ribosomal proteins was organized into one operon under the control of the same promoter as well. As is known, rpoB, rpoC1 and rpoC2 genes encoded β, β′ and β″ subunits of the PEP core, respectively. Notably, there were two rpoB (rpoBa and rpoBb), two rpoC (rpoC1 and rpoC2) in chloroplast genome of P. pectinatus, different from some green algae and land plants in which rpoB and rpoC genes formed a separate operon designated as RPOBC [P. pectinatus chloroplast genome.38 genes of s genome . PEP is s genome . Similaras RPOBC . In contP. pectinatus chloroplast genome, there are only 25 tRNA genes were observed. In addition to orf221 in IRB region, another 12 ORFs were annotated, with one or two ORFs in each gene as follows: orf226 and orf317 in psbC, orf318 and orf386 in rcbL, orf257 and orf428 in psaA. A total of 6 ORFs, including orf698, orf293, orf193, orf290, orf277, orf170, were arranged in a row in a region encoding psbA. P. pectinatus mitochondrial genome is consist of 46 unique genes, including 13 protein-coding genes with no introns, 27 transfer-RNA (tRNA) genes and 6 ribosomalRNA (rRNA) genes  genes . There wl genome . All 13 P. pectinatus chloroplast genome was analyzed. A total of 30,558 codons encoding 69 protein-coding genes were detected in the chloroplast genome. The 25 unique tRNA genes included codons for all 20 amino acids necessary for biosynthesis. The detailed codon usage frequency of chloroplast genome and mitochondrial genome were quantified by RSCU value and integrated into the distribution map as shown in P. pectinatus were shown in Chloroplast and mitochondrial codon usage are biased (Codon Usage Bias), and codon bias will affect gene expression to a certain extent and reflect the evolutionary relationship of species. Using SCGene’s integrated Codon Usage analysis process, the codon usage and codon-anticodon recognition pattern of P. pectinatus, the percentage of AT reached up to 70.06% in the entire chloroplast genome  of the protein-coding gene was calculated as 62.32%.In the most bacterial genomes, mitochondrial genomes, and chloroplast genomes, there are significant differences in base composition between heavy and light chains, which are called “AT (AT-skew)” and “GC (GC-skew)”. GC skew = (G − C)/(G + C), which are used to measure the relative contents of G and C. AT skew = (A − T)/(A + T), which are used to measure the relative contents of A and T. The deviation rate of GC and AT were calculated. In t genome . AT-skewP. pectinatus were determined for use in future studies. These SSRs were nonrandomly distributed across different genomic fractions. A total of 73 perfect microsatellites were identified, with an overall density of 377.05 SSRs/Mb , are short tandem repetitive DNA sequences that are ubiquitously present over genomes. Owing to their abundance and high-degree of polymorphism in the number of repeats, SSR research has found numerous applications such as genetic diversity, construction of genetic linkage map, variety identification and molecule marker assistant breeding . In the  SSRs/Mb . That SSP. pectinatus chloroplast genome, the total amount of the mononucleotides (not shorter than 8 bp) was 71, which represented the highest portion (97.2%) followed by dinucleotide repeats (2.74%). All the mononucleotides were composed of A/T. These results were consistent with previous findings that SSRs in chloroplast generally consist of short polyA/T repeats. The longest poly-A and poly-T structures were a 13 A-repeat and a 13 T-repeat, respectively. Overall, the base composition of the SSR motifs showed a strong bias to AT-rich in the P. pectinatus chloroplast genome, consistent with other chloroplast studies. The strong A/T bias exhibited in chloroplast SSRs also contributed to the A/T enrichment in the P. pectinatus chloroplast genome.Among these SSRs, 3 (4.11%) and 6 (8.22%) were located in the exons and introns regions, respectively, whereas 64 (87.67%) were found in the intragenic regions. Interestingly, there were much more intron-localized SSRs than exon-localized SRRs, indicating the effects of elective pressure on nucleotide. The repeatition length of the whole chloroplast genome ranged in size from 8 to 15 bp, all belonging to class II SSRs (short SSRs < 20 bp). 10 bp and 11 bp were the predominant repeat lengths in different regions. In the Pectinodesmus is available. Most phylogenomic studies have used one or more chloroplast genes or plastid ribosomal RNA operon to resolve the genetic relationships among green algal lineages, but a set of single-copy orthologous genes were also used. In this study, to resolve the phylogeny of P. pectinatus and to examine its chloroplast genome, we constructed a phylogenomic tree among chloroplast genomes of 25 representative species whose chloroplast genomes were reported previously, with commonly shared single-copy genes employing the LG + I + G model and 500 bootstrap replicates under the maximum-likelihood (ML) inference. This ML tree was largely consistent with traditional taxonomy and recent phylogenetic relationships within phylum (Coelastrella sp. MACC-549 chloroplast genome (CM031383.1) in the NCBI database was excluded, considering that it was not yet annotated clearly.Studies on chloroplast phylogenomics of chlorophycean algae have provided valuable insights into the phylogeny of green algae, but only limited information on relationships involving the genus n phylum . The CoeP. pectinatus were falling under Sphaeropleales group. Our broadly sampled phylogenies suggested that T. obliquus appeared as a sister group to P. pectinatus, and Hariotina sp. was placed as a sister of T. obliquus and P. pectinatus. Together, P. pectinatus, T. obliquus and Hariotina sp. formed a monophyletic clade as Scenedesmaceae with almost full support, which displayed a kinship to the clade of Neochloridaceae, which is consist of Chlorotetraedron incus and Neochloris aquatica. Neochloridaceae and Selenastraceae were sister groups of Scenedesmaceae. Moreover, a clear separation between Selenastraceae and Scenedesmaceae among phylogenetic tree were observed, and the Selenastraceae are separated from Chromochloridaceae. Besides, the results showed Hydrodictyaceae is located separately from the Scenedesmaceae lineage with two distinct clusters nested inside, suggesting that Scenedesmaceae lineage independently compared with Hydrodictyaceae. It should be noted that C. reinhardtii, Conium pectorale and Dunaliella salina formed a monophyletic clade located outside as outgroup. In Sphaeropleales, Ankyra judayi is separated as Sphaeropleaceae that is the group most close to outgroup including Chlamydomonas, Conium and Dunaliella. Following, Bracteacoccaceae represented by Bracteacoccus aerius, Bracteacoccus minor and Bracteacoccus giganteus separated from Mychonastaceae, followed with Pseudomuriellaceae. Clearly, all Bracteacoccaceae, Mychonastaceae and Pseudomuriellaceae appear as some far branches to Scenedesmaceae. In conclusion, we concluded that Scenedesmaceae lineages are not monophyletic and diverged considerably deeper than the simple diversification from a single green alga.A phylogenomic tree consisting of chloroplast genomes of 16 species was constructed. In the phylogenetic tree generated from chloroplast genome, P. pectinatus was based on the complete mitochondrial genome data of 15 other green microalga species. In the same way, C. reinhardtii, G. pectorale and D. salina served as the outgroup. As expected, similar patterns were observed when a ML tree was constructed from alignment of mitochondrial genome sequences. In agreement with the genetic relationships observed in analysis of chloroplast genomes, C. reinhardtii, G. pectorale and D. salina constitute a monophyletic clade with outside location. Similarly, P. pectinatus was observed to have a closer genetic relationship with T. obliquus with strong bootstrap support than with any other examined species . It recently formed a new genus separate from Scenedesmus based on the internal transcribed spacer, two consensus sequences, which were distinguished from other members of the family Scenedesmaceae [Scenedesmus, including P. pectinatus [P. pectinatus is a typical cenobium with four or eight cells arranged linearly or twisted up to 90° to each other [P. pectinatus other than its basic morphological characteristics and preliminary taxonomic status. The green algae show extensive variation in chloroplast genome size [P. pectinatus is in agreement with published chloroplast genomes of land plants and green algae, in which identical long IRs containing the ribosomal operons, suggesting it was evolved from the ancestor of chlorophycean algae [The genus esmaceae . There actinatus . P. pectch other . Informaome size . In thisan algae ,33,61.P. pectinatus. It indicates that those genes suffered from election by certain factors. As summarized in P. pectinatus chloroplast genome. It raises a challenge question why P. pectinatus psbA keeps so many introns in the long evolution history. Another two split genes, cemA and rps18, were residents in the SSC region. In addition, one of the three rRNA genes in P. pectinatus chloroplast genome, rrn23S is the only one containing intron in the IRs area. Because the intron was present in both duplicates, it is clear that the rrn23S got copied in chloroplast genome after the intron-exon splicing site evolved.Consistent with previous findings in several green algal plastids, introns were completely present in protein-coding genes and tRNAs of chloroplast genome of P. pectinatus have one gene encoding for components of the cell division protein (ftsH) found in other green algae  but lost in some Chlorodendrophyceae chloroplast genomes [P. pectinatus as well. Experiments on C. reinhardtii knockout mutant have shown that cemA is not essential for photosynthesis or the viability of the cell but its absence increases the light sensitivity [P. pectinatus remain elusive. Among which, 3 ycf genes could be functionally annotated with high similarity, including ycf1 (component of TIC complex), ycf3 and ycf4 (subunits of photosystem I). Apart from these ycf genes, homologous gene of ycf12 is reported as a subunit of photosystem II reaction center and is required for optimal growth in high light in C. reinhardtii [In comparison with other core Chlorophyta ,  genomes ,63. The sitivity . Notablynhardtii .P. pectinatus within Chlorophyceae algae, and we have observed close phylogenetic relationships between P. pectinatus, T. obliquus and Hariotina sp. It was not surprising that similar patterns of clustering were obtained in the ML tree constructed from mitochondrial genome sequences as the one observed from analysis based on chloroplast genomes. However, in this study, the mitochondrial genome ML tree supported the significant separation between P. pectinatus and Selenastraceae. It is different from the sister relationships of chloroplast genomes of Selenastraceae and Scenedesmaceae, clear separated groups were formed for Selenastraceae represented by Ourococcus multisporus and Scenedesmaceae including P. pectinatus, T. obliquus and Hariotina sp. Results clearly showed that P. pectinatus and T. obliquus formed a separate clade within Scenedesmaceae, whereas Hariotina sp. was relatively distant from the other two green algae. Thus, collinearity analysis of mitochondrial genomes was performed on P. pectinatus and other three chlorophycean algae. The results attested that the gene rearrangements between different algal species are universal and showed that the rearrangement of P. pectinatus mitochondrial genome compared with other green algae (P. pectinatus is largely different from that of T. obliquus, which is the closest sister in phylogenic tree. In both evolutionary analyses, C. reinhardtii, Gonium pectorale and Dunaliella salina were classified as outgroup and they served the purpose effectively. Although the relationships of Scenedesmaceae with other major clades from Chlorophyceae remains unclear, our results now showed the genetic position of Scenedesmaceae in all green algae. Our results on P. pectinatus are an add-on in the understanding of the phylogenetic relationship among different Chlorophyceae species.Molecular phylogenetics are the cornerstone of current biodiversity and evolutionary research. Undoubtedly, many features conserved among mitochondrial genomes were observed, but the chloroplast genome organization is more conserved and valuable for understanding the evolutionary pattern and phylogeny of green algae. Besides, to unravel phylogenetic relationships, complete chloroplast genome sequences are a better choice than concentrating only on protein-coding sequences or conserved regions. Here our main focus was on the position of en algae . InteresP. pectinatus F34 in this study. Phylogenetic analysis based on these organelle genomes showed that P. pectinatus was closely related to T. obliquus and Hariotina sp. and also supported the well-known classification of different Chlorophyceae groups and families. Yet it is unclear whether current data in our hands are sufficient to finally obtain a robust tree topology for all the genus of Chlorophyceae, further study is still needed. This phylogeny would require further investigation by a broader sampling of chlorophycean algae. All in all, the sequenced complete chloroplast genome and mitochondrial genome of P. pectinatus F34 provides detailed fundamental molecular data for species identification, Chlorophyceae evolution and genetic manipulation, serving its potential in water-sewage industry.During the endosymbiosis event happened over 1.2 billion years ago, the free-living cyanobacterium started to coexist inside a eukaryotic cell, which turned out to be essential for life. Understanding of chloroplast and mitochondrial genetic machineries is key to a full explanation of the basic process in green algae. With the strong believes that these organelle genomes can play a great tool for taxonomic and phylogenetic analysis, we successfully assembled and analyzed chloroplast and mitochondrial genomes of


Lactobacillus delbrueckii subspecies have low efficiency for identification at subspecies level. Therefore, our objective in this study was to select novel genetic markers for accurate identification and discrimination of six L. delbrueckii subspecies based on pangenome analysis. We evaluated L. delbrueckii genomes to avoid making incorrect conclusions in the process of selecting genetic markers due to mislabeled genomes. Genome analysis showed that two genomes of L. delbrueckii subspecies deposited at NCBI were misidentified. Based on these results, subspecies-specific genetic markers were selected by comparing the core and pangenomes. Genetic markers were confirmed to be specific for 59,196,562 genome sequences via in silico analysis. They were found in all strains of the same subspecies, but not in other subspecies or bacterial strains. These genetic markers also could be used to accurately identify genomes at the subspecies level for genomes known at the species level. A real-time PCR method for detecting three main subspecies  was developed to cost-effectively identify them using genetic markers. Results showed 100% specificity for each subspecies. These genetic markers could differentiate each subspecies from 44 other lactic acid bacteria. This real-time PCR method was then applied to monitor 26 probiotics and dairy products. It was also used to identify 64 unknown strains isolated from raw milk samples and dairy products. Results confirmed that unknown isolates and subspecies contained in the product could be accurately identified using this real-time PCR method.Genetic markers currently used for the discrimination of  Lactobacillus delbrueckii comprises six subspecies, namely delbrueckii, lactis, bulgaricus, indicus, jakobsenii, and sunkii, all of which have historically been differentiated based on their ability to metabolize different carbohydrates [L. delbrueckii subsp. lactis and bulgaricus are usually associated with the manufacture of dairy products such as cheeses and yogurt [L. delbrueckii subsp. bulgaricus is one of the starter culture components for the production of yogurt [L. delbrueckii subsp. lactis is traditionally used in cheese production and can be distinguished from L. delbrueckii subsp. bulgaricus by its extensive carbohydrate metabolizing capabilities [L. delbrueckii subsp. delbrueckii cannot ferment lactose. It is typically associated with fermented vegetables [L. delbrueckii subsp. indicus, jakobsenii, and sunkii are relatively minor subspecies isolated from Indian dairy products, fermented alcoholic beverages, and non-salted pickle as a traditional Japanese food, respectively [hydrates . Among td yogurt . L. delbf yogurt , 3. Thisf yogurt . On the bilities , 5. L. dgetables . L. delbectively -9.L. delbrueckii subspecies in food samples is an important issue to confirm probiotic properties and perform product quality assessment [L. delbrueckii used in PCR-based methods [L. delbrueckii at the species level, they cannot be applied to distinguish L. delbrueckii at the subspecies level [Accurate identification of sessment . Geneticsessment ,10-12. Hsessment . Of thes methods . Althouges level .L. delbrueckii subspecies-specific genetic markers by pangenome analysis to develop a real-time PCR method for rapid identification of bacterial strains. The real-time PCR method we developed was then applied to bacterial strains isolated from raw milk, probiotic products, and dairy products to identify and differentiate three L. delbrueckii subspecies.Recently, the development of whole-genome sequencing (WGS) and the increase in genome sequences have made it possible to rapidly and freely process large-scale sequence data on microorganisms in public repositories . PangenoL. delbrueckii subspecies is illustrated in L. delbrueckii subsp. delbrueckii, lactis, bulgaricus, indicus, jakobsenii, and sunkii were obtained from the National Center for Biotechnology Information (NCBI) (The in silico scheme for selecting the genetic markers of six n (NCBI) . PhylogeL. delbrueckii subspecies was calculated using Bacterial Pan Genome Analysis (BPGA) pipeline ver. 1.3 (identity cut off = 50%) [The pangenome of f = 50%) . The panL. delbrueckii subsp. lactis, bulgaricus, and delbrueckii were designed from their genetic markers using Primer Designer .A total of four genome sequences registered at the species level were obtained from the NCBI. Genetic marker specificity was aligned with 45 genome sequences, including genomes known to the species level using USEARCH ver. 9.0 . AlignmeL. delbrueckii subspecies were obtained from the Korean Agricultural Culture Collection , the Korean Collection for Type Cultures , the Korean Culture Center of Microorganisms , the NITE Biological Resource Center , and the Laboratory Isolates   following the protocol described previously [For specificity testing of primer pairs developed in this study, the reference strains of lactic acid bacteria mainly isolated from probiotic and dairy products were used. A total of 54 strains of lactic acid bacteria including , Korea) . All refeviously . DNA conL. delbrueckii subspecies and 44 other lactic acid bacteria. For the accuracy test, genomic DNA from each reference strain was serially diluted and used for real-time PCR.Real-time PCR assay was conducted to determine the specificity and accuracy of primer pairs using a 7500 Real-time PCR system. Each reaction contained 20 ng of genomic DNA, 10 μl of 2X Thunderbird SYBR qPCR Mix , 500 nM of primer pairs, and distilled water up to 20 μl total volume. Real-time PCR conditions consisted of initiation at 95°C for 2 min followed by 30 amplification cycles of 95°C for 5 s and 60°C for 30 s. Melting curves were obtained at 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. Specificity of primer pairs was tested against a total of 10 strains of L. delbrueckii subspecies were isolated from three raw milk samples and three dairy products. Serially diluted samples were spread onto MRS agar plates  and incubated at 42°C for 48 h under anaerobic conditions. Probiotic and dairy products were obtained from markets around the world. Genomic DNAs were extracted from isolates and products under the same conditions as described in section 2.3. DNA Extraction of L. delbrueckii Subspecies and Lactic Acid Bacteria. For the application test, genomic DNAs of isolates or products were added to wells of 96-well plates containing 2X qPCR mix (Toyobo) and subspecies-specific primer pairs. The real-time PCR condition was the same as that described in section 2.4. Specificity and Accuracy for Specific Primer Pairs.To test the developed real-time PCR method, 64 isolates, 15 probiotic products, and 11 dairy products were used. L. delbrueckii subspecies by phylogenetic analysis based on the pangenome before specific genetic markers were selected. Phylogenetic analysis results based on pangenome frequencies were displayed along with the distribution of subspecies’ specific regions. Each bar represents L. delbrueckii subspecies genomes and each layer presents pangenome distribution , Ser/Thr protein kinase (Accession No. EGD27260.1), choline kinase (Accession No. KNZ37552.1), DNA methyltransferase (Accession No. KNE31255.1), RpoD family RNA polymerase sigma factor (Accession No. EOD03403.1), and hypothetical protein (Accession No. APG74821.1), respectively.As a result of pangenome analysis, a total of 67,178 genes from 41 L. delbrueckii subsp. bulgaricus was present in 19 bulgaricus genomes (95–100% identity), but one genome had the genetic marker for L. delbrueckii subsp. lactis instead of bulgaricus (99% identity). A genetic marker for L. delbrueckii subsp. delbrueckii was present in six delbrueckii genomes (99–100% identity), but one genome had the genetic marker for L. delbrueckii subsp. sunkii instead of delbrueckii (100% identity). These results were the same as those of pangenome analysis. Genetic markers were aligned with their genomes to determine the subspecies of genomes registered at the species level. L. delbrueckii AVK, TJA31, and 328M contained the genetic marker for L. delbrueckii subsp. bulgaricus (96–97% sequence identity). L. delbrueckii LDELB18P1 contained the genetic marker for L. delbrueckii subsp. lactis (100% sequence identity).The specificity of genetic markers was tested using 45 genomes including genomes registered at species level by in silico analysis. The heatmap for identities of genetic markers in genomes is shown with color codes, ranging from blue (region with high identity) to sky blue (region with low identity) . Each geL. delbrueckii subspecies [L. delbrueckii subspecies and other bacterial strains. They were able to accurately identify subspecies level for unknown genomes.In previous studies, genes such as 16S rRNA, 16S–23S rRNA intergenic spacer region, and the elongation factor Tu (tuf) gene have been used to distinguish microorganisms at the species or subspecies level ,9,26-29.bspecies , 13. In L. delbrueckii subspecies with genetic markers selected in this study requires WGS and bioinformatics analysis to confirm the presence of their markers. This method can accurately identify L. delbrueckii subspecies. However, the cost associated with WGS and its informational capacities must be considered. In addition, specialized researchers are needed to handle bioinformatics analysis [L. delbrueckii isolates using relatively simple procedures. This real-time PCR method is designed to identify three main subspecies, L. delbrueckii subsp. delbrueckii, lactis, and bulgaricus [The method to identify analysis , 30. Thelgaricus  that areL. delbrueckii subspecies and lactic acid bacteria did not generate any signal  were greater than 0.998 and all amplification efficiencies were more than 94% and bulgaricus (n = 47)  . These rL. delbrueckii subspecies. These genetic markers were present in all genomes of the same subspecies but absent in genomes of other subspecies and bacterial strains. To rapidly and cost-effectively identify L. delbrueckii subspecies, subspecies-specific primer pairs for three subspecies mainly isolated from food samples were designed. The real-time PCR method using these genes could accurately identify L. delbrueckii subspecies and other lactic acid bacteria with high specificity. The developed real-time PCR method was able to successfully monitor probiotic and dairy products and identify various isolates. Thus, our method can be used to accurately identify L. delbrueckii subspecies and determine the nomenclature of these subspecies. Furthermore, it can contribute to safety in the food industry by ensuring products are labeled to show the correct strain.In conclusion, pangenome analysis was performed to select genetic markers for six


Elizabethkingia anophelis is an emerging species and has increasingly been reported to cause life-threatening infections and even outbreaks in humans. Nevertheless, there is little data regarding the E. anophelis geographical distribution, phylogenetic structure, and transmission across the globe, especially in Asia. We utilize whole-genome sequencing (WGS) data to define a global population framework, phylogenetic structure, geographical distribution, and transmission evaluation of E. anophelis pathogens. The geographical distribution diagram revealed the emerging pathogenic bacteria already distributed in various countries worldwide, especially in the USA and China. Strikingly, phylogenetic analysis showed a part of our China original E. anophelis shared the same ancestor with the USA outbreak strain, which implies the possibility of localized outbreaks and global spread. These closer related strains also contained ICEEaI, which might insert into a disrupted DNA repair mutY gene and made the strain more liable to mutation and outbreak infection. BEAST analysis showed that the most recent common ancestor for ICEEaI E. anophelis was dated twelve years ago, and China might be the most likely recent source of this bacteria. Our study sheds light on the potential possibility of E. anophelis causing the large-scale outbreak and rapid global dissemination. Continued genomic surveillance of the dynamics of E. anophelis populations will generate further knowledge for optimizing future prevent global outbreak infections. Elizabethkingia is a genus of Gram-negative, aerobic, oxidase-positive, weakly indole-positive, non-fermenting, and non-motile bacillus belonging to the family Flavobacteriaceae. It can be widely found in natural environments such as soil and water , were reidentified as E. anophelis bacteria [Elizabethkingia were reidentified as E. anophelis by 16S rRNA gene sequencing [Interests, in previous studies, many stimated ,8,9. Forbacteria . Moreovequencing .E. anophelis infections: multi-drug resistance and high fatality rate [E. anophelis infections, and the arresting case fatality rate was reported to top 31% (20/65) [There are two prominent peculiarities of ity rate ,7,9,17,  (20/65) .E. anophelis were less well understood. The unknown pathogenesis mechanisms and multi-drug resistance limited available genomic information and lack of effective therapeutic regimens challenging the management of E. anophelis infections [E. anophelis data regarding geographical distribution, phylogenetic structure, and transmission globally, especially in Asia. Previously, we had collected 197 E. anophelis strains from different provinces in China. Then, we completed the genome sequencing and downloaded almost all the genomes of this bacteria in the National Center of Biotechnology Information (NCBI) databases. We utilize these WGS data to define a global population framework, phylogenetic structure, geographical distribution and transmission evaluation. Our analysis indicates there may be a potential outbreak and rapid global dissemination of E. anophelis.In contrast to other familiar bacteria, the epidemiology, transmission, and evolutionary mechanism of fections ,19,20. FE. anophelis isolated from clinical samples from the Strain Library of the First Affiliated Hospital of Zhejiang University School of Medicine between January 2010 and April 2019 was performed. Some bloodstream infection strains in the Library came from other hospitals and provinces. A total of 197 E. anophelis isolated from sputum (138 isolates), blood (25), abdominal fluid (7), cerebrospinal fluid (5), bronchoalveolar fluid (5), cardiac catheter (3), bile (2), pleural fluid (2), throat (2), burns (1), secretion (1), drainage (1), swab (1), body fluid (1), heart valve (1), abscess(1), and urine(1) were included in the work. For comparative genomic analysis, 318 E. anophelis strains were used in this study, including 197 newly sequenced and 121 publicly available strains (Supplementary Table 1), comprising the global collection isolates originating from more than 12 countries spanning four continents . At the time of writing this paper, only 121 whole-genome sequences of E. anophelis species, including raw data were available in the NCBI genome sequence repository of GenBank or SRA centre. Full details of the isolates (including download strains) are provided in Supplementary Table 1.A longitudinal survey of E. anophelis we isolated were initially identified using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOF MS) , which may be misidentified. To reconfirm our 197 collected E. anophelis and reduce the impact on our collection, we constructed a heatmap based on the average nucleotide identity (ANI) values. Bacteria E. anophelis NUHP1 and R26, E. meningoseptica G4120 and KC1913, E. miricola EM798-26 and BM10 were as reference genomes.The Bacteria were cultivated in Mueller–Hinton Broth  without antibiotics in aerophilic conditions at 37°C on a shaker for 24–48 h. DNA extraction was performed using the Gentra Puregene Yeast/Bact. Kit  according to the manufacturer’s instructions. The harvested DNA was detected by the agarose gel electrophoresis and quantified by Qubit® 2.0 Fluorometer (Thermo Scientific).E. anophelis was sequenced using Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd. To ensure the accuracy and reliability of the subsequent information analysis results, the Raw Data was filtered to obtain Clean Data. Assembled was performed with three different software: SOAP denovo, SPAdes and Abyss. Then, the three software assembly results were integrated with CISA software, and the assembly result with the least scaffolds was selected.A total amount of 1 µg of DNA per sample was used as input material for the DNA sample preparations. Following the manufacturer's recommendations, sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina , and index codes were added to attribute sequences to each sample. PCR products were purified (AMPure XP system), and libraries were analysed for size distribution by Agilent 2100 Bioanalyzer and quantified using real-time PCR. The whole genome of http://www.repeatmasker.org/). The tandem Repeats were analyzed by the TRF (Tandem repeats finder) [Genome component prediction included the prediction of the coding gene, repetitive sequences, non-coding RNA, genomics islands. We used the GeneMarkS  programm finder) . Transfe finder) . Ribosom finder) . Small n finder) ,27. The  finder)  programmE. anophelis CSID_3015183678, using MUMmer [https://github.com/katholt/RedDog). Then, the concatenated alignment of these SNP alleles was used to generate a maximum likelihood phylogenetic tree for all the E. anophelis isolates using PhyML [https://github.com/katholt/plotTree/#python-code). The globally geographical distribution graph was performed by R software (version 3.5.3) with the rworldmap package. An interactive version of the global phylogeny labelled genotype, country origin, and isolation year was performed by Microreact [ANI values of all sequenced genomes were analyzed by FastANI . To asseng PhyML  with theng PhyML  with theng PhyML  and Pythcroreact .E. anophelis ICEs structures (ICEEa) were predicted for all genome assemblies using ICEberg [ ICEberg  database ICEberg .E. anophelis was performed with BEAST 2.5.2 [E. anophelis were aligned to define a core and accessory genome using Roary [E. anophelis cluster were performed using BEAST 2.5.2 [Temporal analysis of ST 2.5.2 . MoleculST 2.5.2  based onng Roary . The resng Roary  using a ST 2.5.2 , which mE. anophelis were estimated using a flexible coalescent Bayesian skyline incorporated in BEAST V2.5.2 [Population dynamics of the T V2.5.2 , combineElizabethkingia species are presented in Figure S1. The ANI values ranged from 79% (between E. meningoseptica_G4120 and E. anophelis SKLX001947) to 100% (among a coup of newly sequenced E. anophelis). Clearly, the ANI values among all E. anophelis strains are >95.6% , suggesting that they are the same species according to the microbial taxonomy for species delineation (>95% cut-off for ANI) [The values of 203 representative E. anophelis genomes . The picture revealed the emerging pathogenic bacteria already distributed in various countries worldwide, especially in the USA and China. Primary clusters 1, 2, and 4 were broadly distributed across continents , ICEEaII (Type II) and ICEEaIII (Type III) [Integrative and conjugative elements (ICEs), also called conjugative transposons, are a diverse group of mobile genetic elements found in both gram-positive and gram-negative bacteria, with a range of mechanisms to promote their core functions of integration, excision, transfer, and regulation, contributing to bacterial pathogenesis . Recent ype III) .E. anophelis strains worldwide were searched for ICEs , making the strain more liable to mutation and outbreak infection [E. anophelis strains contained Type I ICE (ICEEaI). Therefore, it implied the E. anophelis strains, outside the USA and especially in China, which contain ICEEaI might have the risks of localized outbreaks and rapid global dissemination. We should do a good job of monitoring and corresponding measures to prevent outbreaks of E. anophelis infection. It was an intriguing observation that among those Type I ICE possessed E. anophelis the original isolate came from China (in 2012), years earlier than the outbreak strain in the United States. Thus, there may be a possibility that the strain with the ability to break out of infection firstly originated in China and spread abroad.In this research, all 318 genomes of for ICEs . Based o strains . The rec strains . Neverthd Sweden . ICEEa1 nfection . Indeed,E. anophelis pathogens (ICEEaI) emergence, we constructed temporally resolved phylogenies using Bayesian evolutionary analysis by sampling trees (BEAST 2.5.2) , implying that the ICEEaI carriage E. anophelis strain continued diversifying in a measurable way over the course of the evolution. Based on the BEAST analysis, the MRCA for ICEEaI E. anophelis was dated twelve years ago . The result suggests that China was the most likely recent source of this ICEEaI containing E. anophelis . The increase began approximately seven years ago (2012), and the decreases occurred relatively close to three years ago (2016) but was lesser than the first rapid increase . This observation is consistent with a previous report outbreak of infections in the United States between 2015 and 2016. If confidence intervals are taken into account, we assume that in this study we observed the last main increase in population size.To investigate the structure and history of these 84 potentially explosive infectious T 2.5.2) . Firstlynophelis , which inophelis , the ICElineages . This moE. anophelis transmission networks across the globe. Within samples from various countries and different contemporary, we observed the emerging pathogen inherently has the possibility of global transmission and outbreak. To our knowledge, our study of E. anophelis is the largest and most representative in China to date and one of the largest internationally. We sampled isolates from a geographically dispersed subset of clinics and environments in various countries. Although we found some diﬀerences between the characteristics of the study isolates and those of all diagnoses in China, the absolute diﬀerences were small. Therefore, our study probably provides reliable insights into the molecular epidemiology or transmission of E. anophelis globally.This large molecular epidemiology study showed how sequencing data could be used to gain insights into E. anophelis has recently emerged as a cause of life-threatening infections in humans, particularly in immunocompromised patients [Elizabethkingia  have been proposed in the last decade [E. meningoseptica, E. anophelis is the most prevalent pathogen and has the highest mortality in this genus [Elizabethkingia species remains a considerable challenge in clinical settings, and the cases of E. anophelis must be underestimated  due to the imperfect strain identification method . Indeed, in our research, nearly three hundred E. anophelis isolates were firstly identified by MALDI–TOF MS, but actually, only 197 were reconfirmed after genome sequencing and Nucleic acid alignment. In addition, our isolates number in is sustainable growth in recent years. Fortunately, recently, several studies have focused on the ameliorative identification means of Elizabethkingia species. It was reported MALDI–TOF MS systems with amended databases specific peaks could be used to differentiate Elizabethkingia species. However, these amended databases, either in the VITEK Mass Spectrometry or Bruker Biotyper systems, are merely available for research purposes but are not for clinical application in clinical microbiology laboratories [rpoB) gene sequencing are able to correctly distinguish Elizabethkingia strains at the species level [lepA), have been recently developed for detecting and differentiating Elizabethkingia species [The species patients . Severalt decade . Numerouis genus . Howeverratories . Housekees level . In addi species ,47.E. anophelis occurred in the USA. Unexpected, the case fatality rate was reported high at 31% (20/65) [E. anophelis outbreak in the USA was definitely attributed to the ICEs [E. anophelis strain? Unfortunately, after in silico identification of ICEs, we found the critical pathogenic type I ICE (ICEEaI) also existed in a part of our E. anophelis. It sounded the alarm for us that the E. anophelis might also have the ability to cause the outbreak of infections in China and rapid global dissemination. Expected, if the E. anophelis casing rapid global transmission and outbreaks, the damage will be more serious and invaluable. Therefore, it is necessary to monitor the epidemiology and transmission of the emerging bacterium to control and prevent the global outbreak of infections of the E. anophelis.In 2015–2016, the first shocking outbreak of infection of  (20/65) ,18. In a (20/65) ,12,15. Ithe ICEs . Here, wE. anophelis has the character of multi-drug resistance, which show different degree of antibiotic resistance such as β-lactams, aminoglycosides, quinolones, tetracyclines, chloramphenicol, even carbapenems and vancomycin [E. anophelis resistance, and the multiple resistance mechanisms are still unclear. In recent years, biofilm has increasingly become a hot topic. Generally, biofilms are defined as a community of cells or bacteria encased within an exopolymeric matrix and attached to a surface. They are recognized as being more resistant to antimicrobial therapy and host defences [E. anophelis contained multiple biofilm-forming genes such as flmH, ugd, capL, rmlC, capE, fleQ, cpsO, cap8E, fnlA, VipB/tviC, uppS, motD, fleR, kpsF, wbjD/WecB, rmlD, rmlB, capL, and cap5H . Therefore, we propose the following hypothesis: The related genes carried by E. anophelis bacterium promote the formation of a biofilm, then enter a slow growth lag phase, and acquire antibiotic multiple resistance characteristics. It may contribute to clarifying the antibiotic multiple resistance mechanisms of E. anophelis. Therefore, more and further experiments are deserved to perform.It is documented that ncomycin ,16,17,48defences ,50. Studdefences . IntriguE. anophelis because of obvious sampling bias . This limitation also hinders our ability to account for secular trends. Nevertheless, an important reason is that many E. anophelis strains are not sequenced, and the available genome is limited. Apparently, available genomic data is far less than the number of reported cases. This indicates that people should pay enough attention to this emerging pathogenic bacterium in the future.A limitation of this study exists. We did not establish the true extent of the global distribution of E. anophelis transmission. Our study sheds light on the potential possibility of E. anophelis pathogens causing the large-scale outbreak and rapid global dissemination. Continued genomic surveillance of the dynamics of E. anophelis populations with increased geographical representation and length sampling time will generate further knowledge for optimizing future prevent outbreak infections and rapid global dissemination.In conclusion, genomic, spatial, phylogenetic, and epidemiological data helped us to better understand the complex dynamics of Click here for additional data file.


Halyomorpha halys (Hemiptera: Pentatomidae). Our results may be useful for the improvement of monitoring strategies for early pest detection. Additionally, the higher efficacy of the multimodal traps would allow their use in attract-and-kill or push–pull strategies within integrated pest management.Pest monitoring using traps is a key component of integrated pest management. For several insects, trapping is achieved using visual or olfactory stimuli. Although the combination of both is supposed to provide higher efficacy, this has often been overlooked in trap design. Through laboratory bioassays and field experiments we evaluated the use of UV-A and visible light in combination with olfactory stimuli to improve trapping of the invasive brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), are challenging. Here we developed and evaluated a multimodal trap which combines visual and olfactory stimuli. Visual stimuli consisted of LEDs emitting UV-A and visible light. Olfactory stimuli were comprised of the synthetic aggregation pheromone and odours from trapped H. halys individuals. Stink bug attraction at different wavelengths was evaluated in laboratory two-choice bioassays, and different prototypes of the trap were tested in 2021 in natural, agricultural, and urban settings. Traps with a combination of UV-A and blue or green visible wavelengths provided higher H. halys attraction (up to ~8-fold) compared to traditional sticky or small pyramidal traps. The concurrent presence of synthetic pheromone and LED had a synergistic effect on H. halys positive phototaxis. Further development and implementation of the multimodal trap is discussed for prospective use in attract-and-kill or push–pull strategies.Capture strategies for the brown marmorated stink bug,  Invasive invertebrates that establish in a new area may cause environmental and economic damage to agriculture and society . From 19Halyomorpha halys Stål (Hemiptera: Pentatomidae) is native to Asia and is now widespread in each continent of the Northern Hemisphere [H. halys adults seek shelter within human-made structures to overwinter, thereby being a nuisance to citizens [The brown marmorated stink bug misphere . Its hosmisphere , and, inmisphere ,14,15. Tmisphere . Additiocitizens ,14.H. halys populations is crucial for anticipating further invasion, allowing growers to determine the best control tools and to ensure timely chemical applications [H. halys has resulted in the disruption of IPM programmes for several important crops, with possible non-target effects on beneficial insects, e.g., predators and egg parasitoids notoriously sensitive to lethal and sublethal insecticide doses [H. halys [H. halys [Plautia stali Scott [H. halys direct control, e.g., in attract-and-kill or push-pull strategies [H. halys mating behaviour [H. halys adults compared to classic sticky panels [H. halys [H. halys, we tested traps with the combined effect of different stimuli, i.e., LED lights, synthetic pheromone, and natural odours emitted by trapped insects. Captures of insects other than H. halys were also recorded to possibly evaluate the effect of such traps on non-targets. Multimodal traps were assessed in agricultural, urban, and natural landscapes of northern and central Italy where H. halys is responsible for serious economic losses to crops and citizen nuisance.Assessing the density of ications . In receications . As a code doses ,20,21. BH. halys . Sweep nH. halys  but ofteH. halys . The ideH. halys  and the li Scott , has proli Scott ,27,28. Nli Scott . To overrategies ,31. Vibrehaviour , and theehaviour . Rotatiny panels . In addiy panels . Hence, H. halys ,36. BlacH. halys . Therefohttps://www.mo-el.it/, accessed on 4 May 2022). The trap  is vertically fixed on the plastic body of the trap. Basically, each lamp consisted of four LEDs (peak emission at 365 nm) equidistantly positioned every 2.6 cm in a digital strip and is partially covered by a removable and interchangeable plastic grid (14 × 4.2 × 2.1 cm) made of translucid coloured polycarbonate. LED lamps are powered by an electronic converter for LEDs . When a portion of the UV-A flux intercepts the plastic part of the grid it undergoes a wavelength change, i.e., from 360–380 nm to 380–780 nm, which corresponds to visible light (details in patent EP3909426A1). A second portion of the radiation emitted by LEDs does not intercept the plastic part of the grid and totally consists in UV-A radiation. A motorised fan (details in US20030131525A1) is horizontally placed inside the trap at a central position. By rotating, the fan generates an airstream that sucks the insects surrounding the LED lamp and eventually conveys them inside a polyamide funnel bag placed behind the trap. The trap is completed by a metal grill (approx. 2–4 × 8 cm rectangular holes) externally fixed to prevent the entrance of birds or small mammals into the openings  based on their model “TurbiLED”  was positioned on the uppermost extremity of the trap. Dispensers comprised both the two-component H. halys aggregation pheromone and the synergistic MDT [For our experiments, differently coloured plastic grids were evaluated (provided by MO-EL S.p.A.), allowing emission of wavelengths with a dominance of blue (hereafter “blue-UV”), green (“green-UV”), or yellow (“yellow-UV”). A black grid with no reflectance was also tested for emission of UV-only wavelengths. To increase stink bug captures, a dispenser of the aggregation pheromone of stic MDT . The novH. halys were collected in northern and central Italy from overwintering sites (January-February 2021) or from agricultural and natural ecosystems (spring 2021) and served to establish a laboratory colony. Clear plastic food containers (30 × 20 × 15 cm) with 5-cm round holes covered by a mesh were used to rear adults in a laboratory under environmentally-controlled conditions . A diet of apples, carrots, green beans, hazelnuts, and sunflower seeds was supplied. Diet was replaced twice a week. Vicia faba L. potted plants were placed inside the cages both as a supplementary food source and an oviposition substrate [Adults of ubstrate . Eggs weThe radiation flux emitted by the different combinations of UV lamp and plastic grids was measured in the laboratory by positioning the sensor of a digital spectrophotometer  at close contact or at 2 cm distance from the plastic grid covering the LED lamp. Emission intensity was measured up to 3750 photon count.H. halys phototactic behaviour, bioassays consisted of two-choice tests and were performed in a dark room maintained at a constant temperature of 25 ± 1 °C. Groups of 5 adult females or males were isolated in 50 mL Falcon tubes closed with a small net to allow aeration. Three hazelnut fruits were supplied as food, and insects were kept in the bioassay room for 1 h before being tested. One stink bug at time was assayed. For the evaluation of Bioassays were conducted on the surface of a black table (100 × 80 cm). Two visual stimuli were both provided at one of the longest sides of the table. The two stimuli were positioned at 60 cm distance and were separated by a black cardboard panel  to minimise interference between the two stimuli.Treatments consisted of one UV lamp with four LEDs partially covered by a plastic grid  between July and August 2021. A multimodal trap with 4 lamps emitting blue-UV, green-UV, yellow-UV, or UV-only wavelengths (experiment 1) and a trap with 2 lamps emitting blue-UV or green-UV (experiment 2) light were assessed for H. halys were collected. In experiment 4 , two traps per type were used and both H. halys and other insect groups were assessed. Controls were a pyramidal trap  in experiment 3 and sticky traps  in experiment 4.Experiments 3 and 4 were conducted in a fruit orchard in central Italy near Ponte Pattoli, Perugia  between August and September 2021. The difference in daily capture among the multimodal trap and two different commercial traps as control was evaluated. The multimodal traps were equipped with 4 lamps emitting green-UV or blue-UV light. In experiment 3, a total of 3 samplings were conducted; only one trap for each type was positioned and only data on Experiment 5 was conducted in an urban park in northern Italy near Crevalcore, Bologna  between August and September 2021. Three multimodal traps with different equipment were compared. These were: a multimodal trap with 4 blue-UV lamps and the pheromone lure, a trap with the pheromone lure but with the LED lamps turned off, a trap with the LED lamps turned on but without pheromone lure. Eleven samplings were conducted.For laboratory bioassays, generalised linear models  were fitted to test the first choice, i.e., the area close to the stimulus the stink bug entered first . For allThe measurement of the radiation flux for blue-UV, green-UV, yellow-UV, or UV light revealed that the higher portion of the emission was represented by undisturbed UV-A wavelengths, and only a small fraction constituted visible light B. H. halys were highly attracted to visual stimuli from blue-UV light compared to control (no light) . No differences were detected between blue-UV and yellow-UV, or green-UV (p > 0.05 for both comparisons) light. Male H. halys where highly attracted towards visual stimuli from blue-UV compared to control (p < 0.0001) or to UV-only (p = 0.029). No differences were detected between blue-UV and yellow-UV or green-UV (p > 0.05 for both comparisons). In laboratory conditions, female p = 0.0008). Experiment 2: When only blue-UV and green-UV light were compared in the same trap, the presence of the stink bugs was similar across treatments  and sampling days   but higher for the blue and green traps compared to control trap  was higher in relation to green-UV light compared to all other treatments (results of LMM followed by multiple comparison procedure are reported in  = 0.48) . ExperimH. halys were higher for both blue-UV and green-UV traps compared to sticky traps (p = 0.030).Experiment 4: Captures of ky traps . SimilarH. halys captures were detected for the complete multimodal trap compared to the other treatments (results of LM followed by multiple comparison procedure are reported in p = 0.038) and was higher for the two treatments with light compared to the treatment with pheromone only. Captures of ladybird beetles (Coleoptera: Coccinellidae) were stable across sampling days  and did not differ across treatments . Captures of wasp-waisted wasps (Hymenoptera: Apocrita) were different across sampling days  and were higher for the two treatments with lights compared to the treatments with only pheromone.Experiment 5: When captures were compared among a trap with blue-UV light and pheromone, a trap with pheromone only, and a trap with blue-UV light only, higher H. halys males and females were attracted toward LED lights emitting both UV-A and visible wavelengths. In addition, the combination of such lights with odour stimuli resulted in higher captures compared to pheromone-based traps or light traps alone.In agricultural ecosystems, early detection of a herbivorous insect population is crucial for effective pest management ,44. HencH. halys, as well as herbivorous insects in general, including other stink bugs, are attracted by different light wavelengths [Nezara viridula (L.) (Hemiptera: Pentatomidae) exhibited stronger attraction toward UV compared to visible wavelengths, and secondarily toward blue and green compared to orange light [H. halys showed positive phototaxis toward fluorescent black light (with a UV dominant output) or blue light (with a dominant output in the visible spectrum and no emission in the UV spectrum) [H. halys [H. halys can detect and are attracted by either blue, green, or yellow light. Most insects have three types of pigments that ensure UV-blue-green trichromacy [H. halys of a singleton UV-opsin homolog and duplicates of long-wavelength opsin homologs but lacked the presence of blue-sensitive opsin ortholog [H. halys remains to be investigated [H. halys compared to other wavelengths, including blue-UV. However, subsequent field experiments revealed that green-UV or blue-UV multimodal traps captured similar numbers of H. halys. A speculative explanation is that because H. halys has a marked preference for immature and mature fruits, this species has evolved photoreceptors to better discriminate green and longer (yellow or red) wavelengths, and that such preference is only evident during the photophase . However, further experiments should aim at clarifying such findings.It is already known that elengths . In a frge light . In dualpectrum) . Attractpectrum) , as it sH. halys . Similarchromacy . A recenortholog . In othestigated ,53. It iH. halys trapping in mid-Atlantic sites of the US [H. halys individuals successfully attracted conspecifics, possibly because of the production of natural aggregation pheromone from trapped individuals [H. halys captures compared to a control trap [Riptortus pedestris, H. halys, and Piezodorus hybneri [H. halys trapping [Sesamia nonagroides Lefèbvre [Chilo partellus (Swinhoe) [H. halys similarly respond to UV-A and visible wavelengths must be verified.Although light and pheromone alone were similarly attractive, the simultaneous presence of both stimuli synergistically improved the attractiveness. This result is consistent with a previous investigation where the combination of fluorescent blue light and aggregation pheromone increased mid-season f the US . Modificf the US  and weref the US ). The faividuals . Similarrol trap . In a so hybneri . The addtrapping . In our trapping . AnotherLefèbvre ,57 and CSwinhoe) ). Hence,Coccinella septempunctata L. (Coleoptera: Coccinellidae) exhibited positive phototactic behaviour toward UV and red lights and much lower response to green or blue lights [C. septempunctata [Harmonia axyridis  (Coleoptera: Coccinellidae) was strongly attracted by UV and blue wavelengths [H. axyridis populations in those areas where this species has become a concern for threating insect biodiversity [Non-target insect trapping is a side effect of traditional light traps ,60. In oe lights . Notwithpunctata . Converselengths ,65. Despelengths , it is celengths ,68,69. Hiversity . Notablyiversity ,72. Conviversity . Becauseiversity , the proiversity .H. halys, e.g., through mass trapping [H. halys control, also because it is expected to have low interference with complementary control practices, such as biocontrol with native and exotic parasitoids [Concerning IPM, the multimodal trap that we evaluated could be used not only for pest monitoring, but also for directly controlling trapping . Additioasitoids ,79,80,81H. halys captures. Other research would be needed to better understand the H. halys phototaxis behaviour in different environmental conditions. For example, the response at different light intensities [We have evaluated a multimodal trap that combines visual and olfactory stimuli and greatly enhanced ensities , evaluatensities ), or phyensities . Furtherensities .


Zanthoxylum bungeanum. Here, 109 ZbNAC proteins were identified from the Z. bungeanum genome and were classified into four groups with Arabidopsis NAC proteins. The 109 ZbNAC genes were unevenly distributed on 46 chromosomes and included 4 tandem duplication events and 17 segmental duplication events. Synteny analysis of six species pairs revealed the closely phylogenetic relationship between Z. bungeanum and C. sinensis. Twenty-four types of cis-elements were identified in the ZbNAC promoters and were classified into three types: abiotic stress, plant growth and development, and response to phytohormones. Co-expression network analysis of the ZbNACs revealed 10 hub genes, and their expression levels were validated by real-time quantitative polymerase chain reaction (qRT-PCR). Finally, ZbNAC007, ZbNAC018, ZbNAC047, ZbNAC072, and ZbNAC079 were considered the pivotal NAC genes for drought tolerance in Z. bungeanum. This study represented the first genome-wide analysis of the NAC family in Z. bungeanum, improving our understanding of NAC proteins and providing useful information for molecular breeding of Z. bungeanum.NAC  transcription factors (TFs) are one of the largest plant-specific TF families and play a pivotal role in adaptation to abiotic stresses. The genome-wide analysis of NAC TFs is still absent in  The NACon (CUC) . NAC proon (CUC) ,3. The vOsNAC2 was found to positively affect drought tolerance through ABA pathways in rice [TaNAC071-A in wheat reduced plant drought tolerance, whereas its overexpression significantly enhanced drought tolerance through improved water-use efficiency [PwNAC11 enhanced the drought tolerance in transgenic Arabidopsis by activating EDR1 [VuNAC1 and VuNAC2, improved the drought tolerance of transgenic Arabidopsis lines [NAC TFs have been reported to regulate various developmental processes in many plants, including lateral root construction , cell di in rice . Knockdoficiency . Moreoveing EDR1 . Furtheris lines .Zanthoxylum bungeanum Maxim , a member of the Rutaceae family, is an economically important plant. The Z. bungeanum pericarp is a popular cooking condiment and food additive in most Asian countries because of its unique taste and numbing sensation. Recent years have witnessed a rapidly increasing demand for Z. bungeanum pericarp due to the popularity of Sichuan hotpot, a well-known Chinese dish. In addition, Z. bungeanum pericarp and leaves are traditional herbal medicines for the treatment of numerous diseases; they are reported to promote blood circulation, dispel cold dampness, and diminish inflammation [Z. bungeanum pericarp may also have significant potential in the cosmetics industry due to the high antioxidant activities of its volatile oil [Z. bungeanum is widely cultivated in arid or semiarid areas because of its adaptation to the drought environment. Genome-wide analyses of the NAC family have been performed in a large number of species, including Ziziphus jujuba [Helianthus annuus [Juglans mandshurica [Z. bungeanum owing to the absence of a reference genome. Notably, Z. bungeanum genome information was reported in our recent research [Z. bungeanum plant.ammation . Z. bungtile oil . Z. bungs jujuba , Heliants annuus , Juglansdshurica , peanut dshurica , and sweresearch , enablinZ. bungeanum. Totally, 109 NAC TFs were identified from the Z. bungeanum genome and classified into four groups. Gene structure, conserved motif composition, chromosomal distribution, gene duplication, phylogenetic characteristics, promoter cis-elements, and homologous relationships among the ZbNAC TFs were investigated. In addition, the expression patterns of ZbNAC genes under progressive drought stress were analyzed in two Z. bungeanum cultivars using RNA-seq data and validated by real-time quantitative polymerase chain reaction (qRT-PCR). The results of this research are expected to improve our understanding of NAC TFs and provide valuable clues for molecular-assisted breeding in Z. bungeanum.In the present study, the genome-wide identification and analysis of NAC family was firstly performed in Z. bungeanum (ZbNACs). All were located alone or in clusters on 46 chromosomes, and they were named based on their genetic locations across the Z. bungeanum chromosomes (ZbNAC001–ZbNAC109)  . The numZbNAC genes , T. aestivum (5), and Z. mays (8).To further explore the evolutionary relationships between Zea mays . There wA. thaliana than in Z. bungeanum in groups II–IV: group II contains 9 AtNAC proteins and 24 ZbNAC proteins, group III contains 9 AtNAC proteins and 38 ZbNAC proteins, and group IV contains 8 AtNAC proteins and 41 ZbNAC proteins. ZbNAC and AtNAC proteins with close phylogenetic relationships may have similar gene structures and biological functions.To explore the evolutionary relationships among NAC proteins, a phylogenetic reconstruction was performed based on a multiple sequence alignment using MEGA 7.0 . An unroTo further explore the relationships among ZbNAC family members, the phylogeny, gene structures, and conserved motifs of the ZbNAC TFs were analyzed . Eight cZbNAC genes (67) contained two introns, two  contained one intron, and one (ZbNAC080) contained eight introns. As expected, genes that were closely phylogenetically related shared common motifs and similar motif locations. For example, ZbNAC068, ZbNAC069, and ZbNAC070 are clustered together in the phylogeny tree; all have five exons with similar locations and lengths.Gene structure analysis showed that the ZbNAC encoding genes contained 1 to 8 introns C. Most ocis-elements were distributed unevenly in the 2000-bp upstream regions of the 109 ZbNAC genes (cis-elements were classified into three categories: abiotic stress (6 types), plant growth and development (7 types), and response to phytohormones (10 types). Judging from the numbers of these elements, they are mainly involved in phytohormone pathways and responses to abiotic stress, thus revealing the important role of ZbNACs in the regulation of hormone synthesis and adaptations to environmental stress. With respect to abiotic stress, TC-rich repeats (75) were the most abundant elements; they were located in 45 ZbNAC promoters and were annotated as drought response elements , and salicylic acid pathways. Among these elements, ABRE elements were the most abundant  followed by CGTCA motifs  and TGACG motifs . The numbers and types of cis-elements were similar in ZbNAC genes that had a close phylogenetic relationship. In group I, there were fewer elements, and they were seldom assigned to the plant growth and development category. In group II, the number of cis-elements was highest, and TC-rich repeats, ABRE elements, CGTCA motifs, and TGACG motifs were the most abundant. For example, there were 18 ABRE elements in ZbNAC092, 14 ABRE elements in ZbNAC065 and ZbNAC046, and 8 CGTCA motifs in ZbNAC004 and ZbNAC102, and these elements were annotated as ABA and MeJA responsive. The ZbNAC TFs in group II may therefore be important for drought response and ABA and MeJA regulation.Twenty-three types of AC genes . These celements . With reZbNAC genes of two Z. bungeanum cultivars under drought stress. These genes were divided into four clusters (clusters 1–4) based on their FPKM values  and ZbNAC102 (1.97) in HJ. Thirty-three ZbNACs were placed into cluster 2, and most of them increased in expression with progressive drought stress, with higher expression in HJ than in FJ. The F4/F1 and H4/H1 ratios were greater than one in almost all members of cluster 2 except for ZbNAC081 (0.95) and ZbNAC094 (0.84). Interestingly, the ratios of six ZbNACs from cluster 2  were greater than ten in both cultivars, suggesting that these six genes may play pivotal roles in response to drought stress. There were 19 ZbNACs in cluster 3; most were significantly enhanced in FJ during early drought, but the changes in HJ were less marked, indicating that the genes in cluster 3 were specifically involved in adaptation to drought stress in FJ. In cluster 4, the expression levels of most genes increased in the early drought period and decreased in the later period, suggesting that these TFs may regulate the expression of early drought-responsive genes in Z. bungeanum. In addition, the average FPKM values suggested that the expression levels of ZbNAC057 (99.52) and ZbNAC090 (44.15) were significantly higher than those of other genes, which averaged 4.30. The total expression levels of the 109 ZbNAC genes were displayed in violin diagrams , and only 12 pairs showed negative correlations (p < 0.05). GO analysis assigned GO terms to these genes from three categories: biological process (BP), cellular component (CC), and molecular function (MF)  were identified using the Cytohubba program in Cytoscape 3.9.0  were the most dominant in the network because of their high connectivity with other genes.Correlation analysis was performed for 87 genes in clusters 1, 2, and 3 with FPKM values greater than 1 to explore their possible relationships A. The reion (MF) B. Among pe 3.9.0 C. Three The AtNAC homologs of the ten hub ZbNAC proteins were predicted from the phylogenetic tree  and are Z. bungeanum owing to the absence of a reference genome. Recently, a chromosome-scale assembly of Z. bungeanum genome with high quality was published in our research [Z. bungeanum. Here, we firstly performed the comprehensive genome-wide identification of NAC family members and characterized their gene expression under drought stress in Z. bungeanum.NAC TFs are one of the largest plant-specific TF families and play significant roles in plant growth, development, and adaptation to environmental stress . Genome-research , which pDactylis glomerata [Cucumis melo [Fagopyrum tataricum [Manihot esculenta [O. sativa [Z. mays [Glycine max [n = 68) in the Z. bungeanum genome, owing to frequent fusion/fission events after whole-genome duplication [Based on the presence of the NAC domain structure, 109 non-redundant ZbNAC TFs were identified, close to the 108 NACs in lomerata ; more thmis melo , 80 in Fataricum , and 96 sculenta ; and few. sativa , 152 in [Z. mays , and 152cine max . These rlication . The 109Panicum miliaceum [Musa acuminata [Z. bungeanum, 42 pairs of segmental duplicates and 5 pairs of tandem duplicates were identified, indicating that segmental duplication was more common during evolution of the ZbNACs. Interestingly, five tandem duplicates were found together on chromosome 2. We speculate that these five genes may have a common ancestor and may therefore share similarities in structure and function. Ka/Ks = 1, <1, and >1 are indicative of neutral selection, purifying selection, and accelerated evolution under positive selection, respectively [Ka/Ks <1, indicating that most ZbNACs evolved primarily under purifying selection. C. sinensis, Z. bungeanum, Z. jujube, and Arabidopsis are dicots, whereas maize, rice, and wheat are monocots. Here, the dicots exhibited greater collinearity with Z. bungeanum than the monocots, indicating that NAC TFs experienced extensive evolution and duplication after the divergence of monocots and dicots. Feng et al. [Z. bungeanum shared a close phylogenetic relationship with C. sinensis and that they diverged from their most recent common ancestor approximately 35.3 million years ago. Consistent with this finding, the greatest number of collinear NAC genes were found between these two species.Gene duplication is a potential driving force for species evolution as well as gene functional diversity . NAC TFsiliaceum , the 17 cuminata , and thecuminata . In Z. bectively . In thisg et al.  found thZbNAC genes varied from 1 to 8, similar to numbers reported in chickpea (1–6) [ZbNAC068, ZbNAC069, and ZbNAC070, which were most closely related in the same group, harbored five exons of similar location and length. The two ZbNAC genes with only one intron (ZbNAC073 and ZbNAC047) may have a faster response speed and may be located upstream in the response pathway. ZbNAC080 contained eight introns, and the length of each exon was relatively short. Thus, we suspect that the insertion of introns led to mutation in gene structure and that ZbNAC080 may have specific functions. The motifs in TF proteins are often associated with DNA binding, protein interaction, and transcriptional activity [Exons and introns are important features of gene evolution, and exons are an important part of protein-coding genes . The insea (1–6)  and sunfea (1–6) . Family ea (1–6) , and thiactivity ,43. A toactivity .cis-elements indicate possible responses of these genes to internal and external factors [ZbNACs and were divided into three categories: abiotic stress response elements, hormone response elements, and developmental regulation elements. Numerous elements were involved in abiotic stress and hormone response, consistent with NAC genes in jujube [ZbNACs may be associated with plant hormone signaling and abiotic stress responses. LTR (response to low temperature) and CCTG (response to drought stress) accounted for a large proportion of abiotic stress elements, suggesting a vital role for ZbNACs in low temperature and drought stress. Consistent with this notion, substantial evidence suggests that the NAC gene family plays a critical role in plant responses to abiotic stress. For example, overexpression of ONAC022 improved drought and salt tolerance in rice [MbNAC25 from Malus baccata increased the resistance of transgenic Arabidopsis to cold and salinity [ZbNAC promoters. Likewise, ABA and MeJA elements were also abundant in NAC promoters of Ipomoea trifida [ZbNACs may participate in drought resistance via regulation of ABA and MeJA.The structure and regulatory mechanisms of gene promoters are closely related to specific plant traits . The phy factors . Twenty-n jujube , implyin in rice , and ovesalinity . There w trifida . ABA and trifida . Previou trifida , and exo trifida ,50,51. TZ. bungeanum is a commercial crop that is widely grown in arid and semi-arid regions, and the above analyses revealed the possible function of ZbNACs in drought response. Therefore, exploring the expression of these ZbNACs under drought is necessary for a comprehensive analysis of the ZbNAC family. In the present research, RNA-seq was performed with two Z. bungeanum cultivars of different drought tolerance under progressive drought stress. The 109 ZbNAC genes were divided into four groups based on their expression levels, and three clusters  were upregulated by drought, suggesting that most ZbNAC genes are involved in drought tolerance of Z. bungeanum. Likewise, drought also increased the expression levels of most NAC genes in peanut [ZbNACs are pivotal genes that dominate the interactions of transcription factors, and they may have important regulatory functions in drought response. The qRT-PCR results showed that the expression levels of these ten ZbNACs increased with drought, and most of their expression levels were higher in HJ than in FJ. This result not only verified our speculation but also confirmed the reliability of our RNA-seq data.n peanut  and peppn peanut . Correlan peanut ,55,56. HArabidopsis [ZbNAC007, ZbNAC047, and ZbNAC072, and ZbNAC079 may participate in the regulation of MAPK and ABA signaling transduction pathways, and ZbNAC018 may be involved in PCD in Z. bungeanum under drought. With regard to subcellular localization, these five ZbNAC proteins were all located in the nucleus, further emphasizing their importance in regulating the expression of target genes under drought stress. Taken together, ZbNAC007, ZbNAC018, ZbNAC047, ZbNAC072, and ZbNAC079 are potential candidate genes for improving the drought resistance of Z. bungeanum, but further verification research needs to be performed in the future.Plant drought response mechanisms are complex and include ROS scavenging, stomatal closure, root elongation, production of antioxidant enzymes, and accumulation of antioxidant substances . MAPK anbidopsis ,62. The bidopsis . PCD hasbidopsis . Thus, tbidopsis ,21. ConsZ. bungeanum genome resource was published in our previous research (BioProject ID PRJNA524242) [http://pfam.sanger.ac.uk/, accessed on 12 February 2022). HMMER 3.0 was used to identify the NAC proteins in the Z. bungeanum genome with a cut-off value of 0.01 [https://web.expasy.org/decrease_redundancy, accessed on 12 February 2022). Subsequently, the putative NAC proteins were further confirmed by BLASTP and the Conserved Domain Database . Protein physicochemical parameters, including number of amino acids, molecular weight, and pI value, were predicted using ExPasy website tools . The subcellular localization of the NAC proteins was predicted using WoLF PSORT  [The  of 0.01 . Redundary 2022) .ZbNAC genes were extracted from the GFF file of the Z. bungeanum genome. The chromosomal map with gene positions was produced using TBtools (v. 1.098696) [ZbNAC genes were named according to their chromosomal positions, ZbNAC001–ZbNAC109. MCScanX software with default parameters was used to analyze gene duplication events among 109 ZbNAC genes [http://phytozome.jgi.doe.gov/pz/portal.html#, accessed on 15 February 2022) [Arabidopsis genome file was obtained from the Arabidopsis Information Resource . The genome sequences and annotation files of wheat, maize, and jujube were acquired from NCBI . The synteny analysis between Z. bungeanum and other species was performed using TBtools , and with parameters CPU for BLATP: 4, evalue: 1× 10-10, Num of BlastHits: 5.The chromosomal positions of the .098696) . The ZbNAC genes . The genZ. bungeanum and Arabidopsis were aligned using ClustalW in MEGA 7.0, and an unrooted phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA 7.0 with pairwise deletion and 1000 bootstrap replicates. The resulting phylogenetic tree was further processed with the online tool iTOL .The NAC protein sequences from http://gsds.gao-lab.org/, accessed on 18 February, 2022) was used to determine exon–intron structures by comparing CDSs and genomic sequences of the ZbNACs. Conserved motifs were identified in the ZbNAC proteins using the online program MEME  with a maximum of 10 motifs and a range of motif widths from 6 to 200. The integrative analysis of gene structure and conserved motifs was visualized using TBtools [The online Gene Structure Display Server 2.0  for cis-element analysis. The PlantCARE output file was used to illustrate the cis-element distribution in the ZbNAC promoters using TBtools.The 2000-bp sequences upstream of the Z. bungeanum cv. ‘Fengjiao’) and HJ (Z. bungeanum cv. ‘Hanjiao’) were collected from the Z. bungeanum Experimental Station of Northwest Agriculture and Forestry University in Fengxian, Shaanxi province, China . The seeds were sown in a mixture of perlite, vermiculite, and chernozem, and the resulting seedlings were cultivated at 25 ± 2 °C and 60–70% humidity in an experimental greenhouse at Northwest Agriculture and Forestry University in Yangling, Shaanxi province, China. Three months after germination, healthy seedlings of similar size were selected for drought treatment. After stopping the water supply, leaves were sampled at 0 d (D1), 6 d (D2), 9 d (D3), and 15 d (D4), frozen in liquid nitrogen, and stored in a −80 °C freezer.Mature seeds of FJ  and sequenced on a flow cell of the Illumina HiSeq 2500 platform . The transcripts raw data have been deposited in the NCBI database with a project number PRJNA784034 .Biomarker Technologies Co., Ltd.  performed the RNA-seq analysis of 24 Z. bungeanum leaf samples using the Tiangen RNA Pure kit for plants . The integrity, purity, and concentration of the total RNA were determined using a NanoDrop 2000 spectrophotometer . DNA-free RNA was used for first-strand cDNA synthesis with the PrimeScript RT reagent kit with gDNA Eraser . qRT-PCR was performed on a CFX96 Real-Time System  with using SYBR® Green Premix Pro Taq HS qPCR Kit . The reaction protocol was as follows: 1 cycle at 98 °C for 30 s; 38 cycles at 95 °C for 5 s, 56 °C for 30 s, and 72 °C for 30 s. Primer Premier 6.0  was used to design the specific primers for qRT-PCR  using the NAC proteins from Arabidopsis that were homologous to the NAC proteins in Z. bungeanum.Co-expression relationships were analyzed based on FPKM values from RNA-seq using Cytohubba in Cytoscape (version 3.9.1) with a threshold of 0.25. The protein–protein interaction network was predicted with STRING 11.5  were potential candidate genes for drought tolerance in Z. bungeanum. In general, the genome-wide analysis of NAC family was first performed in Z. bungeanum and the expression pattern of ZbNAC genes was carefully explored under drought in this study. The results improved our understanding of NAC family in plants. Additionally, these candidate ZbNAC genes provide pivotal clues for molecular breeding of Z. bungeanum in the future.In this research, 109 ZbNAC TFs were identified in