Pyrenophora teres f. teres, is a destructive foliar disease of barley with the potential to cause significant yield loss in major production regions throughout the world. The complexity of the host–parasite genetic interactions in this pathosystem hinders the deployment of effective resistance in barley cultivars, warranting a deeper understanding of the interactions. Here, we report on the high-resolution mapping of the dominant susceptibility locus near the centromere of chromosome 6H in the barley cultivars Rika and Kombar, which are putatively targeted by necrotrophic effectors from P. teres f. teres isolates 6A and 15A, respectively. Utilization of progeny isolates derived from a cross of P. teres f. teres isolates 6A × 15A harboring single major virulence loci  allowed for the Mendelization of single inverse gene-for-gene interactions in a high-resolution population consisting of 2976 Rika × Kombar recombinant gametes. Brachypodium distachyon synteny was exploited to develop and saturate the susceptibility region with markers, delimiting it to ∼0.24 cM and a partial physical map was constructed. This genetic and physical characterization further resolved the dominant susceptibility locus, designated Spt1 (susceptibility to P. teres f. teres). The high-resolution mapping and cosegregation of the Spt1.R and Spt1.K gene/s indicates tightly linked genes in repulsion or alleles possibly targeted by different necrotrophic effectors. Newly developed barley genomic resources greatly enhance the efficiency of positional cloning efforts in barley, as demonstrated by the Spt1 fine mapping and physical contig identification reported here.Net form net blotch, caused by the necrotrophic fungal pathogen  Pyrenophora teres is the causal agent of net blotch of barley (Hordeum vulgare) and exists in two forms which, although morphologically indistinguishable under the microscope, can be differentiated by the symptoms observed on susceptible barley genotypes   and P. nodorum (SnToxA), interact with the dominant host sensitivity protein Tsn1 , maize (Zea mays), rice (Oryza sativa), and sorghum (Sorghum bicolor), diverged from a common ancestor ∼55–70 million years ago  clones and incorporation of whole-genome shotgun (WGS) sequence, BAC end sequence (BES), sequence from selected BAC clones, and integration of genetic mapping data . FurtherSpt1.R/Spt1.K (formerly rpt.k/rpt.r), which are hypothesized to confer dominant susceptibility to P. teres f. teres isolates 6A and 15A, appear to operate in a complex fashion. We hypothesize that alleles of Spt1, Spt1.R, and Spt1.K, or several tightly linked genes, confer major effect dominant susceptibility to P. teres f. teres isolates 6A and 15A, respectively. Here, we describe the high-resolution mapping of a dominant susceptibility locus located in the centromeric region of barley chromosome 6H. Mapping was done utilizing markers developed via the exploitation of the syntenic relationship with B. distachyon chromosome 3. Additionally, the identification of barley physical contigs, enabled by the newly developed barley genomic resources, were used to construct a partial physical map of the region. The results of this research will soon facilitate the identification of the dominant susceptibility gene Spt1 and allow for the subsequent functional analysis of its alleles that are hypothesized to function by NETS. This research will fill gaps in knowledge concerning these complex host–parasite genetic interactions and the underlying molecular mechanisms determining compatible interactions in this complex barley-NFNB pathosystem.The dominant susceptibility genes P. teres f. teres isolates 6A and 15A, and progeny identified as containing single virulence QTL for VK1, VK2, and VR2  progeny identified from a population consisting of 118 individuals derived from a Rika and Kombar cross  cereal genotyping center, Fargo, ND, using the high-throughput procedure described by Two previously identified flanking simple sequence repeat (SSR) markers, Bmag0173 on the distal side and Rbah21g15 on the proximal side  with a disposable tissue grinder followed by the addition of 200 µl of chloroform. Samples were vortexed for 10 sec and centrifuged at 15,600 × g for 10 min. The supernatant (∼300 µl) was transferred to a new 1.5 ml tube and 300 µl of isopropanol was added. The samples were mixed by inversion and incubated at room temperature for 5 min followed by centrifugation at 15,600 × g for 10 min. The pellets were rinsed with 70% ethanol, dried, and resuspended in 100 µl of ddH2O. The 15 F2:3 individuals from each F2 family identified with a critical recombination in the Spt1 region were genotyped utilizing the codominant marker rpt-M4 to identify homozygous recombinant individuals, which represent immortal critical recombinants (ICR). The ICR individuals were given a progeny designation and were utilized for all further phenotyping and genotyping.After the FB. distachyon chromosome 3, genetic markers were developed to saturate the Spt1 region through the use of the annotated B. distachyon genome sequence  were identified, utilizing Blastn searches limiting to ESTs other_Hordeum vulgare that correspond to B. distachyon genes within the region. A set of these ESTs were selected at ∼60 kb intervals using predicted function as the criteria for selection, as certain genes have a higher probability of containing polymorphisms compared to others. For example, NBS-LRR genes typically have more diversity than highly conserved house-keeping genes. Oligonucleotide primers were designed from the barley EST sequences and used to produce amplicons from Rika and Kombar genomic DNA by PCR (Utilizing the syntenic relationship between barley chromosome 6H and sequence . OrtholoA by PCR . GenomicA by PCR . If a rohttps://triticeaetoolbox.org/barley/) and 11 primer pairs designed from the predicted coding, intron, or 3′ UTR sequence from barley orthologs of B. distachyon genes within the region with the aim of SNP discovery. Additionally, four previously designed STS markers were included in the panel for regenotyping and internal control. A 22 nucleotide CS1 adaptor (5′-ACACTGACGACATGGTTCTACA-3′) (Fluidigm) was added to the 5′ end of forward primers and a 22 nucleotide CS2 adaptor (5′-TACGGTAGCAGAGACTTGGTCT-3′) (Fluidigm) was added to the 5′ end of the reverse primers. Barcoded adaptor primers were designed containing the Ion Torrent A adaptor sequence, unique 12 nucleotide barcode, and the CS1 adaptor sequence (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG(NNNNNNNNCGAT)ACACTGACGACATGGTTCTACA-3′). A universal reverse primer was designed containing the Ion Torrent P1 adaptor sequence and the CS2 adaptor sequence (5′- CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATTACGGTAGCAGAGACTTGGTCT-3′). Primers were multiplexed by adding 5 µl of each forward and reverse primer (100 µM) into a sterile 1.5 ml Eppendorf tube and 820 µl of nuclease-free H2O for a final primer pool volume of 1 ml. PCR reactions consisted of 1.5 µl genomic DNA, 1 µl of primer pool (100 nM each primer), and 2.5 µl Platinum Multiplex PCR Master Mix (Life Technologies). DNA concentrations were normalized to ∼20 ng/µl and 1.5 µl of each sample was pipetted into individual wells on a 96-well plate. Replicates of Rika, Kombar, RK107, RK121, and RK122 were also included on the plate. The primary PCR reaction was conducted as follows: Initial denaturation at 94° for 10 min, 10 cycles of 94° for 20 sec and 64° decreasing each cycle by 0.8° for 60 sec, followed by 20 cycles of 94° for 20 sec, 57° for 60 sec, and 68° for 30 sec, ending with a final extension of 72° for 3 min. The PCR plate was then briefly centrifuged and 15 µl of nuclease-free H2O was added to each sample. The plate was then sealed, vortexed, briefly centrifuged, and 2 µl of each sample was aliquoted into a new 96-well plate. Additionally, 2 µL of each reaction was separated on a 1% agarose gel containing GelRed to ensure successful reactions. The barcoding PCR reaction consisted of 0.235 µl of H2O, 0.625 µl NEB Standard Taq Buffer (1.25 ×), 0.1 µl dNTPs (500 µM), 1 µl universal reverse primer (1 µM), 1 µl unique barcode adaptor primer (0.4 µM), 0.04 µl of NEB Taq polymerase (0.2 units), and 2 µl template (diluted primary PCR reaction). PCR parameters are the same as the primary reaction. Following PCR, the plate was briefly centrifuged and each sample was diluted by adding 15 µl of H2O. Samples were pooled by aliquoting 5 µl of each sample into a 1.5 ml Eppendorf tube and purified using the E.Z.N.A. Cycle Pure Kit. The pooled library was then amplified to prepare for sequencing. PCR reactions consisted of a 2 µl sequencing library, 13 µl H2O, 2.5 µl NEB Standard Taq Buffer (1 ×), 3 µl ABC1 primer (0.6 µM), 3 µl P1 primer (0.6 µM), 0.5 µl dNTPs (200 µM), and 1 µl NEB Taq polymerase (5 units). PCR parameters were as follows: initial denaturation at 95° for 5 min, followed by 8 cycles of 94° for 30 sec, 62° for 30 sec, and 72° for 30 sec, ending with a final extension at 72° for 7 min. The amplified library was quantified using the Qubit dsDNA High Sensitivity Kit and the Qubit Fluorometer (Life Technologies) and diluted to ∼3 pg/µl. Sequencing was conducted on the Ion Torrent PGM utilizing the Ion PGM Template OT2 200 Kit, Ion PGM Sequencing 200 Kit v2, and an Ion 314 Chip (Life Technologies).A PCR genotyping-by-sequencing (PCR-GBS) panel was also constructed consisting of two previously designed SNP markers from the T3 database . Fungal cultures were incubated at room temperature in the dark for 5 d, placed under light at room temperature for 24 hr, and then returned to the dark at 15° for 24 hr. Conidia were harvested by flooding the plates with H2O and scraping the culture with a sterile inoculation loop. Inoculum was adjusted to a concentration of ∼2000 spores/ml and one drop of Tween 20 was added per 50 ml of inoculum. Three seeds of each ICR line were planted in a single conetainer  and three conetainers were planted for each ICR, totaling nine seedlings per line. Seedlings were grown under greenhouse conditions until the second leaf was fully expanded, approximately 14 d. Inoculum was applied as described by The ICRs were phenotyped with A total of 14 ICRs harboring a recombination between flanking markers Bmag0173 and Rbah21g15 were identified and utilized in the construction of a high-resolution map. Genetic distances were calculated based on recombination frequency between markers and denoted as map units (M.U.).9k Illumina Infinium iSELECT assay was utilized to genotype the parents Rika and Kombar and eight of the ICR lines  with recombination in the Spt1 region. The genotyping was performed as described by The barley http://webblast.ipk-gatersleben.de/barley/viroblast.php). When possible, positions of the WGS contigs on larger fingerprinted contigs (FPCs) were identified. Those markers that did not yield anchored WGS contigs were used to screen the Morex 6H MTP BAC library via PCR (INRA-CNRGV) to identify its position on a FPC. Additionally, a Morex BAC library  requiring 95% sequence homology and minimum contig size of 1 kb for assembly.DNA from the selected BACs 783N21 and 650G13  individual was identified for each F2:3 family.A total of 1488 FPyrenophora teres f. teres isolates 15A, 6A, 15 × 6A #20, 15A × 6A #63, and 15A × 6A #72. Lines that exhibited an average disease reaction of five or less were classified as resistant and lines that displayed average reactions greater than five were considered susceptible. Following inoculations with isolate 15A, Rika and Kombar exhibited average reactions of 1.67 and 7.5, respectively. A total of five ICRs were classified as resistant with individual averages ranging from 1–3.33 and an overall average of 1.77. The remaining nine ICRs exhibited individual average disease reactions ranging from 6.83–7.83 with an overall average of 7.59 and were classified as susceptible  were not polymorphic between Rika and Kombar. However, we were able to identify polymorphism between Rika and Kombar within the amplicon of a single selected ortholog of a  rpt-M14 . AdditioScreening of a barley cultivar Morex BAC library with markers rpt-M8, rpt-M13, and rpt-M12 identified four BAC clones and were confirmed via PCR . Marker P. teres f. teres 6A × 15A pathogen population .Previous studies on host resistance to NFNB in barley have been conducted utilizing diverse arrays of barley lines, as well as fungal isolates from geographically distinct regions of the world. The results of these investigations were the identification of resistance/susceptibility loci distributed throughout the barley genome  at this locus , a domin2 population utilizing markers derived from orthologous B. distachyon genes within the previously delimited rpt.k/rpt.r region, we have delimited a putative dominant susceptibility locus, designated Spt1, to an ∼0.24 cM region near the centromere of barley chromosome 6H. This high-resolution mapping greatly refined the previously delimited ∼3.3 cM region and facilitated the construction of a physical map using the new barley genome sequence. The use of the various newly available barley genomic resources, which are quickly being refined and updated, expedited the genetic to physical map construction and candidate Spt1 gene identification. Although the physical map of the Spt1 region is not completely contiguous, these tools have allowed for the positioning of physical sequences in correlation with our high-resolution map, as well as identification of flanking BAC clones to be used for further marker development and map saturation. Due to the identification of barley contigs containing the syntenic B. distachyon genes, we expect that we have captured the majority of the Spt1 physical region and show that it has been delimited to ∼9.5 Mb of the barley genome which is predicted to contain 49 genes.Through high-resolution mapping of a Rika × Kombar FSpt1 locus within the characterized region confers dominant susceptibility to NFNB isolates 6A and 15A through the recognition of necrotrophic effectors produced by the pathogen and the subsequent induction of PCD pathways in the host. It has recently been shown that P. teres f. teres produces necrotrophic effectors that induce the aforementioned host responses , have been mapped in a P. teres f. teres mapping population derived from a cross of isolates 6A and 15A, identifying two major virulence QTL per isolate in different regions of the P. teres f. teres genome. Progeny isolates containing single virulence loci were identified for VK1, VK2 and VR2, essentially Mendelizing the major virulence QTL from 15A and one from 6A by isolating the specific necrotrophic effector–host interactions. These single QTL progeny isolates were inoculated onto the 6H critical recombinants to map the sensitivity genes, which we expect will encode immunity receptor-like proteins that govern susceptibility mediated by these unique effectors. Susceptibility to the VR2, VK1, and VK2 single major virulence progeny isolates mapped to the same region on 6H and, interestingly, all mapped to the Spt1 locus. This indicates that virulence conferred by isolate 15A may be governed by the recognition of two unique necrotrophic effectors by two tightly linked genes. Alternatively, a single gene at the Spt1 locus may perceive both necrotrophic effectors produced by isolate 15A. The putative necrotrophic effector produced by a progeny isolate harboring VR2, conferring virulence to isolate 6A, may also interact directly or indirectly with a dominant susceptibility factor at the Spt1 locus, which may or may not be alleles of a gene conferring susceptibility to isolate 15A. Alternatively, several effector targets within the ∼0.24 cM Spt1 genetic interval may exist in a “susceptibility island,” each recognizing a unique necrotrophic effector and eliciting PCD. We have delimited the genetic region to ∼9.5 Mb of barley physical sequence which, based on POPSEQ anchoring of barley unigenes and BLAST searches of orthologous B. distachyon genes within the region against the barley genome sequence, contains a predicted 39 high-confidence genes. Based on annotation of the functional domains, six of the genes encode predicted immunity receptor-like proteins. Thus, these genes are candidate Spt1 genes and are being further analyzed via allele analysis and high-resolution association mapping.We hypothesize that the gene/s underlying the Spt1, as well as the effectors that interact directly or indirectly with them, will allow for greater knowledge and understanding of the molecular mechanisms underscoring this pathosystem, as well as how necrotrophic specialist fungal pathogens have evolved to exploit the intricate immunity system of the host to induce beneficial PCD responses. The isolation of individual resistance/susceptibility genes implicated in this pathosystem will fill gaps in the understanding of the molecular basis of necrotrophic specialist–host interactions and allow researchers to effectively utilize identified sources of resistance. This work greatly advances the effort toward the positional cloning and functional analysis of the host gene/s involved in this complex pathosystem.The barley-NFNB pathosystem proves to be genetically complex, consisting of dominant, recessive, and incomplete resistances, which are clustered at the 6H locus. This 6H complexity complicates efforts to incorporate effective resistance into elite barley lines. The cloning of


P = 0.024). Five out of six patients with the loss of 8p11.23 died within 8 months after initial diagnosis with a median survival of 6 months. Several candidate genes were identified in the regions with frequent chromosomal aberrations, including ADAM3A (8p11.23) and GSTT1 (22q11.23). In summary, our studies detected recurrent genetic alterations in NK/T-cell lymphoma, some of which are associated with adverse prognosis. Some candidate genes in these regions may be involved in the pathogenesis and disease progression.Extranodal NK/T-cell lymphoma, nasal type, is an aggressive mature NK-cell/T-cell lymphoma. Using array-based comparative genomic hybridization (array CGH) assays, we screened genomic alterations and potential candidate genes implicated in pathogenesis, progression, and prognosis. Our array CGH analysis detected an average of 83 chromosomal aberrations in 13 cases, ranging from 0 to 387. There were 177 recurrent chromosomal gains and 35 recurrent losses. Eleven gains and 14 losses were detected in more than 30 % of the cases, including gains of 3q26.1, 7q34, and 8q24.3 and losses of 15q24.2, 19q13.32, 5p13.2, and 14q21.1. The most common losses were observed in the 15q24.2 and 19q13.32 regions . Loss of 8p11.23 was associated with significant poor survival ( Extranodal NK/T-cell lymphoma, nasal type (NK/TCL), is a highly aggressive disease. It occurs predominantly in adult male patients and most frequently involves the upper aerodigestive tract . This diPRDM1, ATG5, AIM1, and HACE1 [A few studies have described the cytogenetic alterations based on limited numbers of fresh or frozen tissues, but no specific changes have been achieved –7. Recennd HACE1 , 9. HoweTCRG) rearrangement  were performed in all cases. Reference samples were obtained from FFPE mucosal tissues with chronic inflammation.Thirteen cases of NK/TCL were included in our study. This study was approved by Peking University Bioethics Committee and has therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The diagnosis for each case was confirmed by a panel of expert hematopathologists according to 2008 WHO classification criteria, and these diagnoses were agreed upon by all expert reviewers in all 13 cases. The major antibodies used in this study included CD56, CD3ε, CD2, cytotoxic molecules , CD20 , and TCRβF1 . In situ hybridization for Epstein-Barr virus encoded RNAs (EBER)  and PCR for T-cell receptor gamma gene . Areas with necrosis and without tumor cells were dissected out from the specimen to make sure that the tumor cells accounted for >95 % of the remaining tissue. Genomic DNA concentration and purity were monitored using NanoDrop UV–VIS spectrophotometer , and the intactness of DNA was evaluated by electrophoresis on 2 % agarose gels. The extracted genomic DNA from all 13 cases met the following criteria: A260/A280 ratio within 1.8–2.0, A260/A230 ratio > 1.0, average DNA size > 500 bp, and fragmented DNA > 300 bp according to the published data .Array CGH assays were performed using Agilent SurePrint G3 Human CGH Microarray Kit 2 × 400 K . Briefly, 2-μg genomic DNA and 2-μg reference DNA were labeled with ULS-Cy5 and ULS-Cy3 by ULS™ arrayCGH labeling kit , respectively. After purification, the same amount of labeled sample DNA and reference DNA was combined and then mixed with a provided hybridization solution and human Cot-1 DNA , followed by hybridization at 65 °C for 40 hrs, according to the manufacturer’s instructions. After two washings, the arrays were scanned on an Agilent Microarray Scanner. Feature Extraction Software  was used for data extraction from raw microarray image files. Agilent CGH Analytics Software (v3.4) was used to visualize, detect, and analyze chromosomal patterns with microarray profiles. Chromosome X and Y were excluded because of sex mismatching.Loss of genetic regions was confirmed using Sanger sequencing. Three alteration primers surrounding the identified variants were designed using Primer3 software Table . Primersp value was <0.05.Patient survival was analyzed using a SPSS program (v17.0). The probability of overall survival (OS) was calculated using the Kaplan–Meier method with log-rank test for comparison. Significant difference was considered when the The clinicopathologic features of all 13 patients were summarized in Table All patients received CHOP or CHOP-like chemotherapy or/and radiotherapy. Four patients achieved complete regression (CR) with median survival of 8 months (range 2–66 months)   and 4 patients showed no response (NR) to therapy. In the other 7 cases without 8p11.23 loss, there were 4 CR, 1 PR, and 2 NR.ADAM3A, SIRPB1, SIGLEC14, GSTT1, TAP1, TAP2, and TAPBP, may be related to the pathogenesis and progression of NK/TCL , five regions  did not contain any known genes and protein-coding sequences  lumen and then loading onto newly synthesized MHC class I molecules [TAP by blocking the binding of peptides with ATP to the transporter complex [P = 0.024), and 5 out of 6 patients died within 8 months after initial diagnosis. The chromosomal 8p11.23 region contains ADAM3A gene, a member of disintegrin and metalloproteinase (ADAM) family, which is widely expressed and has many potential functions related to cell–cell and cell–matrix interactions [ADAM3A gene primers. A single-nucleotide mutation at the 108225 base pair (bp) was detected in 5 of the 6 cases with 8p11.23 loss, and one of these 5 cases (Case 12) also showed another single-nucleotide mutation at the 102240 bp  family, which consists of several phase II detoxification enzymes and plays important roles in detoxification of a wide range of substrates, including mutagens, carcinogens, and chemotherapeutic agents, such as alkylating agents, platinum compounds, and anthracyclines. Besides its important roles in protecting cells from environment, genotoxic and oxidative stress, the detoxification capability is also associated with drug resistance [GSTT1 was reported to be associated with an increased risk of Hodgkin lymphoma and some types of non-Hodgkin lymphoma. However, it is intriguing that loss of GSTT1 is associated with a better prognosis in patients treated with substrates for GSTT1 [GSTT1 decreases the detoxification capability so as to decrease the pharmacokinetic and pharmacodynamic profile of chemotherapeutic agents, leading to a better response to treatment. In our study, the aberrations of GSTT1 gene in NK/TCL were various, which presented as loss and gain. We attempted to detect the GSTT1 gene in cases with 22q11.23 loss. There was no amplification of product in any of the 4 cases with 22q11.23 loss (Table GSTT1 sequence was found in the other cases. This GSTT1’s contribution to NK/TCL remains unclear and needs more studies.Aberrations of 22q11.23 were detected in 7 cases, including loss in 4 cases and gain in 3 cases. This region contains sistance . The losor GSTT1 . A possiIn summary, the present array CGH results suggested the involvement of a complex and multiple genetic changes in the development of NK/TCL. A further study to identify the potential candidate genes was required to better understand the pathogenesis of NK/TCL.


SLC2A1, SLC2A3, and HIF-1α genes was noted in 83.9, 82.1, and 71.7 % of SCC specimens and in 34.4, 59.4, and 62.5 % of laryngeal cancer samples. Higher levels of mRNA/protein for GLUT1 and HIF-1α were noted in SCC compared to NCM (p < 0.05). SLC2A1 was found to have a positive relationship with grade, tumor front grading (TFG) score, and depth and mode of invasion (p < 0.05). SLC2A3 was related to grade and invasion type (p < 0.05). There were also relationships of HIF-1α with pTNM, TFG scale, invasion depth and mode, tumor recurrences, and overall survival (p < 0.05). In addition, more advanced tumors were found to be more likely to demonstrate positive expression of these proteins. In conclusion, the hypoxia/metabolic markers studied could be used as molecular markers of tumor invasiveness in laryngeal cancer.Increased glucose uptake mediated by glucose transporters and reliance on glycolysis are common features of malignant cells. Hypoxia-inducible factor-1α supports the adaptation of hypoxic cells by inducing genes related to glucose metabolism. The contribution of glucose transporter (GLUT) and hypoxia-inducible factor-1α (HIF-1α) activity to tumor behavior and their prognostic value in head and neck cancers remains unclear. The aim of this study was to examine the predictive value of GLUT1, GLUT3, and HIF-1α messenger RNA (mRNA)/protein expression as markers of tumor aggressiveness and prognosis in laryngeal cancer. The level of hypoxia/metabolic marker genes was determined in 106 squamous cell laryngeal cancer (SCC) and 73 noncancerous matched mucosa (NCM) controls using quantitative real-time PCR. The related protein levels were analyzed by Western blot. Positive expression of  It is well known that the rapid proliferation of malignant cells causes the formation of hypoxic areas within the stroma of solid human tumors . Hypoxianditions –5. The rnditions .SLC2A) have been identified [Until now, 14 members of the GLUT family coded by genes belonging to the solute carrier 2A family  and isoform 3 (GLUT3), as well as the regulatory HIF-1α gene and the levels of their corresponding proteins, to investigate their influence on tumor aggressiveness and patient prognosis in squamous cell laryngeal cancer.The aim of this study was to determine the messenger RNA (mRNA) expression of The study material constituted 106 fresh biopsy tissue samples obtained from squamous cell laryngeal carcinoma (SCC) cases, and the control constituted 73 tissue samples from a morphologically estimated noncancerous laryngeal mucosa (NCM) from individuals who were qualified for total laryngectomy. According to ethical rules, the adjacent normal epithelium of the larynx in patients qualified for partial laryngectomy was not sampled. The patients  were recruited between January 2003 and December 2011 and were under treatment at the Department of Otolaryngology and Laryngological Oncology, Medical University of Łódź, Poland. All individuals had a confirmed diagnosis of laryngeal carcinoma based on histopathological evaluation and had undergone partial or total laryngectomy, depending on the extent of neoplastic lesions. All fresh samples were stored at −80 °C before the analyses. The tissue specimens collected in the operation room were prepared and evaluated by an experienced pathologist. Normal laryngeal tissues were collected from the sites as far as possible from the margins of the tumor by individually harvesting samples from presumptive noncancerous regions. Hematoxylin and eosin (H&E)-stained sections provided histological confirmation of noncancerous and cancerous tissues. The criteria for patient participation in this study were as follows: (1) a pathologically confirmed diagnosis of squamous cell planoepitheliale carcinoma; (2) primary surgical resection without receiving prior immunotheraphy, radiotheraphy, or chemotherapy; (3) absence of distant metastases and second primary neoplasms; (4) a negative history of previously diagnosed with other types of primary cancers; and (5) a negative history of recurrences of laryngeal cancer. Informed consent was obtained from each subject. The investigations were performed with the approval of the Bioethical Commission of the Medical University of Łódź and the National Science Council, Poland . In all cases, surveys were performed to complete the cancer registry database. The database catalog was queried every 2 months and identified all histopathologically confirmed incident primary squamous cell laryngeal cancer cases reported within 4 months of diagnosis preceding the recruitment. The sociodemographic features of the study subjects are shown in Table Archival formalin-fixed paraffin-embedded tissue samples were used for the histological classification of tumors. All specimens were assessed according to the criteria conducted in accordance with the AJCC TNM classification of 2010 for laryngeal cancers . MorpholThe tissue specimens collected in the operation room were prepared and evaluated by an experienced pathologist. Samples were stored at −80 °C until RNA preparation. Total RNA was isolated using Trizol® Reagent  according to manufacturer’s protocol and quantified spectrophotometrically. RNA was eluted in 20 μl RNase-free water, quantified by spectrophotometry at 260 nm and stored at −20 °C. RNA with a 260/280 nm ratio in the range 1.8–2.0 was considered high quality. First-strand complementary DNAs (cDNAs) were obtained by reverse transcription of 1 μg of total RNA using RevertAid™ first-strand cDNA synthesis kit  following the manufacturer’s protocol.SLC2A1, SLC2A3, and HIF-1α) was performed using TaqMan® Gene Expression Assays  according to manufacturer’s instructions. The hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene was used as internal control. The assay numbers for these genes were as follows: Hs00892681_m1, Hs00359840_m1, Hs00936368_m1, and Hs02800695_m1. Each PCR reaction was performed in a 10-μl volume that included 5 μl of 2× TaqMan Universal PCR MasterMix , 4.5 μl of water diluted cDNA template (50 ng), and 0.5 μl of TaqMan® Gene Expression Assay consisted of a pair of unlabeled PCR primers and TaqMan probe with a FAM™. The RT-qPCR reaction was carried out using the Mastercycler ep realplex (Eppendorf) under the following conditions: denaturation for 10 min at 95 °C followed by 50 cycles of 15 s at 95 °C, 1 min annealing and extension at 60 °C. Relative RNA quantification was performed using the ΔCt method. ΔCt (Ctgene − CtHPRT1) values were recalculated into relative copy number values .Real-time gene expression analysis of target genes  with the glucokinase (GCK) gene used as the reference gene. The real-time PCR primers are as follows: SLC2A1 (f) 5′-TGTGCAACCCATGAGCTAA-3′, SLC2A1 (r) 5′-CCTGGTCTCATCTGGATTCT-3′; SLC2A3 (f) 5′-TTCGTCTCTAGCCTGCACTG-3′, SLC2A3 (r) 5′-ACACAACTTCTCCGGGTGAC-3′; GCK(f) 5′-CGGATGCAGAAGGAGATGGA-3′, and GCK(r) 5′-CATCTTCACACTGGCCTCTTCA-3′. Real-time PCR was performed in 50-μl reaction volumes that contained 2× Power SYBR Green PCR Master Mix  and 0.9 mM forward and reverse primers. PCR conditions were as follows: 5 s at 95 °C followed by 40 cycles consisting of 15 s at 95 °C and 30 s at 60 °C. ΔCt was calculated by a Ct value of GCK taking away that of SLC2A1 and SLC2A3 and three or more ΔCt was defined as amplified.To determine the The protein content of the tissue homogenate fraction was estimated by means of the modified Lowry procedure  using boAll RT-PCR and Western blot reactions were repeated three times for each sample.U test, Spearman’s rank analysis). Kruskal-Wallis test with post hoc multiple comparisons was used according to clinical data. Kaplan-Meier survival analysis was performed to determine the association of SLC2A1, SLC2A3, and HIF-1α mRNA expression with overall survival. The cutoff value was established to be the median of SLC2A1, SLC2A3, and HIF-1α mRNA level. The survival curves were compared between two groups: high  and low  expression using log-rank tests. Distribution of quantitative variables was described using means and standard deviations. A p value <0.05 was considered as statistically significant.The statistical analyses were performed using STATISTICA version 9.0 . Since levels of expression in endometrial and breast cancer specimens did not follow a normal distribution (Kolmogorov-Smirnov test), nonparametrical statistical tests were applied  and 82.1 % (87/106) samples of laryngeal cancer, respectively. In the case of normal tissue, positive expression of GLUT1 and GLUT3 mRNA was demonstrated in 49.3 % (36/73) and 43.8 % (32/73) of samples, respectively. Positive expression of HIF-1α gene was noted in 71.7 % (76/106) of SCC and in 9.6 % (7/73) of NCM, respectively. Thus, positive expression of both GLUT transcripts and HIF-1α was more frequent in SCC than in NCM.The mRNA expression of genes p < 0.001 and p < 0.001, for the SLC2A1 and SLC2A3 genes, respectively). The relative expression of SLC2A3 transcripts was much lower than SLC2A1 in both SCC and NCM. Similarly, mean mRNA expression of the HIF-1α gene in neoplastic tissue was also higher than that in normal laryngeal tissue, but the difference was not significant (p > 0.05). Mean GLUT1, GLUT3, and HIF-1α gene expression in SCC and NCM, as well as the results of statistical analysis, are summarized in Table p > 0.05). However, a significant correlation was found between expression of the HIF-1α gene with SLC2A1 transcripts , but not SLC2A3 transcripts , as determined by real-time PCR. The expression of HIF-1α mRNA in relation to SLC2A1 and SLC2A3 genes in laryngeal cancer is shown in Fig. A significant difference was noted in the levels of GLUT1 and GLUT3 mRNA in laryngeal cancer tissue compared to adjacent normal laryngeal tissue (SLC2A1 gene amplification was observed in 2 % (2/106) of laryngeal cancer cases, but in none of the normal tissue samples. In the case of SLC2A3 gene, amplification was confirmed neither in cancerous nor normal tissues.Gene copy number analysis revealed that SLC2A1, SLC2A3, and HIF-1α transcripts with regard to relevant clinicopathological parameters in laryngeal cancers is shown in Table p = 0.0005 and p = 0.003, respectively). Significant differences were noted between grade 1 and grade 3 tumors as well as between grade 1 and grade 2 positive cancers with regard to SLC2A1 expression , as well as between grade 3 and both grade 1 and 2 positive cancers with regard to SLC2A3 expression . No differences in SLC2A1 and SLC2A3 gene level were noted with respect to other pTNM parameters (p > 0.05). However, the results showed that more advanced tumors  were more likely to be more frequently positive and to have a higher mean levels for both SLC2A1 and SLC2A3 transcripts than less advanced cancers .The expression of SLC2A1 mRNA expression was significantly higher in laryngeal TFG cancers with a total score ≥14 points than less aggressive ones (p = 0.021). Moreover, SCC characterized by deep invasion with submucosa or cartilage infiltration (3–4 points) and by diffuse growth or invasion with no distinct borderlines (3–4 points) demonstrated significantly higher levels SLC2A1 mRNA in comparison with less invasive tumors . Mean SLC2A3 gene expression was significantly higher in more disseminated invasion type cancers (p = 0.0001). Furthermore, SLC2A1 transcript level was also significantly related to disease-free survival when compared <5- and ≥5-year categories. Increased GLUT1 expression was found to be associated with a survival time of less than 5 years (p = 0.012). SLC2A1 expression was found to have no connection with the incidence of local and nodal recurrences (p > 0.05). No significant differences were found between SLC2A3 mRNA level and either recurrence or 5-year disease-free survival (p > 0.05). The association between clinicopathological parameters and the expression of SLC2A1 and SLC2A3 transcripts, as well as with the results of the statistical analysis, are shown in Fig. HIF-1α mRNA level significantly increased with the size of the primary tumor (pT3-4) (p < 0.001), the presence of nodal metastases (pN1-3) (p < 0.001), and tumor grade (p = 0.004). Moreover, laryngeal cancers with TFG total score ≥14 points, deep invasion (3–4 points), and disseminated growth (3–4 points) were noted to have significantly higher HIF-1α gene level compared to less invasive tumors . In addition, increased expression of the gene coding HIF-1α was found to promote a survival time of less than 5 years (p < 0.001) and the presence of local and nodal recurrences . The relationship between various clinicopathological parameters and HIF-1α gene expression, as well as with the results of the statistical analysis, is shown in Fig. The  tumor pT-4 . However, the results revealed a trend of worse overall survival rate in cases with a high expression of the GLUT1 and GLUT3. Mean overall survival time was 76.8 months in low HIF-1α mRNA expression and 52.5 months in high HIF-1α expression. A significant shorter overall survival for a higher HIF-1α gene expression was estimated (p < 0.001).Moreover, overall survival was analyzed through the Kaplan-Meier plots, as shown in Fig. p = 0.032). Mean GLUT1, GLUT3, and HIF-1α protein expression in SCC and NCM and statistical analysis results are shown in Table In the next stage, expression of GLUT1, GLUT3, and HIF-1α was examined on the protein level using Western blotting analysis, in homogenate samples of both SCC and NCM specimens. The 32 tissue samples selected for quantitative densitometric analysis were representative for whole group studied and characterized by similar distribution, based on the pTNM classification. Of the carcinoma tissue samples, 34.4 % (11/32) and 59.4 % (19/32) showed GLUT1- and GLUT3-positive expression, respectively. In the case of matched normal laryngeal tissue, GLUT1 protein expression was observed in 31.3 % of samples and GLUT3 in none. The results of the quantitative densitometric analysis of the intensity of GLUT1 and GLUT3 bands in homogenates of normal and cancerous tissues are shown in Fig. GLUT and HIF-1α expression was also compared with the pTNM classification of laryngeal cancer cases, since the tumor stage is currently the only accepted prognostic marker. Despite the relatively small size of positive results for the presence of proteins studied and the small proportion of subcategories of pTNM classification, the results show a clear relationship between quantitative densitometric results and stage parameters.The results reveal a trend of more advanced tumors to have more common positive expression of GLUT1, GLUT3, and HIF-1α proteins, according to Western blot analysis. The positive expression of GLUT1 and GLUT3 proteins in SCC samples according to pTNM categories was as follows: 33.3 % 5/15) in pT1-T2 and 35.3 % (6/17) in pT3-T4 tumors, 18.8 % (4/22) in pN0 and 70 % (7/10) in pN1-3 for GLUT1, and 46.7 % (7/15) in pT1-T2 and 70.6 % (12/17) in pT3-T4 tumors, 50 % (11/22) in pN0 and 80 % (8/10) in pN1-3 for GLUT3. It should be also emphasized that higher graded tumors were found to demonstrate higher expression of the GLUT1 protein, but not GLUT3. The results concerning GLUT1 and GLUT3 expression in homogenate samples of laryngeal cancer tissues in relation to tumor grade are shown in Fig. /15 in pTSimilar trends were demonstrated for the HIF-1α protein. The levels of positive HIF-1α expression according to pTNM categories and histological grade was as follows: 26.7 % (4/15) in pT1-T2 and 94.1 % (16/17) in pT3-T4 tumors, 54.5 % (12/22) in pN0 and 80 % (8/10) in pN1-3 and 10 % (1/10) for well-differentiated tumors, 75 % (9/12) for moderately-differentiated cancers, and 100 % (10/10) for poorly-differentiated tumors.In the last decade, a growing body of evidence indicates that increased level of glycolytic activity observed under aerobic conditions (the Warburg effect) needed to meet the energy requirements of tumor cells and the endogenous markers of metabolism/hypoxia may contribute to the development, proliferation, angiogenesis, and progression of various types of head and neck cancers –5. StudiOur study documents for the first time the relationship between the expression of GLUT1 and GLUT3 isoforms in the fresh human biopsy material and the dynamics of tumor growth in laryngeal cancer, according to a precise, multifactorial histological analysis of tumor front aggressiveness. Importantly, the material studied constitutes a homogeneous and numerous groups of head and neck cancers sharing the same origin. This composition of the study groups adds to the value of this research, when the difficulties in obtaining fresh human biopsy tissues such as cancerous and matched normal tissues are taken into consideration. It should be also noted that, due to these difficulties, the overwhelming majority of previous studies have used cell cultures and paraffin-embedded archival material, often also limited in number, which doubtlessly have an impact on the final results. In this study, the protein expression in less material due to this limitation complements research on the genes studied.SLC2A1 and SLC2A3 genes in fresh tissues from the head and neck region have yet been performed. Since copies of the studied genes were found in only 2 % cases of laryngeal carcinoma, it can be concluded that SLC2A1 and SLC2A3 amplification does not affect the expression of GLUT1 and GLUT3 in this type of neoplasm.A literature search indicates that no studies on the amplification of the The resulting data concerning expression of GLUT isoforms in laryngeal cancer resembles the findings of other researchers , 38–40. In addition, a literature review reveals a wide range of findings concerning expression rate and distribution of GLUTs in various head and neck cancers. The data concerning the expression of GLUT1 and GLUT3 transcripts or, most often, protein immunoexpression identified in laryngeal cancer cases were found to be very similar to ours: the mean frequency of GLUT1 was in the range 50.3–98 % while GLUT3 positive expression was less so , 24–26. Studies have confirmed the role of GLUT isoforms, mainly GLUT1, in head and neck neoplastic lesions and both cancer development, tumor progression and patient prognosis, but many demonstrate equivocal and divergent results –26. ImpoMost studies confirm the increased expression of GLUTs in more advanced head and neck cancers, where a positive association was found for clinical variables including tumor grade and stage , 26, 43.Nevertheless, contrary results for GLUT1 and GLUT3 immunoexpression related to such clinical parameters as primary tumor size, extrathyroidal extension, and lymph node metastases has also been reported by Kaida et al. . This daUnfortunately, the relationship between GLUT level and prognosis was also found to be divergent in the head and neck cancer populations. The obtained data confirms that GLUT isoform expression was unrelated to mortality. The prognosis results described by Müssig et al.  were almHowever, other researchers report different findings and conclude that GLUT activity may be implicated in patient survival time. For instance, Ohba et al.  suggesteLess controversy and fewer conflicting results concern the clinical and prognostic value of HIF-1α and its relationship with glucose metabolism molecules in head and neck cancers , 45. OurA few studies concerning HIF-1α expression in relation to tumor behavior and prognosis in head and neck carcinomas also confirm our findings –31. Li eUnfortunately, a few studies present inconsistent data regarding the status of HIF-1α as a biomarker of tumor invasiveness and prognosis , 33. ForFinally, it should be also stressed that there are limitations to our study. While GLUT1 and GLUT3 expression could be considered as useful potential biomarkers for tumor behavior in laryngeal cancer, discrepancies exist for other tumors of this region due to variation of tumor types, histological differentiation status, and proliferative index, which cause differences in their biology. Admittedly, the results indicate the issue role of GLUTs and interactions with HIF-1α as their key regulator in cancers, but it is not clearly understood and there is a need for future research to clarify the relevance of GLUT proteins in carcinogenicity and the behavior of various tumors. Also, because of the limited amount of biological material in our study, GLUTs and HIF-1α were compiled with regard to protein expression only, with pTNM classification, the currently accepted prognostic parameters, being used in a smaller number of laryngeal cancer cases.SLC2A1 and SLC2A3 genes, as well as the related GLUT1 and GLUT3 proteins, can affect the behavior of laryngeal cancer. Moreover, our findings indicate the importance of the expression of GLUTs as a significant factor in determining tumor aggressiveness, which should be taken into consideration in choosing alternative and optimal treatment modalities, i.e., extension of primary surgical procedure, and can contribute to prognosis in patients with cancer of the larynx. However, additional data on SLC2A1 and SLC2A3 genes and their products in head and neck carcinomas is needed to elucidate the biological function of GLUTs in carcinogenesis, tumor progression, and patient outcome.In conclusion, our findings suggest that both the


T1 mapping by cardiovascular magnetic resonance (CMR) provides tissue-dependent relaxation times in line with the underlying myocardial composition. In this study, we examined the value of native T1 in differentiation between acute viral myocarditis and chronic stages of healing.Fifty-three patients with a clinical diagnosis of acute viral myocarditis were compared to fifty-six subjects in clinical convalescence. All subjects underwent assessment of myocardial oedema, function and scar by CMR at 1.5 and 3-Tesla. T1 values were measured in midventricular short-axis slice prior to and after administration of 0.2 mmol/kg of gadobutrol.Compared to controls (n = 40), native T1 values were increased in both acute and chronic myocarditis at both field strengths : 940 ± 17 vs. 1070 ± 34 vs. 998 ± 28, p < 0.001; 3T: 1045 ± 18 vs. 1182 ± 66 vs. 1094 ± 15, p < 0.001). Native T1 values were significantly higher in acute vs. chronic myocardium (p < 0.001). Native T1 was identified as the independent discriminator between health and disease, as well as acute and chronic myocarditis  comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital National Health Service Foundation Trust. Dr. Rocio Hinojar was supported by the Spanish Society of Cardiology.


Unreduced gametes.Triticum aestivum L. × Secale cereale L., 4x = 28, ABDR). By using in situ hybridization with a centromere pAet6-09 probe and immunostaining with H3Ser10ph-, CENH3-, and α-tubulin-specific antibodies, we distinguished four chromosome behaviour types. The first one is a mitotic-like division that is characterized by mitotic centromere architecture, robust bipolar spindle, one-step loss of arm and centromere cohesion, and sister chromatid separation in the first and only meiotic division. The second type involves a monopolar spindle formation, which appears as a hat-shaped group of chromosomes moving in one direction, wherein MT bundles are co-oriented polewards. It prevents chromosome segregation in meiosis I, with a bipolar spindle distributing sister chromatids to the poles in meiosis II. These events subsequently result in the formation of unreduced microspores. The other two meiotic-like chromosome segregation patterns known as reductional and equational plus reductional represent stand-alone types of cell division rather than intermediate steps of meiosis I. Only sterile pollen is produced as a result of such meiotic-like chromosome behaviours. Slightly variable meiotic phenotypes are reproducibly observed in hybrids under different growth conditions. The 2R(2D)xR genotype tends to promote reductional division. In contrast, the genotypes 1Rv(1A)xR, 5R(5D)xR, and 6R(6A)xR promote equational chromosome segregation and monopolar spindle formation in addition to reductional and equational plus reductional division types.The absence of a strict pachytene checkpoint in plants presents an opportunity to study meiosis in polyhaploid organisms. In the present study, we demonstrate that meiosis is coordinated in hybrids between disomic wheat–rye substitution lines 1Rv(1A), 2R(2D), 5R(5D), 6R(6A) and rye (The online version of this article (doi:10.1007/s00497-016-0279-5) contains supplementary material, which is available to authorized users. Polyploidy is widespread among flowering plants, and allopolyploidy is one of the major speciation pathways in plant evolution  species within tribe Triticeae: Triticum urartu (AA), an extinct or yet undiscovered species from Aegilops speltoides (BB) lineage, and Ae. tauschii (DD) ; the second allopolyploidization event between emmer wheat and the D genome donor formed modern hexaploid bread wheat (AABBDD) (Feldman and Levy Ae. speltoides and T. urartu (AA) gave rise to the allotetraploid emmer wheat  by polyploidization. Bread wheat originated by allopolyploidization from a third hybridization (less than 0.8 MYA), between emmer wheat and Ae. tauschii (DD). Despite being polyploid, bread wheat displays diploid-like meiotic behaviour , which leads to full fertility and disomic inheritance  IGWSC . The priModern studies aiming at genome reconstruction of the bread wheat have shown that as soon as two parental genomes have joined to form an allopolyploid genome, this resulted in a “genomic shock”. Specifically, the issues of distinct genome sizes, chromosome numbers, regulation, and cell cycle progression must have been resolved  date back to 1930s and are still actively pursued , T. turgidum L. (AABB genome) and Aegilops tauschii Coss. (DD genome) 2, 2R(2D)3, 5R(5D), 5R(5A), 6R(6A) (2n = 42) has provided evidence for a genetic control of chromosome behaviour  and 6R(6A) with rye, about 40 % of meiocytes displayed equational distribution of chromosomes and omission of the second division. The hybrids 5R(5D) with rye had high proportions of cells with equationally dividing chromosomes (about 25 % of meiocytes), dyad formation, and partial fertility. In androgenic haploids of the line 6R(6A), half of the meiocytes showed an equational division of the 21 chromosomes present and a failed second division  the monopolar spindle assembles in meiosis I; (2) chromosome segregation fails; and (3) the bipolar spindle is formed followed by segregation of sister chromatids in meiosis II.T. aestivum L. cv. Saratovskaya 29 ; the rye cultivar, S. cereale L. cv. Onokhoiskaya ; and wheat–rye F1 hybrids . The parental plants of wheat–rye hybrids included four disomic single chromosome wheat–rye substitution lines (2n = 42): 1Rv(1A)  and 5R(5D), 6R(6A) , 2R(2D)  , Russia , and under greenhouse conditions during the autumn–winter seasons in 2012, 2013, and 2014 with temperature 24/18 °C day/night and under a day/night cycle of 16/8 h.This study used the wheat cultivar, For meiotic studies, young spikes at the appropriate stages were fixed in a (3:1) mixture of 96 % ethanol and acetic acid for 24 h and then stored in 70 % ethanol in a refrigerator. Pollen mother cells (PMCs) were stained with and squashed in 3 % acetocarmine. All of the anthers with PMCs at metaphase I–anaphase I and anaphase II–telophase II were analysed. Each anther was analysed individually, assaying all PMCs in each anther.For fluorescence in situ hybridization (FISH), spikes were fixed in 45 % acetic acid for 2–4 h at room temperature, anthers with meiocytes at MI–AI were selected, squashed, and slides were frozen in liquid nitrogen, dehydrated through a series of alcohols with increasing concentrations of 70, 90, and 96 %, and stored at −20 °C until needed. Each anther was examined individually, and all scorable PMCs were assayed. A total number of 689 meiocytes in 21 plants were examined. For the analysis of mitotic stages, root tips were fixed in a solution of ethanol–acetic acid  and stored at −20 °C. Slides were frozen in liquid nitrogen and then cover slips were removed. Slides were dehydrated in a graded series of 70, 90, and 96 % (v/v) ethanol and air-dried.Ae. tauschii pAet6-09 probe specific for rye, wheat, rice, and barley centromere repeats , which specifically recognized histone H3 phosphorylated at Ser 10; anti-CENH3 , which specifically recognized the centromeric histone H3 variant; and anti-α-tubulin , which detects the α-tubulin of microtubules. The secondary antibodies to anti-phH3S10 and anti-CENH3 were anti-rabbit IgG conjugated with rhodamine ; the secondary antibody to anti-α-tubulin was anti-mouse IgG conjugated with FITC .The method reported by Manzanero et al.  was usedl-lysine-coated slides. After freezing for 15 min at −70 °C and blocking for 30 min in 3 % bovine serum albumin (BSA)/PBS/non-fat milk, incubation with the primary antibodies was completed overnight at 4 °C. Then, slides were washed 4 × 15 min in PBS and incubated with the secondary antibody at room temperature for 1 h. After 4 × 15 min washes in PBS, the slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted in anti-fade Vectashield medium. A total number of 3776 meiocytes (including control wheat and rye plants) in 83 plants were examined.The material was disaggregated on poly-All slides were examined under an Axio Imager M1 (Karl Zeiss) microscope. Images were recorded with a ProgRes MF camera  and processed using the Adobe Photoshop CS2 software.Meiosis in 2R(2D)xR is invariably a two-step process unlike that in 1Rv(1A)xR, 5R(5D)xR, and 6R(6A)xR where meiosis is a mixed, one-step and two-step process.1 hybrids, univalents were observed to be randomly scattered between the poles at metaphase I (MI) xR FThe first meiosis in 1Rv(1A)xR, 5R(5D)xR, and 6R(6A)xR hybrids displayed a number of peculiar features Fig. .The ratio of the three types of meiocytes varied. Overall, greater than 1/3 meiocytes in hybrids 1Rv(1A)xR, 5R(5D)xR, and 6R(6A)xR characterized by a separation of sister chromatids in the first meiosis, but a small number meiocytes (from 0.42 to 2.19 %) had chromosomes arranged as a ring or circle .Ascribing the exact stage of division in hybrid meiocytes is a challenging task because chromosome behaviour in these cells is distinct from that observed in normal meiosis. Stages were defined based on the microtubule (MT) dynamics and distribution of anti-H3Ser10ph and anti-CENH3 signals. At prometaphase I, anti-H3Ser10ph signal covered the entire length of the chromosomes . Chromosomes undergoing this type of division displayed dense pAet6-09 hybridization spots near the poles. The remainder of the chromosomes lied at the equational plate, and their pAet6-09 hybridization sites appeared as stretched diffuse bands across the chromosomes . As division proceeded to late anaphase, each of the stretched diffuse sites turned into two independent signals, which was indicative of separated sister centromeres . In the first meiotic division, anti-H3Ser10ph signal was detected throughout the entire chromosome body . We interpreted this phenomenon as the completion of division.In these cells, the H3Ser10ph signal spanned the entire length of each chromosome Fig. b–e, or, An exclusive type of chromosome behaviour producing a monopolar spindle was uncovered. Upon nuclear envelope breakdown, the chromosomes were observed tightly clustered together, and MT nucleation at kinetochores began Fig. a, m. Latx = 28) amphihaploids. The 2R(2D) genotype tends to promote reductional division, which ultimately produces sterile gametes. In contrast, in the genotypes 1Rv(1A), 5R(5D), and 6R(6A) in addition to reductional and equational plus reductional division types, an equational chromosome segregation pattern and monopolar spindle formation are observed. The latter two types of chromosome behaviour result in the formation of unreduced gametes in the hybrids.Our observations indicated that the genotypes of disomic wheat–rye substitution lines contribute to the regulation of meiosis in wheat–rye  in hybrids with rye  and Ae. tauschii  line  with Ae.tauschii had a high frequency of equational division at the first meiosis. Fertility analysis of F1 hybrids between durum wheat (T. durum Desf.) Langdon and its 10 disomic substitution lines with Ae. tauschii accession AS60 showed increased selfed seedset rates in the hybrids of 1D(1A), 1D(1B), 3D(3A), 4D(4B), 7D(7A), and 2D(2B) with AS60, with lower rates in the hybrids of 3D(3B) + 3BL, 5D(5A) + 5AL, 5D(5B) + 5B, and 6D(6B) + 6BS with AS60 compared with the hybrids of Langdon with AS60  and with a restitution nucleus stage (FDR) , 5R(5D), 6R(6A) lines and rye. During SDM, equational segregation of univalents in the first and only meiotic division occurs. Upon FDR, monopolar spindle is formed at meiosis I, which blocks chromosome segregation, and so no cytokinesis stage follows; then, at meiosis II, sister chromatids segregate.In the present work, we provide evidence for coexistence of FDR and SDM in the meiosis of partially fertile FDuring SDM, double anti-CENH3 signals mark functional sister kinetochores attached to the MTs of opposing spindle poles. Diffuse and “stretched” hybridization signals observed for the centromeric probe pAet6-09 analogous to the pattern observed in mitotic chromosomes and chromosomes undergoing the second meiotic division xR, 5R(5D)xR, and 6R(6A)xR hybrids, one could observe meiocytes with monopolar spindles. Chromosomes were present as univalents with unsplit kinetochores and were not locked into stable positions. Instead, they oscillated towards and away from the single pole. Most of the studies of the mechanisms of monopolar spindle formation and subsequent block of mitotic cell cycle address the down-regulation or absence of kinesin 5   and a bipolar orientation of sister kinetochores attached to the bipolar spindle MTs. Chromosomes located at the equator divide and form sister chromatids that segregate to the poles where they join the chromosomes with monopolar orientation. Equatorial convergence of all chromosomes was never observed in such meiocytes, and a phragmoplast was formed upon segregation of sister chromatids.T. turgidum × Ae. tauschii hybrids corresponds to the reductional segregation in T. aestivum × S. cereale hybrids, whereas equatorial travelling of chromosomes as their “kinetochores switch from monopolar to bipolar orientation” in T. turgidum × Ae. tauschii . The other mechanism involves formation of a monopolar spindle in the first meiotic division followed by the segregation of sister chromatids during the second meiotic division (FDR).Overall, these chromosome behaviours should not be taken as strict abnormal, as the meiotic modifications observed are perfectly compatible with each other and are genetically controlled in wheat–rye hybrids. In our opinion, this adds yet another piece to the puzzle of meiotic control in partially fertile amphihaploids. OS conceived of the study, designed, performed experiments, analysed, interpreted data, and wrote the manuscript; DL performed experiments, interpreted data, and contributed to Figs. Supplementary material 1 (PDF 334 kb)Supplementary material 2 (AVI 30558 kb)Supplementary material 3 (AVI 35114 kb)Below is the link to the electronic supplementary material.


There are errors in S1 FigBacteria were cultured in LB medium at 37°C to OD600 0.5. The recombinant proteins were induced for 3 h by supplementation with 0.5 mM IPTG. Cells were harvested, washed in PBS, and then resuspended in 10 mM PBS . After several freeze/thaw cycles, the cell suspension was sonicated for 3 min with an interval of 4 s between pulses, and then centrifuged at 5000 g for 10 min at 37°C. Twenty microliter supernatant samples were analysed by 12% SDS-PAGE.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Cucumis sativus L.) is a typical parthenocarpic plant but the QTLs controlling parthenocarpy in cucumber were not mapped on chromosomes, and the linked markers were neither user-friendly nor confirmed by previous studies. Hence, we conducted a two-season QTL study of parthenocarpy based on the cucumber genome with 145 F2:3 families derived from a cross between EC1 (a parthenocarpic inbred line) and 8419 s-1 (a non-parthenocarpic inbred line) in order to map novel QTLs. Whole genome re-sequencing was also performed both to develop effective linked markers and to predict candidate genes.Parthenocarpy is an important trait for yield and quality in many plants. But due to its complex interactions with genetic and physiological factors, it has not been adequately understood and applied to breeding and production. Finding novel and effective quantitative trait loci (QTLs) is a critical step towards understanding its genetic mechanism. Cucumber , a QTL on chromosome 2, was a major-effect QTL with a logarithm of odds (LOD) score of 9.0 and phenotypic variance explained (PVE) of 17.0 % in the spring season and with a LOD score of 6.2 and PVE of 10.2 % in the fall season. We confirmed this QTL using a residual heterozygous line97-5 (RHL97-5). Effectiveness of linked markers of the Parth2.1 was validated in F3:4 population and in 21 inbred lines. Within this region, there were 57 genes with nonsynonymous SNPs/InDels in the coding sequence. Based on further combined analysis with transcriptome data between two parents, CsARF19, CsWD40, CsEIN1, CsPPR, CsHEXO3, CsMDL, CsDJC77 and CsSMAX1 were predicted as potential candidate genes controlling parthenocarpy.A genetic linkage map, employing 133 Simple Sequence Repeats (SSR) markers and nine Insertion/Deletion (InDel) markers spanning 808.1 cM on seven chromosomes, was constructed from an FA major-effect QTL Parth2.1 and six minor-effect QTLs mainly contribute to the genetic architecture of parthenocarpy in cucumber. SSR16226 and Indel-T-39 can be used in marker-assisted selection (MAS) of cucumber breeding. Whole genome re-sequencing enhances the efficiency of polymorphic marker development and prediction of candidate genes.The online version of this article (doi:10.1186/s12870-016-0873-6) contains supplementary material, which is available to authorized users. Parthenocarpy is defined as fruit set in the absence of fertilization or other stimulation . It has DefH9-iaaM auxin-synthesizing gene into cucumber [SLTIR1 (an auxin receptor) [SLARF7 (Auxin Response Factor 7) [SLIAA9 (a subfamily of Aux/IAA gene) transgenic tomatoes [Controlling parthenocarpy3.1 (Cop3.1) and Cop8.1, respectively [Parthenocarpy can be influenced by environmental, physiological, and genetic factors. Environmental conditions such as low temperatures and short day lengths promote parthenocarpy. Parthenocarpy has been shown to be dependent certain hormones. For instance, endogenous IAA concentrations in parthenocarpic ovaries or on fruits have been found to be higher than in pollinated organs in cucumbers –5. Therecucumber , eggplancucumber  can stimeceptor) , down-reactor 7)  and SLIAtomatoes  also givtomatoes , 21. In ectively .Parthenocarpy is widespread in cucumber germplasm resources, and so cucumber is a promising model plant for the study of parthenocarpy. Genetic studies of parthenocarpy in cucumber started in 1930. Hawthorn , Juldash2:3 families from an EC1 × 8419 s-1 cross. The major-effect QTL was confirmed with RHL97-5 . The effectiveness of linked markers to this QTL was validated in F3:4 plants and in 21 inbred lines. Whole genome re-sequencing allowed us to develop polymonrphic markers and predict candidate genes. The ascertainment of the major-effect QTL of parthenocapy will provide a good foundation for its fine mapping with large segregating population and the linked markers to this QTL will be useful for molecular breeding of parthenocarpy in cucumber.In this study, we performed a two-season QTL study for parthenocarpy in cucumber in F1 derived from these two parents exhibited medium PP (37.11 and 31.37). Results from ANOVA and variance component analysis for parthenocarpy from the F2:3 population are presented in Additional file 2:3 family in two seasons both revealed significant difference between F2:3 families , seasons , and family × season interactions . The broad sense heritability estimate (h2) for parthenocarpy was 78.3 %. A significant positive correlation  . The map covered a total of 808.1 cM and contained 7 chromosomes. The number of markers on each chromosome was between 14 and 26, and the average marker interval of this map was 5.7 cM . Some markers that didn’t show good amplification products or segregate in F2:3 families in spring and fall 2013  of chromosome 1, Parth2.1 at 6.5 cM  of chromosome 2, Parth3.1  at 93.8 cM of chromosome 3, Parth5  at 58.0 cM of chromosome 5, Parth7  at 23.4 cM of chromosome 7 (Table R2 = 10.2 %), Parth2.2 at 50.3 cM  of chromosome 2 and Parth3.1 at 57.5 cM  of chromosome 3. Parth2.1 flanked by SSR00684 and SSR22083 was considered as a major-effect QTL since it was the only QTL detected in two seasons and could explain more than 10 % of the phenotypic variance  were statistically significantly higher than those containing homozygous 8419 s-1 alleles but significantly lower than those with homozygous EC1 alleles at P < 0.05. These results confirmed the QTL effect, with 8.07 % higher PP for plants containing the homozygous EC1 alleles over plants with homozygous 8419 s-1 alleles at Parth2.1. Moreover, PP of the donor parent EC1 (61.11 ± 6.57) was significantly higher than plants having homozygous EC1 alleles in the Parth2.1 QTL region (P < 0.05), implying that the other QTLs also contributed to parthenocarpy in addition to Parth2.1.We confirmed the presence of Parth2.1 with 161 plants of RHL97-5 segregating for Parth2.1 Fig. . Plants 2 population (17.1 cM) and the mean distance between two neighboring markers was 1.09 cM. Linkage mapping analysis showed a major-effect QTL of parthenocarpy with a PVE of 24.4 %. The highest LOD score of 9.1 located between SSR16226 and Indel-T-39 according to a 2-LOD drop for a confidence interval of the QTL , χ2 = 19.20 > χ20.05,8(15.51), χ2 = 25.73 > χ20.01,8(20.09) and χ2 = 17.59 > χ20.05,8(15.51) respectively indicating that these markers were significantly related to parthenocarpy. The PP means of plants with homozygous EC1 alleles at loci Indel-T-32, Indel-T-34, SSR16226 and Indel-T-39 were 26.84 ± 11.86, 26.89 ± 11.76, 26.80 ± 11.78 and 27.89 ± 11.41 respectively which were significantly higher than those plants with homozygous 8419 s-1 alleles  at P < 0.01. The PP means of plants with heterozygous genotype at loci Indel-T-32, Indel-T-34 and Indel-T-39 were significantly lower than those with homozygous EC1 alleles at P < 0.05 but not significantly different with those with homozygous 8419 s-1 alleles whereas at locus SSR16226 showed the opposite way  and two gynoecious non-parthenocapic inbred lines (14418 and 14435) after electrophoresis are shown in Fig. We also collected phenotype data of 11 gynoecious and 10 monoecious cucumber inbred lines . The polymorphic nucleotide sequences between EC1 and 8419 s-1, including InDels, were obtained by comparing the whole genome sequences of EC1 and 8419 s-1 with the reference ‘9930’ sequence. There were 83,119 SNPs and 14,772 InDels in EC1, 52,278 SNPs and 9462 InDels in 8419 s-1 on chromosome 2 , 241 genes located within the Parth2.1 region. By comparing the whole genome sequences of EC1 and 8419 s-1 with the reference 9930 sequence, we found 57 candidate genes containing the polymorphic SNP/Indels in the coding sequence regions that led to missense or frameshift mutations  databases. Most of them have been functionally characterized , Csa2M070230 (CsWD40) and Csa2M070880 (CsEIN1) were identified as phytohormone related genes. Csa2M068680 (CsARF19) encodes AUX/IAA like protein, which functions in various biological processes, e.g. lateral root development, fruit development [Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set and development [CsWD40) encodes WD-40 repeat family protein, which functions in cytokinin responses [CsEIN1) encodes prokaryote sensory transduction proteins, which functions in ethylene binding and has ethylene receptor activity [Referring to the cucumber genome database  also expressed differentially. Moreover, qRT-PCR suggested that transcription of Csa2M070230 (CsWD40), Csa2M070330 (CsPPR) and Csa2M073000 (CsHEXO3) were continuously up-regulated whereas Csa2M055050 (CsMDL), Csa2M058620 (CsDJC77) and Csa2M058620 (CsSMAX1) were continuously down-regulated during the parthenocarpic fruit set  encodes a pentatricopeptide repeat protein involved in mitochondrial RNA editing. Csa2M073000 (CsHEXO3) encodes a protein with beta-hexosaminidase activity. Csa2M055050 (CsMDL) encodes VHS domain-containing protein or GAT domain-containing protein involved in cyanide biosynthetic process. Csa2M058620 (CsDJC77) encodes DNA heat shock N-terminal domain-containing protein involved in protein folding. Csa2M058640 (CsSMAX1) encodes heat shock related-protein involved in protein metabolic process. Compared to 8419 s-1, Csa2M070330 (CsPPR) and Csa2M073000 (CsHEXO3) showed significant expression at P < 0.01 at 2 dpa in EC1, Csa2M070230 (CsWD40) and Csa2M058640 (CsSMAX1) showed significant expression at P < 0.05 and 0.01 at 2 and 4 dpa respectively in EC1  × season interaction differences (P < 0.001) as well, which is consistent with the conclusions drawn by Sun [2:3 families in two seasons also exhibited wide genetic variations (low PP means with large standard derivation among F2:3 families)  and moderate marker density that provide less opportunities for recombination and subsequently limit the precision of QTL detection. To improve this situation, a high resolution map in the target region and an advanced population segregating only in this region will be beneficial.We identified five significant QTLs in spring and three in fall in this study. Five of these QTLs showed positive additive effects, which indicated that alleles increasing PP come from high parthenocarpic parent EC1. However, parent 8419 s-1 also carried the alleles increasing PP on two QTLs of Parth5.1 and Parth7.1 that could explain why 8419 s-1 produced parthenocarpic fruits in some plants although PP is pretty low. Therefore, the linked markers at Parth5.1 and Parth7.1 from 8419 s-1 should be used during MAS for parthenocarpy in cucumber. The QTL Parth2.1 on chromosome 2, which contributed over 10 % of PVE and expressed in both seasons, was a stable and major-effect QTL. The rest of QTLs were environment-specific with low PVE, indicating that a major and many minor effects mainly contribute to the genetic component of parthenocarpy in cucumber. A study has been carried out for QTL mapping of parthenocarpy in cucumber. Sun  detectedQTL Fig. . The reaP < 0.05. Likewise, PP of plants with homozygous EC1 alleles was significantly higher than those with the heterozygous genotype in the QTL region, suggesting a dominance effect, in contrast to the original QTL study which showed a larger additive effect for Parth2.1.QTL confirmation is an indispensable step to make sure a target QTL that can be further studied and to measure its effect more accurately. Using a segregated population, RHL97-5, the major-effect QTL Parth2.1 was confirmed in a homozygous background at other QTLs Fig. . Parth2.Based on the re-sequencing information of two parents, we developed new InDel markers to construct a high-resolution linkage map in Parth2.1 region. Linkage mapping analysis revealed a major QTL with higher PVE of 24.4 % compared to the original QTL study (17.0 and 10.2 %), demonstrating that the more homozygous the background was, then the higher phenotypic variance could be explained. However, parthenocarpy is a complex trait that phenotypic data of a target individual can be influenced when fertilization is being conducted at the same time. Therefore, segregating population construction from one target individual can only be attained by cuttings, which make it difficult to produce enough seeds for further study before the coming planting season and fine mapping of this trait will take longer time. Currently we are developing a large segregating population by cuttings from the target individual to fine map this QTL.3:4 plants. It was also applied to 11 gynoecious and 10 monoecious cucumber inbred lines to test its accuracy. Among 11 gynoecious inbred lines, the extreme phenotype of parthenocarpic lines all showed the same genotype with corresponding parents, which demonstrated that the major-effect Parth2.1 does exist and play roles in extreme parthenocarpy materials. Whereas, all monoecious cucumber inbred lines showed low PP  acts as a negative regulator of fruit set and transgenic plants with decreased SlARF7 mRNA levels forms seedless (parthenocarpic) fruits [CsARF19) is homologous to a member of Arabidopsis ARFs, ARF19, this indicates that it is a promising candidate gene involved in auxin signaling and it may trigger parthenocarpy. Another gene, Csa2M070230 (CsWD40), is an ortholog of Arabidopsis WD40 that plays a role in cytokinin responses [CsEIN1) is an ortholog of Arabidopsis ETHYLENE INSENSITIVE 1(EIN1) that negatively regulates ethylene-activated signaling pathway [CsEIN1 is also a promising candidate gene possibly involved in ethylene signaling pathway, and may result in parthenocarpy.Mutations between the genes of EC1 and 8419 s-1 in CDS sequences have the potential for transcriptional or functional differences that can regulate parthenocarpic/non-parthenocarpic fruit set. In the present study, we found that 57 genes located in parth2.1 contains missense or frameshift mutations  fruits . Since Cesponses , 39. It esponses , 55. Moresponses . Csa2M07 pathway . This inCsPPR, CsHEXO3, CsMDL, CsDJC77 and CsSMAX1 as candidate parthenocarpy genes because of their different expression patterns during parthenocarpic fruit set and fruit abortion . Each family planted 10 plants which were put next to each other. One day prior to anthesis, on each plant, we trapped eight female flowers from the fifth node above the main stem and eight more from the laterals with colorful metal wire. Well-developed  estimate was calculated from variance components. The broad sense heritability was estimated using h2B = σ2F/(σ2F + σ2FS/Rs + σ2E/RsRn), where σ2F was the family variance, σ2FS was the family × season interaction (F × S) variance, and σ2E was the residual variance, respectively. Rs was the number of seasons and Rn was the mode of individuals in each family. Correlations between PP in spring and fall were estimated using the PROC CORR function on the basis of PP means for each F2:3 family.Statistical analysis of phenotypic data was conducted with the software Statistical Analysis System (SAS V8). Analysis of variance (ANOVA) was performed with PROC VARCOMP function to estimate the genetic and season effects with a model like YDNA extraction of EC1 and 8419 s-1 was performed by the CTAB method. We constructed 500 bp paired-end sequencing libraries using genomic DNA ≥ 5ug from each parent, and sequenced these libraries using an Illumina Hiseq™ 2000. The raw data obtained by re-sequencing were processed to obtain clean data. The quality of these clean data was evaluated based on reads quantity, data output, error rate, and the content of Q20, Q30 and GC  procedure within Windows QTL Cartographer v2.5 software [P ≤ 0.05. An LOD score of 2.5 was used to determine the presence of QTL. Nomenclature of a QTL was an abbreviation of the trait, followed by relevant chromosome number then QTL serial number on this chromosome.QTL detection for parthenocarpy in cucumber was performed using the arcsin transformed PP means of each Fsoftware . The par2:4 families, each consisting of ten individuals, were planted in Pailou Experimental Greenhouse of Nanjing Agricultural University in winter 2013 in order to screen residual heterozygous plants. RHL97-5 segregated from a residual heterozygous plant 97–5 that is heterozygous for the major-effect QTL region between SSR marker SSR00684 and SSR22083 but homozygous for the other minor-effect QTLs. The RHL97-5 containing 161 plants was used to confirm the major-effect QTL. All markers in the target area (SSR00684-SSR22083) were used to genotype the 161 plants. Moreover, phenotype data collections of these plants were conducted as well. Linkage mapping analysis was performed based on high resolution linkage map and parthenocarpic phenotype data of the 161 plants. These plants were classified into three groups such as homozygous EC1, 8419 s-1 genotype and heterozygous ones based on the genotype in the target area (SSR00684-SSR22083), and ANOVAs were conducted among these three classes.One hundred and thirty five F3:4 plants derived from F2:3 family in the spring of 2014 and genotyped them with Indel-T-32, Indel-T-34 and two flanking markers, SSR16226 and Indel-T-39. Genotypes of these four markers  and plant numbers of each groups based on PP  were used to conduct the test for independence of 3 × 5 table (χ2 test) in order to explore the relationship between these markers and parthenocarpy. ANOVAs of PP among groups in terms of marker genotypes were also performed with significance at P < 0.05. Meanwhile, twenty-one different geographic origins and sexual type cucumber inbred lines . Ovary samples of trapping-treated EC1 and 8419 s-1 at two days post anthesis (dpa) were harvested for RNA-seq analysis. The details about how the transcriptomics experiment was carried out have been presented by Li [Genes located within the confidence interval of Parth2.1 were analyzed based on the whole genome parental re-sequencing and transcriptome data. Coding sequences in Parth2.1 region were searched to detect mutated sequences between EC1 and 8419 s-1 using the SAMTOOLs. Only those genes causing amino acid changes were considered as candidate genes. ed by Li . There wOvary samples of trapping-treated EC1 and 8419 s-1 at 0dpa, 2dpa, 4dpa were collected respectively for qRT-PCR. For each sample, 20 individual ovaries were ground into powder and mixed in liquid nitrogen (three replicates). Total RNAs were isolated using Trizol (Invitrogen) according to the manufacturer’s protocol and Rnase-free DNase I was used to remove DNA in RNA samples. cDNA was prepared with 2 μg of total RNA, using a cDNA Synthesis Kit (Fermentas). Quantitative real-time PCR was conducted with the SYBR Premix Ex Taq™ Kit (TAKARA) following the manufacturer’s instructions on a Bio-Rad CFX96 Real-Time PCR machine. The PCR program is: denaturation at 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Primers were designed using Primer Premier 5.0 software and Actin (GenBank ID: AB010922) was used as the internal control gene. The relative expression levels of each gene for different treatments were normalized to Actin gene and calculated with the 2-△△Ct method. The primers used for qRT-PCR are listed in Additional file


In the original supplemental materials, the last two column headings were titled Temperature (°C) and PM2.5 (µg/m3), but should have been PM2.5 (µg/m3) and Temperature (°C), respectively. The authors also noticed the following misspelling: Saint Diego, CA, should be spelled San Diego, CA. These corrections to Table S1 appear in this erratum.In Table S1, the authors incorrectly inverted the titles of the column headers for temperature and PMThese typographical errors do not affect the information presented in the article. The authors regret the errors.


We sought to determine whether genomic polymorphism in collagen IX genes (COL9A) was associated with Kashin-Beck disease (KBD).Twenty seven single nucleotide polymorphisms (SNPs) in COL9AI, COL9A2 and COL9A3 were genotyped in 274 KBD cases and 248 healthy controls using the Sequenom MassARRAY system. Associations between the COL9A polymorphism and KBD risk were detected using an unconditional logistic regression model. Linkage disequilibrium (LD) and haplotypes analysis were performed with the Haploview software.X2 = 16.74, df = 2, P = 0.0002). Regression analysis showed that the allele “C” in SNP rs6910140 had a significant protective effect on KBD . The frequencies of alleles and genotypes in rs6910140 were significantly different among subjects of different KBD stages . However, haplotype analysis did not detect any significant association between KBD and COL9A1, COL9A2 and COL9A3.After Bonferroni correction, the frequency distribution of genotypes in rs6910140 in COL9A1 was significantly different between the KBD and the control groups (We observed a significant association between rs6910140 of COL9A1 and KBD, suggesting a role of COL9A1 in the development of KBD. Kashin-Beck disease (KBD) is a chronic osteochondropathy affecting the bones and joints that is endemic to certain geographical areas. A key pathological feature of KBD is chondrocyte necrosis in the deep zone of the growth plate of cartilage and articular cartilage ,2]. Cli. Cli2]. The etiology of KBD remains unclear. In the past 150 years, three environmental hypotheses have been proposed: selenium deficiency, mycotoxins from contaminated storage grains, and organic matter  in drinking water –5. RecenType IX collagen, a trimer of three different gene products, a1(IX), a2 (IX) and a3 (IX) chains, are encoded by the COL9A1, COL9A2, and COL9A3 genes, is quantitatively a minor component that functions structurally by covalently cross-linking to the surface of type II collagen fibrils . The sket = 1.29, P>0.05) and sex  . Patients with clinical symptoms or radiographic changes of other osteochondropathy were excluded. The healthy control was defined as no KBD and OA. The controls were randomly selected and were frequency-matched by age  , and casBased on the information of SNPs in COL9A that predispose for OA –20. 27 S2O, 0.5 μL PCR buffer (20 mM MgCl2), 0.4 μL 25 mM MgCl2, 0.1 μL 25 mM dNTP mix, 1 μL primer mix (500 nM each), 0.5 μL QC competitor, 0.5 μL QA spike, 0.2 μL PCR enzyme and 1 μL sample DNA. The PCR was performed in a GeneAmp PCR System 9700 thermal cycler  under the following conditions: denaturation at 95°C for 2 min, 45 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 1 min, and extension at 72°C for 5 min. The PCR products were treated with a cocktail of 1.53 μL H2O, 0.17 μL 10×shrimp alkaline phosphatase (SAP) buffer and 0.3 μL SAP (1.7 U/m L) in a GeneAmp PCR System 9700 thermal cycler at 37°C for 40 min followed by 85°C for 5 min. The sing base extension (SBE) reaction contained 7 μL of the SAP-treated PCR products and 2 μL of iPLEX Pro mix (Sequenom). The iPLEX Pro mix contained 0.2 μL 10×iPLEX Pro buffer, 0.2 μL iPLEX Pro termination mix, 0.94 μL primer mix , 0.041 μL iPLEX1 enzyme, 0.5 μL EXT QA spike, and 0.119 μL H2O. The SBE reaction was performed in a GeneAmp PCR System 9700 thermal cycler under the following conditions: denaturation at 94°C for 30 s, 40 cycles of 94°C for 5 s and five cycles of 52°C for 5 s and 80°C for 5 s, and extension at 72°C for 3 min. A total of 41 μL of molecular-grade H2O and ion exchange resin (Sequenom) was then added to each sample and the results were visualized using a MassARRAY Analyzer 4 system (Sequenom) using autorun settings.Genomic DNA was extracted from the peripheral blood of the 274 KBD patients and 248 healthy controls using a blood DNA extraction kit . Genotyping was performed using the Sequenom MassARRAY system. Primers were designed using Sequenom SNP Assay Design software version 3.0 for iPLEX reactions. The PCR contained: 0.8 μL Hx2 test to compare the expected frequencies of genotypes in controls, SNPs with P>0.05 were considered to be in HWE [The Hardy-Weinberg equilibrium (HWE) of each SNP was tested by the goodness-of-fit e in HWE . The Inde in HWE ,24.To act = 1.29, P>0.05) and sex . In addition, the clinical stages of the 274 KBD patients were divided into I, II and III stages and the constituent ratio was 57.66%(158/274), 31.02%(85/274) and 11.32%(31/274) respectively  . The distribution frequencies of genotypes in rs1135056 and rs6910140  were significantly different between the two groups (P>0.0018).A case-control comparison of both the genotype and allele frequencies for the 27 SNPs is presented in 14–0.95) . When tho groups . HoweverP = 0.03), compared with the homozygous wild type (GG). Dominant model and recessive model were also applied for detecting association between COL9A and KBD. Adjusted ORs for rs1135056 in COL9A1 in the recessive model and rs6910140 in COL9A1 in the dominant model were statistically significant . But after Bonferroni correction, only the SNP rs6910140 in the dominant model remained. The allele “C” of rs6910140 in COL9A1 had a significant protective effect on KBD.ORs and 95% CIs for KBD were calculated from unconditional logistic regression model for evaluating relative risks . A weak Linkage disequilibrium structure is shown in It is not clear whether rs6910140 contributes to the severity of KBD. The association of rs6910140 in COL9A1 with the severity of KBD was monitored in the KBD group. The etiology of KBD remains unclear, genetic factors may particularly account for an individual’s susceptibility to KBD . KBD andIn the present study, fifteen SNPs in COL9A1 were included to investigate the association between the gene COL9A1 and risk of KBD in the Chinese Han population. The distribution frequencies of genotypes in rs6910140 were significantly different between the two groups. The subjects carrying allele “C” have lower risk of KBD than the subjects without carrying allele “C”. Additional analysis showed that rs6910140 was significantly associated with the clinical diagnose of KBD stages. These results suggest that polymorphism of the COL9A1 plays an important role in the risk and severity of KBD in the Chinese Han population. Gene COL9A1 located on the long arm of chromosome 6. Several studies have provided evidences of association between COL9A1 gene and arthritis disease ,28,29. OHowever, our findings did not support an association between the COL9A2, COL9A3 genes and KBD in our population, even though the heterozygosity of rs3765462 in gene COL9A3 showed a weak association with an evaluated KBD risk. However, the significance did not survive Bonferroni correction. Extensive studies have provided evidence for the associations between the genes COL9A2, COL9A3 and osteochondropathy ,30,31. FIn conclusion, we found that the rs6910140 in COL9A1 was associated with the susceptivity of KBD. The allele “C” of rs6910140 in COL9A1 had a significant protective effect on KBD. Further validation of the contribution of COL9A to the development of KBD in other populations is warranted.S1 Dataset(XLS)Click here for additional data file.