High-throughput SNP Genotyping Research Articles

Identification of Canadian barley varieties by high-throughput SNP genotyping

Barley is a major grain crop in Canada and is used for generating malt, in addition to being used as human food and animal feed. Differentiating barley varieties is important for malt quality assurance and grain handling. Here, we present a DNA-based testing method for the identification of Canadian barley varieties. The method uses 24 custom TaqMan genotyping assays, which are analyzed using the high-throughput SmartChip system (Takara Bio Inc.). Using this method, we are able to distinguish 124 barley varieties commonly grown in Canada.

Identification of genetic loci for early maturity in spring bread wheat using the association analysis and gene dissection.

Early maturity in spring bread wheat is highly desirable in the regions where it enables the plants to evade high temperatures and plant pathogens at the end of the growing season. To reveal the genetic loci responsible for the maturity time association analysis was carried out based on phenotyping for an 11-year period and high-throughput SNP genotyping of a panel of the varieties contrasting for this trait. The expression of candidate genes was verified using qPCR. The association between the SNP markers and the trait was validated using the biparental F2:3 population. Our data showed that under long-day conditions, the period from seedling to maturity is mostly influenced by the time from heading to maturity, rather than the heading time. The QTLs associated with the trait were located on 2A, 3B, 4A, 5B, 7A and 7B chromosomes with the 7BL locus being the most significant and promising for its SNPs accelerated the maturity time by about 9 days. Gene dissection in this locus detected a number of candidates, the best being TraesCS7B02G391800 (bZIP9) and TraesCS7B02G412200 (photosystem II reaction center). The two genes are predominantly expressed in the flag leaf while flowering. The effect of the SNPs was verified in F2:3 population and confirmed the association of the 4A, 5B and 7BL loci with the maturity time.

Inconsistent estimates of hybridization frequency in newts revealed by SNPs and microsatellites

Hybridization between the European smooth and palmate newts has recurrently been mentioned in the literature. The only two studies that attempted to quantify the frequency of hybridization and gene admixture between these two species came to strikingly opposite conclusions. According to Arntzen et al. (1998, 42 allozymes), hybrids are rare in nature and introgression negligible, while according to Johanet et al. (2011, 6 microsatellites), introgressive hybridization is significant and widespread across the shared distribution range. To clarify this question, we implemented high-throughput SNP genotyping with diagnostic biallelic SNPs on 965 specimens sampled across Europe. Our results are in line with Arntzen et al., since only two F1 hybrids were identified in two distinct French localities, and no further hybrid generations or backcrosses. Moreover, reanalysis of 78 of the samples previously studied by Johanet et al. (2011) using our SNPs panel could not reproduce their results, suggesting that microsatellite-based inference overestimated the hybridization frequency between these two species. Since we did not detect methodological issues with the analyses of Johanet et al., our results suggest that SNP approaches outperform microsatellite-based assessments of hybridization frequency, and that conclusions previously published on this topic with a small number of microsatellite loci should be taken with caution, and ideally be repeated with an increased genomic coverage.

Marker-assisted introgression of wild chromosome segments conferring resistance to fungal foliar diseases into peanut (Arachis hypogaea L.).

Fungal foliar diseases can severely affect the productivity of the peanut crop worldwide. Late leaf spot is the most frequent disease and a major problem of the crop in Brazil and many other tropical countries. Only partial resistance to fungal diseases has been found in cultivated peanut, but high resistances have been described on the secondary gene pool. To overcome the known compatibility barriers for the use of wild species in peanut breeding programs, we used an induced allotetraploid (Arachis stenosperma × A. magna)4x, as a donor parent, in a successive backcrossing scheme with the high-yielding Brazilian cultivar IAC OL 4. We used microsatellite markers associated with late leaf spot and rust resistance for foreground selection and high-throughput SNP genotyping for background selection. With these tools, we developed agronomically adapted lines with high cultivated genome recovery, high-yield potential, and wild chromosome segments from both A. stenosperma and A. magna conferring high resistance to late leaf spot and rust. These segments include the four previously identified as having QTLs (quantitative trait loci) for resistance to both diseases, which could be confirmed here, and at least four additional QTLs identified by using mapping populations on four generations. The introgression germplasm developed here will extend the useful genetic diversity of the primary gene pool by providing novel wild resistance genes against these two destructive peanut diseases.

Design and validation of a 63K genome-wide SNP-genotyping platform for caribou/reindeer (Rangifer tarandus)

BackgroundDevelopment of large single nucleotide polymorphism (SNP) arrays can make genomic data promptly available for conservation problematic. Medium and high-density panels can be designed with sufficient coverage to offer a genome-wide perspective and the generated genotypes can be used to assess different genetic metrics related to population structure, relatedness, or inbreeding. SNP genotyping could also permit sexing samples with unknown associated metadata as it is often the case when using non-invasive sampling methods favored for endangered species. Genome sequencing of wild species provides the necessary information to design such SNP arrays. We report here the development of a SNP-array for endangered Rangifer tarandus using a multi-platform sequencing approach from animals found in diverse populations representing the entire circumpolar distribution of the species.ResultsFrom a very large comprehensive catalog of SNPs detected over the entire sample set (N = 894), a total of 63,336 SNPs were selected. SNP selection accounted for SNPs evenly distributed across the entire genome (~ every 50Kb) with known minor alleles across populations world-wide. In addition, a subset of SNPs was selected to represent rare and local alleles found in Eastern Canada which could be used for ecotype and population assignments - information urgently needed for conservation planning. In addition, heterozygosity from SNPs located in the X-chromosome and genotyping call-rate of SNPs located into the SRY gene of the Y-chromosome yielded an accurate and robust sexing assessment. All SNPs were validated using a high-throughput SNP-genotyping chip.ConclusionThis design is now integrated into the first genome-wide commercially available genotyping platform for Rangifer tarandus. This platform would pave the way to future genomic investigation of populations for this endangered species, including estimation of genetic diversity parameters, population assignments, as well as animal sexing from genetic SNP data for non-invasive samples.

Identifying Parameters for Defining "Essentially Derived Varieties" of Maize Inbred Lines Using High-Throughput Genome-Wide SNP Markers.

Well-developed maize reference genomes and genotyping technology along with fast decreasing detection costs have enabled the chance of shifting essentially derived varieties (EDV) identification to high-throughput SNP genotyping technology. However, attempts of using high-throughput technologies such as SNP array on EDV identification and the essential baseline parameters such as genetic homozygosity and/or stability in EDV practices have not been characterized. Here, we selected 28 accessions of 21 classical maize inbreds, which definitely form a pedigree network from initial founders to derivatives that had made huge contribution to corn production, to demonstrate these fundamental analyses. Our data showed that average residual heterozygosity (RH) rate of these 28 accessions across genome was about 1.03%. However, the RH rate of some accessions was higher than 3%. In addition, some inbreds were found to have an overall RH rate lower than 2% but over 8% level at certain chromosomes. Genetic drift (GD) between two accessions from different years or breeding programs varied from 0.13% to 13.16%. Accessions with low GD level showed cluster distribution pattern and compared with RH distributions indicated that RH was not the only resource of GD. Both RH and GD data suggested that genetic purity analysis is an essential procedure before determining EDV. Eleven derivative lines were characterized with regard to their genome compositions and were inferred as their breeding histories. The backcross, bi-parental recycling, and mutation breeding records could be identified. The data provide insights of underlining fundamental parameters for defining EDV threshold and the results demonstrate the EDV identification process.

Genome-wide association study reveals growth-related markers and candidate genes for selection in Chinese longsnout catfish (Leiocassis longirostris)

Growth-related traits are economically important in fish because they directly affect fish production. The Chinese longsnout catfish (Leiocassis longirostris) is an important aquaculture species and its consumption is growing in China. In this study, using 200 individuals and high-throughput SNP genotyping technology, we performed the first genome-wide association study (GWAS) in L. longirostris to identify putative genomic regions and candidate genes associated with growth traits (including body mass, total length, standard length, head width, snout length, and caudal peduncle depth). The morphometrics of these traits were highly correlated (Pearson's correlation coefficients = 0.88–0.96, p < 0.05). GWAS detected 12, 5, 6, 9, 4, and 8 SNPs loci that were significantly associated (-log10P ≥ 5.21) with body mass, total length, standard length, head width, snout length, and caudal peduncle depth, respectively. Subsequently, within the 100 kb upstream and downstream regions surrounding these significant SNPs, 96, 31, 39, 61, 22, and 36 candidate genes for the 6 growth traits, respectively, were detected. Interestingly, chromosome (Chr) 16 had the most SNPs significantly associated with growth traits, and the majority of those were shared among multiple growth-related traits. In addition, multiple candidate genes surrounding these SNPs have been shown to serve essential functions in biological processes closely-related to growth, such as enox2, ky, and coma1. These results indicated that Chr16 may be central to growth characteristics. Other Chrs had genes involved in bone development, cell proliferation, and fatty acid metabolism, including matn1, sdc3, if2b3, kif3a, h1b, cadh7, rerg, and fabph. Our findings not only help understand the genetic architecture of growth traits in fish, but facilitate the identification of candidate genes for marker-assisted selection towards breeding faster growing L. longirostris in the future.

Gene-set enrichment analysis of selective sweeps reveals phenotypic traits in Nguni cattle

Adaptation of animals to different environments is typically associated with structural and functional genomic variations. High throughput SNP genotyping and next-generation sequencing (NGS) have made it possible to study positive selection footprints and adaptation traits. Nguni is a small frame-size breed, mostly horned, and well known for being adapted to diverse South African environmental conditions. This study used previously identified selective sweeps to perform functional analysis of genes related to phenotypic characteristics in Nguni. Two hundred and sixty-four candidate selective sweeps were used for gene-set enrichment analysis in molecular functional categories (KEGG pathways) using the database for annotation, visualization, and integrated discovery (DAVID). In total, 107 genes were identified across all the chromosomes with 74 genes associated with eight phenotype queries, including fat content, milk production, walking ability, heat tolerance, meat production, reproduction, and bone and muscle development. Gene CRHR2 was associated with meat quality (juiciness and flavour). The IRAK3 gene was associated with decreased body size, feed intake and fatness in cattle, and CARD15 with disease resistance. Gene annotation using phenotype queries identified four genes (SPI, YWHAZ, RGS4, and RGS5) that were associated with myometrial relaxation in cattle. Genes such as NOD2 and IL21R were associated with inflammatory bowel diseases in cattle, whereas CPLS gene was associated with fat content. These genes are important to the phenotypic and adaptive characteristics present in South African Nguni cattle and hold potential for selection for traits of economic importance.

Development and Application of a Target Capture Sequencing SNP-Genotyping Platform in Rice.

The development of efficient, robust, and high-throughput SNP genotyping platforms is pivotal for crop genetics and breeding. Recently, SNP genotyping platforms based on target capture sequencing, which is very flexible in terms of the number of SNP markers, have been developed for maize, cassava, and fava bean. We aimed to develop a target capture sequencing SNP genotyping platform for rice. A target capture sequencing panel containing 2565 SNPs, including 1225 SNPs informative for japonica and 1339 SNPs informative for indica, was developed. This platform was used in diversity analysis of 50 rice varieties. Of the 2565 SNP markers, 2341 (91.3%) produced useful polymorphic genotype data, enabling the production of a phylogenetic tree of the 50 varieties. The mean number of markers polymorphic between any two varieties was 854. The platform was used for QTL mapping of preharvest sprouting (PHS) resistance in an F8 recombinant inbred line population derived from the cross Odae × Joun. A genetic map comprising 475 markers was constructed, and two QTLs for PHS resistance were identified on chromosomes 4 and 11. This system is a powerful tool for rice genetics and breeding and will facilitate QTL studies and gene mapping, germplasm diversity analysis, and marker-assisted selection.

Comparative analysis of inbreeding parameters and runs of homozygosity islands in 2 Italian autochthonous cattle breeds mainly raised in the Parmigiano-Reggiano cheese production region

Reggiana and Modenese are autochthonous cattle breeds, reared in the North of Italy, that can be mainly distinguished for their standard coat color (Reggiana is red, whereas Modenese is white with some pale gray shades). Almost all milk produced by these breeds is transformed into 2 mono-breed branded Parmigiano-Reggiano cheeses, from which farmers receive the economic incomes needed for the sustainable conservation of these animal genetic resources. After the setting up of their herd books in 1960s, these breeds experienced a strong reduction in the population size that was subsequently reverted starting in the 1990s (Reggiana) or more recently (Modenese) reaching at present a total of about 2,800 and 500 registered cows, respectively. Due to the small population size of these breeds, inbreeding is a very important cause of concern for their conservation programs. Inbreeding is traditionally estimated using pedigree data, which are summarized in an inbreeding coefficient calculated at the individual level (FPED). However, incompleteness of pedigree information and registration errors can affect the effectiveness of conservation strategies. High-throughput SNP genotyping platforms allow investigation of inbreeding using genome information that can overcome the limits of pedigree data. Several approaches have been proposed to estimate genomic inbreeding, with the use of runs of homozygosity (ROH) considered to be the more appropriate. In this study, several pedigree and genomic inbreeding parameters, calculated using the whole herd book populations or considering genotyping information (GeneSeek GGP Bovine 150K) from 1,684 Reggiana cattle and 323 Modenese cattle, were compared. Average inbreeding values per year were used to calculate effective population size. Reggiana breed had generally lower genomic inbreeding values than Modenese breed. The low correlation between pedigree-based and genomic-based parameters (ranging from 0.187 to 0.195 and 0.319 to 0.323 in the Reggiana and Modenese breeds, respectively) reflected the common problems of local populations in which pedigree records are not complete. The high proportion of short ROH over the total number of ROH indicates no major recent inbreeding events in both breeds. ROH islands spread over the genome of the 2 breeds (15 in Reggiana and 14 in Modenese) identified several signatures of selection. Some of these included genes affecting milk production traits, stature, body conformation traits (with a main ROH island in both breeds on BTA6 containing the ABCG2, NCAPG, and LCORL genes) and coat color (on BTA13 in Modenese containing the ASIP gene). In conclusion, this work provides an extensive comparative analysis of pedigree and genomic inbreeding parameters and relevant genomic information that will be useful in the conservation strategies of these 2 iconic local cattle breeds.

Genome-wide scans for signatures of selection in Mangalarga Marchador horses using high-throughput SNP genotyping

BackgroundThe detection of signatures of selection in genomic regions provides insights into the evolutionary process, enabling discoveries regarding complex phenotypic traits. In this research, we focused on identifying genomic regions affected by different selection pressures, mainly highlighting the recent positive selection, as well as understanding the candidate genes and functional pathways associated with the signatures of selection in the Mangalarga Marchador genome. Besides, we seek to direct the discussion about genes and traits of importance in this breed, especially traits related to the type and quality of gait, temperament, conformation, and locomotor system.ResultsThree different methods were used to search for signals of selection: Tajima’s D (TD), the integrated haplotype score (iHS), and runs of homozygosity (ROH). The samples were composed of males (n = 62) and females (n = 130) that were initially chosen considering well-defined phenotypes for gait: picada (n = 86) and batida (n = 106). All horses were genotyped using a 670 k Axiom® Equine Genotyping Array​ (Axiom MNEC670). In total, 27, 104 (chosen), and 38 candidate genes were observed within the signatures of selection identified in TD, iHS, and ROH analyses, respectively. The genes are acting in essential biological processes. The enrichment analysis highlighted the following functions: anterior/posterior pattern for the set of genes (GLI3, HOXC9, HOXC6, HOXC5, HOXC4, HOXC13, HOXC11, and HOXC10); limb morphogenesis, skeletal system, proximal/distal pattern formation, JUN kinase activity (CCL19 and MAP3K6); and muscle stretch response (MAPK14). Other candidate genes were associated with energy metabolism, bronchodilator response, NADH regeneration, reproduction, keratinization, and the immunological system.ConclusionsOur findings revealed evidence of signatures of selection in the MM breed that encompass genes acting on athletic performance, limb development, and energy to muscle activity, with the particular involvement of the HOX family genes. The genome of MM is marked by recent positive selection. However, Tajima’s D and iHS results point also to the presence of balancing selection in specific regions of the genome.

Genome-wide association study reveals genomic regions and candidate genes for head size and shape in bighead carp (Hypophthalmichthys nobilis)

Head-related traits are economically important in fish because they will essentially affect fillet yields. Bighead carp (Hypophthalmichthys nobilis) is an important aquaculture fish, and interestingly its head has higher culinary and market values than its fillet in China. In this study, using 776 bighead carp individuals and high-throughput SNP genotyping technology, we performed genome-wide association study (GWAS) to identify genomic regions and candidate genes potentially associated with head-size and head-shape traits such as head length (HL), head width (HW), head height (HH), head length/head height ratio (HL/HH), head length/head width ratio (HL/HW) and head length/body length ratio (HL/BL). The morphometrics of most of these traits were significantly correlated (P < 0.01). GWAS detected eleven significant SNPs and twelve suggestive SNPs associated with head size of bighead carp, which were mainly located on linkage group (LG) 16 and four of these SNPs were commonly shared among three head-size traits. For HL, four significant and three suggestive SNPs were on LG16. For HW, five significant and four suggestive SNPs were identified on LG3, LG16 and LG21, respectively. For HH, two significant and five suggestive SNPs were detected on LG11 and LG16. Genes involved in cell proliferation and bone development, such as cc2b, prdm1b, fndc5b, lbr, bmp8a and ATP6v1cb, were found surrounding SNPs in the QTL regions. For head shape, seventeen and six SNPs were significantly related to HL/HH and HL/HW, respectively, and underlying candidate genes were identified from genomic regions of bighead carp surrounding these SNPs, including ptch1, col9a1a, tgfbr2, hecw2a, zbtb42 and sema7a. This study shed lights on understanding genetic architecture of head size/shape traits in fish and facilitated to identify candidate genes for marker-assisted selection towards breeding bigger headed carp in future.

Different loci control resistance to different isolates of the same race of Colletotrichum lindemuthianum in common bean.

Linkage and genome-wide association analyses using high-throughput SNP genotyping revealed different loci controlling resistance to different isolates of race 65 of Colletotrichum lindemuthianum in common bean. Development of varieties with durable resistance to anthracnose is a major challenge in common bean breeding programs because of the extensive virulence diversity of Colletotrichum lindemuthianum fungus. We used linkage and genome-wide association analyses to tap the genomic regions associated with resistance to different isolates of race 65. Linkage mapping was done using an F2 population derived from the cross between the Mesoamerican common beans BRS Estilo x Ouro Vermelho, inoculated with two different isolates of race 65. Association genetics relied on a diversity common bean panel containing 189 common bean accessions inoculated with five different isolates of race 65 as an attempt to validate the linkage analysis findings and, eventually, identify other genomic regions associated with resistance to race 65. The F2 population and diversity panel were genotyped with the BARCBean6K_3 Illumina BeadChip containing 5398 SNP markers. Both linkage and genome-wide association analyses identified different loci controlling resistance to different isolates of race 65 on linkage group Pv04. Genome-wide association analysis also detected loci on Pv05, Pv10 and Pv11 associated with resistance to race 65. These findings indicate that resistance to race 65 can be overcome by the virulence diversity among different isolates of the same race and could lead to the loss of resistance after cultivar release. We identified 25 resistant common bean cultivars to all five isolates of race 65 in the diversity panel. The accessions should be useful to develop cultivars combining different resistance genes that favor durable resistance to anthracnose in common bean.

Construction of a Genetic Map of RILs Derived from Wheat (T. aestivum L.) Varieties Pamyati Azieva × Paragon Using High-Throughput SNP Genotyping Platform KASP—Kompetitive Allele Specific PCR

The main purposes of the study were (i) to develop a mapping population, (ii) to construct its genetic map for further identification of genes associated with important agronomic traits. To the best of our knowledge this is the first segregating population and genetic map developed for Kazakh bread wheat. The work is an example of how plant breeding programs in Kazakhstan have started successfully using next generation plant breeding methods to carry out systematic plant breeding to enhance final grain harvest. The KASP technology and SNP DNA-markers have been exploited to genotype and build a genetic map. The total length of the map was 1376 cM. A total 157, which spanned A, B and D subgenomes, out of the initial 178 SNP markers used formed 26 linkage groups leaving 1 duplicated and 20 unassigned markers. As threshold distance between markers was set ≤30 cM, two linkage groups were obtained for chromosomes such as 2А, 2В, 2D, 3A, 5A, 6B and 7A. Kosambi mapping function was employed to calculate recombination units. RILs were developed through SSD method up to F4 generation. Almost 97% of identified alleles were useful in evaluating the population’s genetic diversity; the remaining 3% showed no outcome. As a result, 77 DNA markers were mapped for A, 74 for B and 27 for D genomes. The mapping population will be genotyped using a high marker density array platform such as Illumina iSelect to obtain a genetic map with a relatively high coverage. Then, the population and high-resolution genetic map will be used to identify genes influencing wheat adaptation in Kazakhstan.

A 3K Axiom SNP array from a transcriptome-wide SNP resource sheds new light on the genetic diversity and structure of the iconic subtropical conifer tree Araucaria angustifolia (Bert.) Kuntze.

High-throughput SNP genotyping has become a precondition to move to higher precision and wider genome coverage genetic analysis of natural and breeding populations of non-model species. We developed a 44,318 annotated SNP catalog for Araucaria angustifolia, a grandiose subtropical conifer tree, one of the only two native Brazilian gymnosperms, critically endangered due to its valuable wood and seeds. Following transcriptome assembly and annotation, SNPs were discovered from RNA-seq and pooled RAD-seq data. From the SNP catalog, an Axiom® SNP array with 3,038 validated SNPs was developed and used to provide a comprehensive look at the genetic diversity and structure of 15 populations across the natural range of the species. RNA-seq was a far superior source of SNPs when compared to RAD-seq in terms of conversion rate to polymorphic markers on the array, likely due to the more efficient complexity reduction of the huge conifer genome. By matching microsatellite and SNP data on the same set of A. angustifolia individuals, we show that SNPs reflect more precisely the actual genome-wide patterns of genetic diversity and structure, challenging previous microsatellite-based assessments. Moreover, SNPs corroborated the known major north-south genetic cline, but allowed a more accurate attribution to regional versus among-population differentiation, indicating the potential to select ancestry-informative markers. The availability of a public, user-friendly 3K SNP array for A. angustifolia and a catalog of 44,318 SNPs predicted to provide ~29,000 informative SNPs across ~20,000 loci across the genome, will allow tackling still unsettled questions on its evolutionary history, toward a more comprehensive picture of the origin, past dynamics and future trend of the species' genetic resources. Additionally, but not less importantly, the SNP array described, unlocks the potential to adopt genomic prediction methods to accelerate the still very timid efforts of systematic tree breeding of A. angustifolia.

Utilization of KASP technology for wheat improvement

Hundreds of experiments involving genotyping and phenotyping of the plants are planned daily by plant researchers worldwide for wheat improvement. There are a plethora of molecular markers available that are being widely used in wheat genetic studies but have their own limitations. Developments in the areas of next-generation sequencing and bioinformatics applications have aided in the development of SNP-based markers. SNP-based assays have revolutionized the method of genotyping as these consume less time and have high accuracy and effectiveness. Among the SNP genotyping platforms, kompetitive allele-specific PCR (KASP) assay is a promising technology for high throughput SNP genotyping which is based on the fluorescence resonance energy transfer (FRET) method. KASP genotyping mainly involves two allele-specific forward primers, one common reverse primer and FRET cassettes which results in the identification of respective alleles having a particular SNP or InDel. It is playing a significant role in performing various genetic studies such as genetic diversity analysis, genome-wide association studies and marker assisted selection in wheat. In this review, for the first time, we attempt to give an overview of the KASP assays developed for various biotic and abiotic stresses, agronomic, physiological and quality traits for wheat improvement and the assays developed for genetic diversity analysis and genotyping by sequencing.

Development of high throughput SNP genotyping approach using target sequencing in Pacific white shrimp and its application for genetic study

The Pacific white shrimp Litopeneaus vannamei (L. vannamei) is a worldwide aquaculture species and selection of high performance germplasm is urgently needed. Application of molecular breeding method, such as marker assisted selection (MAS) or genomic selection (GS), is an efficient approach to accelerate the breeding program for high-quality germplasm. However, lack of cost-effective genotyping method is the main obstacle for their applications in shrimp breeding program. In this study, we developed a high throughput SNP genotyping method based on AmpliSeq target sequencing. A total of 581 amplicons including the genome-wide distributed markers, immune-related genes, WSSV resistance related SNPs, growth and sex related SNPs were selected and could be genotyped simultaneously. Totally 10 samples were used for genotyping and 2130 high quality SNPs were genotyped. Through analyzing the genotyped SNPs, the genetic relationship of samples could be accurately reconstructed. Besides, the pooling genotyping method using SNP panel was evaluated. It illustrated that the pooling genotyping method was reliable with read depth at >200. Based on the read depth of each amplicons, the panel was further optimized to 508 amplicons, which will enhance the sequencing uniformity and improve the efficiency of the panel. This study provided a useful genetic tool and a new method for genetic study. It will accelerate the application of MAS and GS in shrimp breeding program.

Genetic mapping and localization of major QTL for bitterness in melon (Cucumis melo L.)

Melon (Cucumis melo L., 2n=2x=24) is a eudicot diploid plant species belongs to the Cucurbitaceae family, which is famous for its specific biological properties and economic importance. Melon with high cucurbitacin contents taste unpleasant and are not preferred by consumers, but the underlying molecular mechanism for the bitterness related trait is not fully known. Therefore, the study of bitterness in melon has important practical value. Exploring the genetic rules of bitterness related traits and the key genes in the process of biosynthesis and metabolic regulation, as well as developing the molecular markers related to the bitterness of melon can provide guidance for the selection of non-bitter taste melon varieties and furnish reference for related studies of cucurbitacin. Here, we constructed a genetic linkage map using type IIB endonucleases restriction-site associated DNA (2b-RAD) based high throughput SNP genotyping data of a BC1 mapping family. The total length of the linkage map was 851.53 cM, with an average distance of 1.11 cM between adjacent markers. Genetic analysis of F2 and BC1 populations derived from two parental lines (C68 with non-bitter taste and C69 with bitter taste) revealed that the melon bitterness related trait is controlled by dual locus, and bitterness is dominant to non-bitterness. QTL mapping identified 3 significant QTLs in 2 linkage groups (LGs). Then, based on the InDel markers obtained from deep re-sequencing of the two parents, we verified the accuracy of the mapping interval. Especially, the Polymerase Chain Reaction (PCR) based co-separation markers on chromosome 2 and chromosome 5 revealed that the genotypes perfectly matched with the phenotypes in F2 and XC6 population, which further proves the accuracy of our mapping results for melon bitterness. Our present study will provide fine mapping of melon bitterness related trait and facilitate marker-assisted selection for the development of non-bitterness cultivars.

Association between PON2 gene polymorphisms and susceptibility to noise-induced hearing loss

Objective: To investigate the relationship between SNP and noise-induced hearing loss (NIHL) susceptibility in occupational noise exposure population in China. Methods: From 6297 for a certain steel works in contact with noise, contact length of 3 years or more and workplace noise exposure intensity of 80 dB (A) , ears or high frequency (3 000, 4 000, 6 000 Hz) average of hearing acuity 40 dB (HL) , or high frequency loss in both ears, on the basis of single whisper frequency (500, 1, 000, 2 000 Hz) average threshold of 26 dB (HL) or object as case group. A case-control study was designed with 1:1 matching. Subjects with the same gender, the same type of work, age ±5 years old, and working age ±2 years after noise exposure were selected as the control group. Subjects with any whisper frequency (500, 1, 000, 2, 000 Hz) whose hearing threshold in any frequency band was ≤25 dB (A) and whose average high-frequency hearing threshold in pure tone hearing test was <35 dB (A) were selected as the control group. Four sites of PON2 gene were genotyped by medium-and high-throughput SNP genotyping. Univariate logistic regression was used to analyze the relationship between single SNP polymorphism and NIHL. Results: A total of 286 case-control pairs were included. Smoking was statistically significant difference between cases and controls (P<0.001) . Conclusion: No statistical difference has been found between single SNP polymorphism and NIHL. At the level of greater than 92 dB of high noise exposure, rs7785846 (CT+TT) genotype is a risk factor for occupational noise deafness, and its OR is 2.74 (95%CI: 1.09-6.89) compared with wild homozygous type (CC) . Conclusion. The rs7785846 (CT+TT) genotype carriers of PON2 gene are more susceptible to hearing impairment when exposed to high noise intensity.

Effects of the last glacial period on genetic diversity and genetic differentiation in Cryptomeria japonica in East Asia

To understand how historical and environmental factors have shaped genetic diversity and spatial genetic patterns of tree species, we characterized the genetic diversity and the genetic structure of a coniferous species, Cryptomeria japonica, which is distributed in Japan (two taxonomic groups) and some parts of southeastern China (C. japonica var. sinensis). We investigated population samples for three groups using high-throughput SNP genotyping, sequencing of multiple genes, and then estimated level of genetic diversity, the genetic differentiation, and the divergence time between the three groups. We also investigated the natural distribution during LGM using species distribution modeling. The genetic variation in the Chinese population, var. sinensis, was lower compared to Japanese populations but it harbored some unique genotypes. The species distribution modeling showed that the Chinese population has decreased since Last Glacial Maximum (LGM) because of lower precipitation than present time, but in Japan the favored environment have remained available for this species even in the LGM. Estimated divergence times of the three groups mostly correspond with the time since the Last Glacial. Our data supports the hypothesis that the glacial period, particularly limitations associated with precipitation, strongly influenced the distribution of C. japonica populations. Our findings suggest that climate change plays an important role in speciation processes of C. japonica.