Development of a target capture sequencing SNP genotyping platform for genetic analysis and genomic breeding in rapeseed

Rapeseed(Brassica napus)is an oil crop grown worldwide,making it a key plant species in molecular breeding research.However,the complexity of its polyploid genome increases sequencing costs and reduces sequencing accuracy.Target capture coupled with high-throughput sequencing is an efficient approach for detecting genetic variation at genomic regions or loci of interest.In this study,588 resequenced accessions of rapeseed were used to develop a target capture sequencing SNP genotyping platform named BnaPan50T.The platform comprised 54,765,with 54,058 resequenced markers from the pan-genome,and 855 variant trait-associated markers for 12 agronomic traits.The capture quality of BnaPan50T was demonstrated well in 12 typical accessions.Compared with a conventional genotyping array,BnaPan50T has a high SNP density and a high proportion of SNPs in unique physical positions and in annotated functional genes,promising wide application.Target capture sequencing and wholegenome resequencing in 90 doubled-haploid lines yielded 60%specificity,78%uniformity within tenfold coverage range,and 93%genotyping accuracy for the platform.BnaPan50T was used to construct a genetic map for quantitative trait loci(QTL)mapping,identify 21 unique QTL,and predict several candidate genes for yield-related traits in multiple environments.A set of 132 core SNP loci was selected from BnaPan50T to construct DNA fingerprints and germplasm identification resources.This study provides genomics resources to support target capture sequencing,genetic analysis and genomic breeding of rapeseed.

Automated SNP Genotype Clustering Algorithm to Improve Data Completeness in High-Throughput SNP Genotyping Datasets from Custom Arrays

High-throughput SNP genotyping platforms use automated genotype calling algorithms to assign genotypes. While these algorithms work efficiently for individual platforms, they are not compatible with other platforms, and have individual biases that result in missed genotype calls. Here we present data on the use of a second complementary SNP genotype clustering algorithm. The algorithm was originally designed for individual fluorescent SNP genotyping assays, and has been optimized to permit the clustering of large datasets generated from custom-designed Affymetrix SNP panels. In an analysis of data from a 3K array genotyped on 1,560 samples, the additional analysis increased the overall number of genotypes by over 45,000, significantly improving the completeness of the experimental data. This analysis suggests that the use of multiple genotype calling algorithms may be advisable in high-throughput SNP genotyping experiments. The software is written in Perl and is available from the corresponding author.

Improved Allele-specific Polymerase Chain Reaction for Single Nucleotide Polymorphism Genotyping

An improved allele-specific PCR（AS-PCR） approach was applied to investigating -55C/T polymorphism in promoter region of the uncoupling protein 3（UCP3）gene. AS-PCR is a competitive PCR method which is based on positioning the 3＇ base of a PCR primer to match one single nucleotide polymorphism（SNP） allele and accurately extend only the correctly matched primer. But it is limited in use because of its poor specificity. In this study, we improved the specificity of AS-PCR by introducing additional mismatch at the penultimate base of 3＇ end of AS-PCR primer in combination with decreasing the level of dNTP in the reaction mixture. Sensitivity, specificity and reliability of this method were assessed for both simple plasmid model and complex human genomic SNP targets. The -55C/T（rs1800849） polymorphisrn of the UCP3 gene was analyzed via this AS-PCR and restriction fragment length polymorphism（RFLP）, the latter was used as a gold standard. The results suggest that the increase in AS-PCR discrimination with this method should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping application.

Fertility,genome stability,and homozygosity in a diverse set of resynthesized rapeseed lines

Rapeseed(Brassica napus,AACC)was formed by hybridization between progenitor species Brassica rapa(AA)and Brassica oleracea(CC).As a result of a limited number of hybridization events between specific progenitor genotypes and strong breeding selection for oil quality traits,rapeseed has limited genetic diversity.The production of resynthesized B.napus lines via interspecific hybridization of the diploid progenitor species B.rapa and B.oleracea is one possible way to increase genetic variation in rapeseed.However,most resynthesized lines produced so far have been reported to be meiotically unstable and infertile,in contrast to established B.napus cultivars.This hinders both maintenance and use of this germplasm in breeding programs.We characterized a large set of 140 resynthesized lines produced by crosses between B.rapa and B.oleracea,as well as between B.rapa and wild C genome species(B.incana,B.hilarionis,B.montana,B.Bourgeaui,B.villosa and B.cretica)for purity(homozygosity),fertility,and genome stability.Self-pollinated seed set,seeds per ten pods as well as percentage pollen viability were used to estimate fertility.SNP genotyping was performed using the Illumina Infinium Brassica 60K array for 116 genotypes,with at least three individuals per line.Most of the material which had been advanced through multiple generations was no longer pure,with heterozygosity detected corresponding to unknown parental contributions via outcrossing.Fertility and genome stability were both genotypedependent.Most lines had high numbers of copy number variants(CNVs),indicative of meiotic instability,and high numbers of CNVs were significantly associated with reduced fertility.Eight putatively stable resynthesized B.napus lines were observed.Further investigation of these lines may reveal the mechanisms underlying this effect.Our results suggest that selection of stable resynthesized lines for breeding purposes is possible.

CDH13 Genetic Polymorphisms, Adiponectin and Ischemic Stroke： a Chinese Family-based Sib-pair Study

Objective To understand the relationships between CDH13（T-cadherin） genetic polymorphisms, adiponectin levels and ischemic stroke, and possible interactions between CDH13 polymorphisms and other risk factors. Methods We recruited 342 Chinese ischemic stroke sib pairs. We genotyped rs4783244 and rs7193788 on CDH13 using time-of-flight mass spectrometry genotyping technology and measured total and high-molecular weight（HMW） adiponectin levels. We investigated associations between SNPs and ischemic stroke, and interactions between SNPs and other risk factors using multi-level mixed-effects regression model. Results In individuals without ischemic stroke, CDH13 rs4783244 was associated with total adiponectin levels（per T： Coef =-0.257, P = 0.001）. CDH13 rs7193788 was associated with total adiponectin levels（per A： Coef =-0.221, P = 0.001） and HMW adiponectin levels（per A： Coef =-0.163, P = 0.003）. rs7193788 was significantly associated with ischemic stroke（GA/AA vs. GG： OR = 1.55, 95% CI： 1.07 to 2.24, P = 0.020） after Bonferroni correction（α = 0.025）. There was an interaction between rs7193788 and diabetes（P = 0.036）. Compared to diabetes-free individuals with rs7193788 GG genotype, diabetes patients with rs7193788 GA/AA genotypes had higher risks for ischemic stroke（OR = 2.64, 95% CI： 1.58-4.40, P 〈 0.001）. Conclusion CDH13 genetic polymorphisms are associated with adiponectin levels and ischemic stroke. An interaction is found between CDH13 SNP and diabetes for ischemic stroke.

Genomic selection of orange-spotted grouper(Epinephelus coioides)based on multiplex PCR enrichment capture sequencing

Orange spotted grouper(Epinephelus coioides)is an important mariculture fish,and genomic breeding of this grouper species has been hindered due to lack of efficient genotyping tools.Here,we developed a single nucleotide polymorphism(SNP)genotyping technology based on multiplex PCR enrichment capture sequencing,which mainly aims at target area for high-throughput sequencing,and 741 SNPs were designed for genomic selection(GS)of growth and ammonia tolerance traits at the same time.The multiplex PCR enrichment capture sequencing assay showed that the genotyping efficiency was more than 99%in the orange-spotted grouper and the predictive accuracy of body weight and ammonia tolerance traits was 82%and 96%,respectively.More importantly,the average identity of the sequences with these SNPs aligned to the genomes of giant grouper(E.lanceolatus)and brown-marbled grouper(E.fuscoguttatus)were both over 96%.Test data showed that the SNP genotyping efficiency was more than 94%in both giant grouper and brown-marbled grouper.In summary,these results indicated that the development of SNP loci and genotyping approach based on the multiple PCR enrichment capture sequencing are suitable for GS of growth and ammonia tolerance traits in various grouper species,and it would provide technical support for practical grouper breeding.