Development And Application Of TNGS Genotyping Platform Based On Faba Bean Transcriptome Data

Faba bean(Vicia faba L.,2n = 12)is an important food legume crop that is widely grown for multiple uses worldwide.However,the current progress in genomics and genetics research is relatively slow compared to other legumes due to its very large genome size(approximately 13 Gb)and limited single nucleotide polymorphism(SNP)markers as well as highly efficient genotyping tools have not been reported for faba bean.Based on the flowers and leaves transcriptome sequencing of 102 global faba bean accession resources,243,120 unigenes were de novo assembled and functionally annotated.A total of 1,579,411 SNPs were identified by bioinformatics and were further filtered according to a selection pipeline to develop a high-throughput,flexible and low-cost Faba＿bean＿130K Targeted Next-Generation Sequencing(TNGS)genotyping platform.A set of 69 Chinese faba bean accessions were genotyped with the TNGS genotyping platform,and the captured reads were mapped to the reference transcripts to calculate the targeted capture rate.The accuracy of TNGS genotyping results were validated by PCR amplification and Sanger sequencing.Comprehensive population genetic analysis was performed on the 69 Chinese faba bean accessions and identified four genetic subgroups correlated with geographic distribution.This study provide valuable genomic data and a reliable genotyping tool that could be implemented in genetic and molecular breeding studies to accelerate new cultivar development and genetic improvement in faba bean.Main results of this research are as follows:1.RNA-seq libraries of the flowers and leaves of 102 global faba bean accessions were constructed and sequenced by Illumina paired-end sequencing.A total of 2.1T clean bases were obtained.287,450 transcripts and 243,120 unigenes were obtained with length N50 of 889 bp and 954 bp,respectively.Comprehensive function annotations were performed to these unigenes with seven public databases including NT,NR,KO,Swiss Prot,PFAM,GO and KOG.A total of 149,990(61.69%)unigenes were annotated by at least one of the seven databases,and 15,025 unigenes can be matched with all the seven databases.The largest number of 103,479(42.56%)unigenes were annotated by NT database while the least number of 31,156(12.81%)unigene was annotated by KOG database.204 RNA-seq data were aligned to the assembled reference transcripts,and a total of 1,579,411 SNP markers were identified through bioinformatics method.2.High quality SNP markers were screened through strict filtering criteria,including single-copy gene filtering,individual deletion rate and genotype deletion rate filtering,minor allele frequency filtering and the combination of linkage markers.After filtering,a total of 130,514 SNP markers were obtained to design the Faba＿bean＿130K TNGS liquid-phase probes.In the validation of the Faba＿bean＿130K TNGS liquid-phase probes,69 faba bean accessions were genotyped.The average mapping rate of reads captured by Faba＿bean＿130K TNGS to the reference transcripts was 93.14%,of which 52.24% were located in the target regions.In addition,genotyping results could generate different SNPs datasets under different filtrations.In order to verify the accuracy of the target captured results of the Faba＿bean＿130K TNGS liquid-phase probes,15 unigene containing SNP were randomly selected and verified by PCR amplification and Sanger sequencing in 10 of the 69 faba bean accessions.Comparing the results of Sanger sequencing with the variant sites of the target captured results using the Faba＿bean＿130K TNGS liquid-phase probes,the average consistency was up to 93.6%.Therefore,it showed that the genotyping result of Faba＿bean＿130K TNGS was highly reliable.3.TNGS liquid-phase probes were applied to population genetic analysis of faba bean accessions.SNP markers were used to construct phylogenetic tree,principal component analysis and population structure analysis.The 69 faba bean accessions were obviously divided into four groups,with clear population boundaries and close relationship with geographical sources.Spring faba bean resources and winter faba bean resources in China were also separated from each other,indicating that the studied faba bean accessions had narrow ecological adaptability and strong regional ecological characteristics.