Evaluation And QTL-allele Detection Conferring Seed-flooding Tolerance Using A Half-sib Recombinant Inbred Lines Population Of Soybean[Glycine Max(L.)Merr.]

Soybean[Glycine max(L.)Merr.]is an important legume crop worldwide for providing food for human and feed for livestock.It is grown on 110 million hectares worldwide.Soybean consumption is increasing day by day because of its edible seed protein and oil content.However soybean is facing flooding as one of the major constraints.The waterlogging stress during the vegetative stage of growth causes a reduction in the grain yield by approximately 17-40%and 40-57%during the reproductive stage.The flooding damage to crop depends upon stress duration and temperature,resulting in the replanting of the crop,depending upon the severity of the stress.Three major soybean producing regions in China are Northeast China,Huang Huai Valleys and Southern China.In Northeast China unusual heavy rain and flooding had negative impact on China's total soybean crop production.The impact of flooding on soybean crop is still undetermined,but preliminary estimates by Chinese sources elaborated that flooding has affected more than 2 million hectares of cropland in the region.The flooding has been an important problem to soybean at various vegetative and reproductive stages in China.Recent changes in the climate resulted in the shift of the flooding stress to the very early stage of crop.It is necessary to explore the methods to screen for seed-flooding tolerant soybean germplasm and the genetic basis of seed-flooding tolerance for early stage management of the crop.Therefore,the objectives of the current study were proposed as 1)Development of favorable methodology and clear indicators for seed-flooding tolerance,2)Identification of the QTLs conferring seed-flooding tolerance 3)Establishment of the QTL-allele matrix and identification of optimal crosses among the three parents as well as QTL allele structure of half-sibs and 4)Prediction of the candidate genes and elaboration of their putative functions.The results in this study will be utilized in the seed-flooding breeding programs for production and deployment of the cultivars with strong tolerance to flooding stress.1.Establishment of testing procedures and indicators for seed-flooding tolerance in soybeanWe selected 11 soybean accessions from various resources to develop the new methodology and suitable indicators to evaluate seed-flooding tolerance.Our experimental results suggested that 48 hours seed-flooding duration is the best one among the four tested durations i.e.24h,48h,72h and 96h due to highest F-value to explore the variation among accessions with great efficiency and moderate seed-damage.Paper roll method performed better than Petri dish and towel media to explore the effects of seed-flooding on the extensive range of traits including previously reported germination rate.The relative germination rate(RGR),seedling length(RSL),root length(RRL),root fresh weight(RRFW),root dry weight(RRDW),shoot length(RShL),shoot fresh weight(RShFW)and shoot dry weight(RShDW)were evaluated for seed-flooding tolerance.RGR,RSL,RRL,RRFW and RRDW were found to be flooding responsive indicators(due to highest heritability,GCV and correlation among them),while RShL,RShFW and RShDW were regarded as flooding non-response indicators(low GCV,h2 and narrow variation among accessions for the traits).Among the flooding responsive indicators,RSL was chosen as the major indicator due to its higher values of heritability(h2),genotypic coefficient of variation(GCV),correlation with other indicators and easier measuring procedure while RRL was recommended as the subsidiary indicator to seed flooding tolerance due to having highest correlation with RSL.Superior seed flooding-tolerant cultivars identified were M8206,NN1138-2 and ZXD.2.QTL mapping and establishment of QTL-allele matrices conferring RSL and RRL indicators of seed-flooding toleranceA soybean half-sib recombinant inbred lines population(LZM)was used to map the quantitative trait loci(QTL)system of the relative seedling length(RSL)and relative root length(RRL)as indicators for seed-flooding tolerance using an innovative restricted two stage multi-locus genome-wide association study procedure(RTM-GWAS).The LZM population of 230 lines was evaluated for flooding tolerance under two environments,and sequenced using RAD-seq to obtain 55,936 single nucleotide polymorphisms(SNPs)and organized into 6,137 SNPLDBs for GWAS.The detected 33 QTLs with 78 alleles on 17 chromosomes explained 51.87%of the total phenotypic variation of RSL with 42 positive and 36 negative allele effects.Fifteen QTLs on the 12 chromosomes with 37 alleles were detected for relative root length(RRL)comprising 19 positive and 18 negative alleles were organized into QTL allele matrix.Total contribution on the 15 loci was 41.79%.There is one QTL overlapped between RSL and RRL.All the lines comprised both positive and negative alleles employing a great potential for recombination within the population for both the RSL and RRL indicators.Furthermore,it was observed that top ten tolerant lines evaluated by RSL were possessing 238 positive alleles while in the top ten sensitive lines had only 126.The RRL evaluated material possessed 100 positive alleles for top ten tolerant lines while on 47 positive alleles were observed in the top ten sensitive lines.Analysis of the molecular variance(AMOVA)at the whole genome level and for the seed-flooding loci shows that there exists significant difference.It shows that the difference between the two populations exists for seed-flooding between the two sub-populations as well.3.Prediction of optimal recombinants among the three parents and exploration of candidate gene systemThe optimal crosses were predicted from matrices showing transgressive potential in LZM population.Recombination potential of parents was optimized by RTM-GWAS and a cross between the Meng8206 and Linhe is concluded to be the best one to get the highest allele effect for RSL while the RRL indicator is observed to get highest improvement in the parental cross of the Meng8206 and Zhengyang.LZM QTL-allele structure depicts that each allele at a locus is either contributed by the single parent or a combination of the parents.The parental lines were found to be having both the positive and negative allele effects on the detected loci suggesting that the potential of optimal crosses exists among the parents to collect the favorable alleles in the progeny.So the materials observed can be the best innovative source in the development of the tolerance resource for seed-flooding.In optimal cross design,population genetics and genomic study have provided a solution to match the breeding by design strategy at both QTL and gene levels in breeding programs.A number of the genes were evaluated in the regions detected in the LZM half-sib RILs.This number of candidate genes was reduced to the 34 and 31 for RSL and RRL indicators by the Chi-square test in the LZM half-sib recombinant inbred lines.From the QTL system 34 candidate genes of the LZM half-sib RILs related to various biological processes were analyzed by ?2 test for seed-flooding candidate gene system.The candidate genes were further annotated with the gene ontology categories of biological process,cellular component and molecular function indicating that diverse genes are involved in seed-flooding tolerance.