Fine Mapping Of QTLs And Function Validation Of A Candidate Gene,Glyma.19g192300,a Major QTL For Plant Height In Soybean

Soybean[Glycine max?L.?Merr]contain complete protein and oil,providing all the essential amino acids necessary for the human diet.Hence,a great effort has been made to increase soybean yield,while maintaining a high level of quality characteristics.Recently,with the rapid development of high-throughput sequencing technology,single nucleotide polymorphism?SNP?markers have emerged as new molecular markers of choice because of their high-density and relatively even distribution across plant genomes.Further,they have resolved many of the problems associated with the efficiency and accuracy of QTL mapping.Restriction-site associated DNA sequencing?RAD-seq?,was one of the next generation sequencing?NGS?methods,has been effectively applied in high-throughput SNP marker discovery and quantitative trait loci?QTL?analysis including the mapping of quality and agronomic trait loci in soybean.In this study,we genotyped a recombinant inbred line?RIL?population?Zhonghuang 24×Huaxia 3?using a restriction-site associated DNA sequencing?RAD-seq?approach,some novel QTLs and important QTL clusters for both yield-related and quality traits were identified based on a new,high-density bin linkage map.A gene was selected as candidates that likely have a direct influence on plant height in soybean.All the results were shown as follows:1.Sequencing of Zhonghuang 24 and Huaxia 3 was conducted to map resistant genes.The total reads were 96.57 M and 90.04 M,total base were 8.69 G and 8.10 G,mapped based were 7.94 G and 7.30 G,respectively.The coverage of sequencing were 96.33%and94.92%,average depth were 8.19 X and 7.51 X.Based on 0.24x RAD-seq?restriction-site associated DNA sequencing?of the Zonghuang 24 and Huaxia 3 RIL population,57.40 G sequence reads were obtained and the average read number was 311.97 M.Half of them have more than 200 M reads.According to this data,a total of 47,472 were integrated into a recombination bin unit,and 2639 recombinant bins were obtained.Based on the genotypes of 2639 bins,a high-density bin linkage map was constructed covering 2638.24 cM,with an average distance of 1.00 cM between adjacent markers.For each chromosome,the average genetic distance between adjacent bins ranged from 0.67 to 1.51 cM.2.Based on the genotypic and phenotypic data from the two parents and their RILs population,a major QTL for SMV strain SC15 was detected on 27801314-27864010 bp of chromosome 13.We further analyze this loci by qRT-PCR,and four candidate resistance geneswerefoundwhichwerepossiblyinvolvedinregulating SMV-resistance.Glyma13g24461 showed highest levels of up-regulation at 1 h and then significantly down-regulated at 4 h in Zhonghuang24.However,the relative expression levels in Huaxia3 were approximately suppressed at all the time points.Thus,this gene is most likely to be involved in response to SC15 infection.3.Most of the traits of Huaxia 3 showed higher values compared with those of Zhonghuang 24,providing ideal material for population construction and QTL analysis.Phenotypic values were found to be continuous with normal or skew normal distributions.Transgressive segregation in the RILs was shown for yield-related traits,suggesting that alleles with positive effects on the measured traits are distributed among the parents.The correlation analysis showed that most of the yield-related traits were correlated with each other in both years.PH was positively correlated with NN,BN,EP,IP and SW,except for EP and IP in 2012 and SW in 2015 where it was not significant.NN also showed significant positive correlations with BN and EP in both years,but no correlation was detected with SW.Significant negative correlations were found for SW with BN and EP,ranging from r=-0.215^**to r=-0.327^**in both years.4.Forty-seven QTLs associated with yield-related traits including PH,NN,BN,EP,IP and SW,were cluster on four chromosomes?Chr04,Chr06,Chr11,Chr19?.A single QTL explained 3.78%-32.56%of phenotypic variance.Among the QTLs,28 were identified on ten chromosomes in 2012.Nineteen QTLs on nine chromosomes were detected in 2015,and qPH19b-2 has the most significant LOD score?10.34?,explaining 24.49%of phenotypic variation and showed negative additive alleles from the male parent Huaxia 3.Of these QTLs,24 were in agreement with earlier reports and 23 QTLs were found to be novel.A total of 18 QTLs were stable across both years.Thirty-four of these QTLs had a positive additive effect,which were contributed from the female parent Zhonghuang 24,whereas thirteen QTLs had a negative effect,with additive alleles from the male parent Huaxia 3.5.The data fom SPSS 17.0 showed that there were more differences in protein and oil content traits between Zhonghuang 24 and Huaxia 3.A highly significant negative correlation(r=-0.775^**,r=-0.761^**)was observed between Pro and oil in both years.Phenotypic values were found to be continuous with normal distributions.Transgressive segregation in the RILs was shown for quality-related traits.6.A total of 13 QTLs were associated with quality traits on ten different chromosomes in both growing seasons.Three QTLs for protein content were identified on Chr07,Chr10and Chr13 in 2012,respectively.Five QTLs for oil content were identified on Chr01,Chr06,Chr10,Chr11 and Chr20.Four QTLs showed positive additive effects ranging from 0.27to0.42,while the other four QTLs showed negative additive effects that were from-0.27 to-0.47.Within these QTLs,three of them had positive additive effects,indicating that the female parent,Zhonghuang 24,contributed the trait for increased oil content.A total of ten QTLs were reported in prior studies,and three new QTLs were identified for the first time in the present study.7.According to mapping QTLs,we found that the QTLs associated with plant height were all detected in a physical position between 44931101 and 45081885 bp on Chr19,explaining 3.78%to 28.01%of phenotypic variation.A total of 9 genes were found within this region using Glyma.Wm82.a1.v1.1 gene model from soybase.According to gene functional annotation,we choose the candidate genes Glyma.19 g192300 for further analysis.This gene is a member of the Trihelix GT-2 subfamily gene.We isolated Glyma.19g192300 from the soybean cultivar Zhonghuang24 and Huaxia 3 which was 1737 bp in length.In soybean,the expression of Glyma.19 g192300 could be detected in all organs,especially in root,stem,flower and apical meristem.Over-expression of Glyma.19g192300in Arabidopsis resulted in increasing the height of plant and number of pod.