Identification Of Loci Regulating Rice Protein Components And Excellent Glutelin Haplotypes

Protein is one of the most important nutrition in rice.Its content is inferior to starch in rice and ranking in the second place,so rice is regarded as the main resource to absorb the protein.With the improvement of people's living standards,food therapy has become a life style that people choose to live.People who having kidney disease and diabete eating rice with high protein content will pose a heavier burden on themselves.However,the healthy people can eat the rice with high protein content to increase protein absorbance.Therefore,to study the molecular mechanism which controlling the protein content in rice,to identify relative molecular sites which regulating rice protein content and relative carrier materials,to improve rice protein content using breeding methods subsequencely,to cultivate the rice variety that satisfying different requests of different people,has become a meaningful target in rice breeding.Previous studies on rice protein mainly focused on QTL mapping of brown rice protein and polished rice protein using linkage analysis.Little research has focused on various protein components in rice.Association analysis is one of the main methods that using to analysis the genetic basis of intricate quantitative traits.It can be divided into genome-wide association study(GWAS)and candidate-gene association analysis.Therefore,this study used a natural population constructed by 329 rice tested accessions with wide geographic resources and 154 pairs of SSR marker that covering the whole rice genome evenly to analyze four protein components in rice using genome-wide association analysis.Furthermore,to identify excellent alleles and carrier materials related to various protein phenotypic traits,this is to provide theoretical basis in rice protein improvement.Subsequently,using the natural population constructed by the 128 accessions selected from the former tested population to do the candidate-gene association analysis.The sequences of GluA and GluB1 genes were detected.To identify the SNP sites,InDel sites and haplotypes associated with rice glutelin content at a significant level to provide theoretical references of molecular breeding in rice glutelin content.The main results of the research are as follows:(1)Coomassie Brilliant Blue G-250 method was used to detect four protein components among 329 rice materials in the tested natural population.Obvious variations exist in the tested population and the range of variation is wide.Various traits of tested population conform to the normal distribution and show typical genetic characteristics of quantative traits.(2)154 pairs of SSR loci that covering 12 chromosomes evenly were used to do genetic diversity analysis,kinship analysis,population structure analysis and linkage disequilibrium analysis in tested population.A total of 845 alleles ranging from 2 to 9 were amplified in 329 tested materials and the average of alleles was 5.49;Kinship analysis showed that 58.6% accessions had zero estimated kinship values,while 90.6% kinship estimates ranged from 0 to 0.1.It indicated that most accessions in tested population had no or weak kinship;The results of linkage disequilibrium showed that 41.35% marker pairs had significant LD(P<0.01).The LD level of each subpopulation was higher than that of the whole population;The population structure analysis showed that all the accessions could be divided into three subpopulations and one MIX subpopulation.The number of each subpopulation was 29,81 and 147,respectively.(3)The genome-wide association analysis between four protein components in rice and 154 pairs of SSR loci was performed using both general linear model(GLM)and mixed linear model(MLM)in TASSEL3.0 in two consecutive years.The results showed that a total of 15 SSR loci were detected under both GLM and MLM.The number of SSR loci associated with albumin,globulin,glutelin and prolamine are 5,5,2 and 3,respectively.(4)The excellent alleles of four protein components were identified.The excellent allele related to albumin,globulin and prolamine were RM233,RM253 and RM1284,respectively.The excellent allele related to high glutelin content was RM415.The excellent allele related to low glutelin content was RM241.(5)The sequences of rice glutelin synthesis genes GluA and GluB1 were both detected to obtain the sequences.In GluA,128 nucleotide polymorphic sites were detected in the whole gene region.To exclude the polymorphic sites with the frequency less than 5%,a total of 46 SNPs and 8 InDels were detected in the coding and non-coding regions;In GluB1,269 nucleotide polymorphic sites were detected in the whole gene region.To exclude the polymorphic sites with the frequency less than 5%,a total of 10 nucleotide variations were detected in the coding and non-coding regions with 6 SNPs and 4 InDels.(6)The software TASSEL5.0 was used to do the neutral selection analysis and linkage disequilibrium analysis of two candidate genes.In GluA,all the regions deviated from neutral evolution model significantly except the first exon,the second exon and the first intron;In GluB1,all the regions deviated from neutral evolution model significantly except the second intron.The linkage disequilibrium was analyzed for two genes,respectively.In GluA,almost all the polymorphic sites were in linkage disequilibrium(including the sites with the frequency more than 5%)with a significant level;In GluB1,no significant linkage disequilibrium was observed in the detected 10 SNPs and InDels(6 SNPs and 4 InDels).(7)The population structure of tested accessions was analyzed using 154 pairs of SSR loci.The results showed that all the accessions could be divided into three subpopulations.The number of each subpopulation was 35,11 and 57.One MIX subpopulation separated alone.This situation of accession division was consistent with the previous accession division basically.(8)The haplotype analysis was done for both two genes.In GluA,all the accessions were divided into 37 haplotypes using the polymorphic sites with the frequency more than 5%.The haplotype diversity(Hd)was 0.461.The unbalanced distribution was observed in the allocation of accessions in different haplotypes.GluAHap-1(The first haplotype of GluA)contained 73 tested accessions;In GluB1,all the accessions could be divided into 21 haplotypes using the detected polymorphic sites and the haplotype diversity was 0.341.The unbalanced distribution of accessions in haplotypes was also observed in GluB1.GluB1Hap-1(The first haplotype of GluB1)contained 84 tested accessions.The haplotype diversity of both genes was relative low.(9)The protein haplotype was analyzed for both two genes.In GluA,most SNP sites located in the first exon.Finally,all the accessions were divided into 27 protein haplotypes using the 43 SNP sites;In GluB1,only one SNP site located in the exon region,so all the accessions could be divided into two protein haplotypes.The unbalanced accession distribution of protein haplotypes was observed in both genes.CDS_GluAHap-1(The first protein haplotype of GluA)contained 89 tested accessions and CDS_GluB1Hap-1(The first protein haplotype of GluB1)contained 121 tested accessions.(10)The candidate-gene association mapping was analyzed using both general linear model(GLM)and mixed linear model(MLM)in software TASSEL5.0.In GluA,five SNP sites were detected to be associated with rice glutelin content at a significant level in both two models.The positions of these associated sites were 10 bp,22 bp,55 bp,78 bp and 87 bp of the GluA sequence;In GluB1,two polymorphic sites(one SNP and one InDel)were detected to be associated with rice glutelin content at a significant level in both two models.The positions of two sites were 1426 bp and 1489 bp of the GluB1 sequence.In GluA,GluAHap-10 and GluAHap-34 were identified to be the low glutelin content haplotypes;In GluB1,GluB1Hap-5 and GluB1Hap-12 were identified to be the low glutelin content haplotypes.(11)The detected sites were verified using high-resolution melting curve(HRM).The results showed that the melting curve image was consistent with the detected polymorphic sites.The results of polymorphic sites were proved to be accurate.