Genome-wide Association Study For Pre-harvest Sprouting Resistance In A Large Germplasm Collection Of Chinese Wheat Landraces

Wheat(Triticum aestivum L.)is one of the major cereal crops belonging to the genus Triticum L.of Gramineae family.Wheat can adapt to a wide range of climate while maintaining stable grain yield.Wheat grain is rich in starch,protein and flour from wheat can be used to make bread,noodles,biscuits,buns,etc.In addition,beer and liquor can be made from fermented grains.Wheat therefore has great nutritional and commercial value.Chinese wheat landrace is an important source of germplasm for breeding due to high variations,adaption,affinity,and resistance to disease,pest,and pre-harvest sprouting(PHS).With the high speed development of productivity,genetic diversity of many cultivars,recent wheat breeding studies are more focused on wheat landrace.Pre-harvest sprouting(PHS)is generally known as the germination of cereals before harvest.PHS in wheat is an important factor for yield loss and quality and yield loss of production due to the physiological and biochemical changes that occur in wheat grain.In this study,717 Chinese wheat landraces were evaluated for PHS and accessions with high PHS resistance were selected.Landraces were grown across six environments in China.Germination test of harvest-ripe grain was used to calculate a germination rate(GR)for each accession at each site.The 717 wheat landraces were genotyped using the DArT-seq(Diversity Arrays Technology,Canberra,Australia)genotyping-by-sequencing(GBS)platform.Out of the 717 landraces 272 that represent genetic diversity,were selected and genotyped using the Axiom(?)Wheat660 SNP array(Affymetrix,Santa Clara,CA,USA).The genomic diversity,population structure,and linkage disequilibrium(LD)were analyzed by two high resolution chips.Genome wide association studies(GWAS)for PHS was also carried out.Moreover,in order to test the relationship between the PHS resistance lociand other agronomic traits,five traits in 4 different sites were analyzed.The result is as follows:1.A collection of 717 wheat landraces from 10 major wheat-growing zones in China were phenotyped for PHS over four years(2012-2015)at three locations.Landraces were grown across six environments in China and germination testing of harvest-ripe grain was used to calculate a germination rate(GR)for each accession at each site.Germination rate(GR)highly correlated with PHS(r = 0.542-0.805)across all environments and showed mean value at 38.927%.Notably,white-grained accessions displayed a significantly higher GR(mean range 47.2%-79.6%)compared to red-grained accessions(mean range 19.1%-56.0%)across six environments.Landraces from mesic growing zones in southern China(zones?-YTS,IV-SAS,V-SWAS)showed lower levels of germination rate than those sourced from xeric areas in northern and north-western China.Zone IX-Q&T showed the highest germination rate.Environmental factors(i.e.altitude,frost-free day,annual sunlight,accumulated temperature and precipitation amount)were also found to have a high correlation with germination rate.A large number of landrace(194)displayed high levels of grain dormancy(i.e.mean GR<20%),these included nine white-grained accessions,which could be very helpful for genetic analysis and modern breeding of PHS resistance in wheat.2.A total of 27,933 DArT-seq and 312,831 Wheat660 SNP markers weremapped on Chinese survey sequence.The genetic diversity showed large differences among homologous chromosomes in Chinese wheat landraces,with fewer regions on the D genome than on A and B.Differences in genetic diversity among the three genomes was observed;the B genome showed the highest diversity followed by A genome while the D genome showed the lowest.There was wide variation in diversity among wheat landraces from different growing zones;landraces from zone IV-SAS showed the lowest diversity,followed by zone III-YTS while zone V-SWAS showed the highest diversity.The ecogeographical factors(altitude,annual sunlight,frost-free days,temperature,and precipitation)explain a significant proportion of the diversity;Structure and the NJ trees separated all the landraces into five groups 4 subgroups(GP1-GP4)and a MIX group,which positively correlated with geographic eco-type.GP1 mainly presented landraces from zone I-NW and zone II-Y&H.GP2 was mainly made up of landraces zone III-YTS zone IV-SAS.GP3 was mainlyrepresented by landraces from zone V-SWAS.GP4 mainly presented landraces from zone VII-NSzone VIII-NWS and zone IX-Q&T.LD analysis revealed that there was a short LD decay distance(approximately 7.42Mb or 4.73cM)in Chinese wheat landrace,which is similar to that in U.S.winter wheat.These results are expected to provide valuable information for future genome wide association study(GWAS)in Chinesewheat landraces.3.Genome-wide association studies(GWAS)using 9,740 DArT-seq and 178,803 SNP markers detected three main quantitative trait loci(QTL):QTL1 positioned at 173.81Mb on Chromosome 3A,which explained 11.49%to 25.12%of the phenotypic variation;QTL2 positioned at 110.99-113.89Mb on Chromosome 3D,which explained 4.35%to 19.28%of the phenotypic variation;QTL3 positioned at 39.36Mb on Chromosome 5D,which explained 11.47%to 12.03%of the phenotypic variation.QTL3 appeared to be novel while QTL1 and QTL2 is known to be associated with the grain color transcription factor Tamyb10.Further investigation of the origin of haplotypes associated with the three main QTL revealed that favorable haplotypes(QTL1-HAP-A,QTL2-HAP-CACTT and QTL3-HAP-A)for grain dormancy were more common in accessions from higher rainfall zones in China.Thus,a combination of natural and artificial selection likely resulted in landracePHS resistance in China.Another GWAS was carried out in 77 white-grained and 186 red-grained wheat landraces separately,and no grain color related QTL(GCR-QTL)was detected in white-grained landraces.While an additional 32 GCR-QTLs including 12 novel QTLs were detected in red-grained landraces.GCR-QTL occurred at high frequencies in the red-grained accessions and a strong correlation was observed between the number of GCR-QTL and GR(R2 = 0.62).This present a crucial information for maintaining high levels of grain dormancy and present targets for introgression into white-grained wheat cultivars.4.To understand the relationship between PHS resistance and other agronomic traits,five agronomic traits were evaluated in 717 wheat landraces at four environments(2012Ya'an,2013Wenjiang,2014Wenjiang and 2015Wenjiang).Low correlation values were detected between germination rate and the plant height,heading date,flowering time,the number of grains per spike and thousand kernel weight(r =-0.267-0.315).In a 2Mb genomic region of 3 PHS related QTLs,only QTLs associated with heading date(HD)and plant height(PH)were identified.Six GCR-QTLs were identified next to these HD and PH related QTLs.GCR-QTL2,GCR-QTL17,GCR-QTL31 and GCR-QTL32 were closer to the QTLs related to PH.GCR-QTL3,GCR-QTL17,GCR-QTL19,GCR-QTL31 and GCR-QTL32 were nearer to the QTL related to HD.Analysis of combination between favor haplotype of PHS resistance and various haplotpyes for PH and HD was carried out.Six correlated positively with PH and HD related QTL.Favor haplotypes for GCR-QTL3,GCR-QTL19,and GCR-QTL32 are more likely to be associated with haplotpyes for late heading date while those for GCR-QTL17 and GCR-QTL31 are more likely to be association with haplotypes for early heading date.The selection of favor haplotype for CR-QTL2,GCR-QTL17 and GCR-QTL31 could be linked to selection of haplotpyes for tall plant,while for GCR-QTL32 is more likely as a result of short plant selection.There were not many overlaps of QTL region between PH,HD,and PHS resistance.QTLs associated to PHS resistance could be selected independently in breeding programs.