QTL Analysis Of Flowering-times And Candidate Genes Association Analysis In Orchardgrass

Orchardgrass(Dactylis glomerata L.)is one of the top four most economically important perennial forage grasses and is native to northern Africa,Europe,and temperate Asia.Early-flowering cultivars tend to have higher seed yield,while late-flowering species are desirable in pastures..In this study,we constructed a high-density genetic map by combining SLAF(specific-locus amplified fragment sequencing)and SSR(Simple sequence repeat)markers,and we identified QTLs associated with heading date(HD)and FT using these markers.According to the annotation from the QTLs,we selected four candidate genes associated with heading and flowering date to sequence all 150 samples from an association population,and made the association analysis between heading date and four candidate genes,and the results showed polymorphisms in the central circadian gene.The result is,to our knowledge,the densest genetic map in orchardgrass.In addition,our analysis was based on field evaluations over two years and at two different locations.This map may serve as a platform for future identification and genetic dissection of many other complex and important traits.Our main results following:1.Comparative genomics analysis by specific-locus amplified fragment sequencing(SLAF-seq).In this study,we developed 447,177 markers based on SLAF-seq and used them for a comparative genomics analysis.Perennial ryegrass sequences were the most similar(5.02%)to orchardgrass sequences,followed by barley(4.30%),B.distachyon(3.24%),and rice(1.21%).It showed that orchardgrass is more close relationship with perennial ryegrass and more further with rice.2.Construction of high-density genetic linkage map by SLAF-seq.Based on the allies numbers and the differences between each sequence,we had three types of SLAF markers,Polymorphic,Non-Polymorphic,Repetitive.And during the 447117 SLAF markers,there are 19.91%polymorphic markers,78.13%non-polymorphic markers and 1.96%repetitive markers.We filtered the markers and had 2467 markers used for mapping.A high-density linkage map was constructed using 2,467 SLAF markers and 43 SSRs,which were distributed on 7 linkage groups spanning 715.77 cM.The average distance between adjacent markers was 0.37 cM.3.QTL anaylsis for flowering time.Based on phenotyping in four environments,11potentially significant(QTLs)for two target traits–heading date and flowering time–were identified and positioned on the linkage groups LG1,LG3,and LG5.All significant QTLs were able to explain 8.20%–27.00%of the total phenotypic variation,with the LOD ranging from 3.85–12.21.Marker167780 and Marker139469 were associated with phenotyping at the same location(Ya'an)over two years.The utility of SLAF markers for rapid generation of genetic maps and QTL analysis has been demonstrated for heading date and flowering time in a global forage grass.4.Candidate genes association analysis for flowering time.Association between heading date and polymorphisms in the CONSTANS(DgCO1),FLOWERING TIME(DgFT1),a VRN1 like MADS-box(DgMADS),and PHOTOPERIOD(DgPPD1-like)containing genes.A diverse population of 150 genotypes was measured for heading date across three years,genotyped,and candidate genes sequenced.Although pairwise population kinship values were generally low,the genotypes fit into a two-group structure model.Linkage disequilibrium decayed rapidly,reaching r~2 levels below 0.2 within the500bp of each gene.SNPs significantly associated with heading date were detected in equal-dose and tetraploid dosage models.The DgCO1 gene had the most significant polymorphisms and those with the largest effects.These polymorphisms can be used for further selection,and development of breeding lines of orchardgrass.