| Elymus sibiricus is a perennial,high quality grass of the genus Elymus?Poaceae?,which has excellent characteristics such as high grass yield,good grass quality,and strong cold and drought tolerance.It is widely used in grassland grazing,artificial grassland planting,and ecological restoration.However,seed shattering of E.sibiricus greatly reduced the seed production,which adversely affected the breeding and utilization of new varieties.The research on genetic basis of seed shattering is an important field that is rarely studied at home and aborad,but urgently needs to be strengthened in forage grass.Based on the development of specific EST-SSR markers and the genetic diversity evaluation of E.sibiricus accessions with different seed shattering,we constructed a genetic mapping population in the study.SLAF-seq was used to construct the first genetic linkage map of E.sibiricus,and the candidate genes of seed shattering were identified by combining QTL and GWAS.This provides a theoretical basis for analyzing the genetic mechanism of seed shattering,accelerating molecular genetic improvement,and breeding noval materials with low seed shattering in E.sibiricus.The main results were as follows:1.We have developed specific EST-SSR molecular markers for E.sibiricus and studied their transferability to other Elymus species.A total of 8,871 putative EST-SSRs were identified from 6,685 unigenes,and 200 primer pairs were developed.Sequencing of PCR amplification products showed that the sequenced alleles from different Elymus species were homologous to the original designed SSR locis.There were 43.5%of the primers showed good transferability across different Elymus species.Thirty polymorphic primer pairs were used for genetic diversity and genetic relationship analysis among 480 individuals of 95 accessions in 17 Elymus species.The cluster analysis showed that all the studied accessions were grouped into three major clusters based on their genomic composition and geographical origin.This study provides marker resources for evaluating the genetic diversity of Elymus species.2.Fourty EST-SSRs were used to evaluate the genetic diversity of 36 E.sibiricus accessions,and the seed shattering was measured in the field as well.The results showed that the molecular genetic diversity and seed shattering variation were rich.Two genetically distant genotypes among Y1005?high seed shattering?and ZhN06?low seed shattering?were used to generate seven F1 hybrids.The molecular and morphological diversity analysis of seven F1 individuals showed a higher-parent heterosis in some traits,including seed shattering,leaf size,culm number,spike length,and awn length.Genetic diversity analysis showed that the female parent?ZhN06?shared more bands with the F1 lines than male parent?Y1005?,and the heritability is higher.After the identification of hybrids and the selection of low seed shattering individuals,the F2 population,containing 200 individuals,was constructed by self-pollinating the F1-7 individual.3.SLAF sequencing was used to F2 population.A high-density genetic linkage map of E.sibiricus was first constructed,including 1,971 markers on the 14 linkage groups?LGs?and was 1,866.35 cM in total.The length of each linkage group varied from 87.67 cM?LG7?to 183.45 cM?LG1?,with an average distance of 1.66 cM between adjacent markers.The marker sequences of E.sibiricus were compared to two grass genomes and showed 1,556?79%?markers mapped to wheat,1,380?70%?to barley.Phenotypic data of seed-related traits?2016–2018?were used for QTL identification,including spike length,floret number per spike,seed shattering,awn length,width of seed,and 1000 seed weight.A total of 29 QTLs were identified in eight seed-related traits among 14 linkage groups.Six seed shattering QTLs were associated in 2,3,6,and 11 linkage groups.Based on annotation with wheat and barley genome,we identified 30 candidate genes for seed shattering,of which 15,7,6 and 2 genes were involved in plant hormone signal transcription,transcription factor,hydrolase activity and lignin biosynthetic pathway,respectively.4.By using SLAF-seq and fifteen phenotypic traits measurement,213 E.sibiricus accessions were used for genome-wide association analysis?GWAS?.The results of SNP linkage disequilibrium?LD?analysis showed that the LD decayed fast in E.sibiricus,with the distance of 0.291kb.The kinship of all individuals was weak and it was suitable for GWAS.There was high level of genetic variation among measured traits in two consecutive years,with an average coefficient of variation of 30.49%.The heritability of seed shattering was 85.13%.A total of 41 significant loci were detected in seed shattering,and this explained the average phenotypic variation of 26.3%.Fourteen candidate genes were annotated for seed shattering,which were mainly related to the regulation process of galacturonidase,hydrolase,and cytokinin glucosyltransferase.In addition,66 and 1,715 significant loci were detected in seed and yield-related traits,respectively.Further,we found 31 and 110 candidate genes,respectively. |
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