Screening Of Low Seed-shattering Germplasm And Identification Of Candidate Genes For Seed Shattering In Elymus Nutans

Elymus nutans,is a perennial,cool-season,self-pollinating,and allohexaploid(StStYYHH(2n=6x =42))forage grass.Due its good forage yield and quality as well as excellent cold and drought tolerance,it has been widely used as forage crops in cultivated pastures and natural grassland.Despite economic importance,it is difficult to grow for commercial seed production due to serious seed shattering,which negatively affect the breeding and utilization of new varieties E nutans.Based on the collection and evaluation of germplasm resources,we analyzed histological and molecular mechanisms of seed shattering in E.nutans using abscission zone and nonabscission zone tissues,and developed and identified candidate gene-based EST-SSR markers for seed shattering evaluation.This study will provide a foundation to better understanding of seed shattering.The present study obtained the following results:1.A total of 127 individual plants of 38 accession were used,and 21 agronomic traits including leaf,stem and seed shattering were evaluated for two consecutive years.The results showed that all tested traits have different variations,among which the coefficient of variation(CV)of the breaking tensile strength(BTS)was larger(2017,CV=37.66%;2018,CV=40.03%).Moreover,13 accessions were chosen for seed shattering measurement.The corrclation between seed shattering and morphological traits were analyzed.The results showed that the PI619516 had the lowest degree of seed shattering(BTS=70.05 gf)among the 13 accessions,while the seed shattering degree of PI639855(BTS=13.99 gf)was the highest.Correlative analysis revealed BTS and spike length,stem diameter were significantly negative correlated(P < 0.05).2.The abscission layer(AL)was found in E.nutans by histological analysis of abscission zone.Abscission zone(AZ)and non-abscission zone(NAZ)tissues of E.nutans were selected for RNA sequencing.A total of 111,667 Unigenes were annotated and 7,644 differentially expressed transcripts(DETs)were predicted.We identified 489 candidate genes related to transcription factor,cell wall hydrolysis or modification,hydrolase activity,phytohormone signaling and response,lignin biosynthesis,and signal transduction or protein turnover.3.Based on RNA-sequence data,a total of 52 polymorphic EST-SSR markers were developed for genetic diversity analysis of 127 individuals of 38 accessions.The polymorphism information content(PIC)each primer varied from was 0.114 to 0.331,with an average of 0.199.Cluster analysis showed that 127 individuals were clustered into two groups.In addition,14 candidate genes-based molecular markers were developed and used for the genetic diversity analysis of 48 individuals of 12 accessions with different seed shattering habits.The cluster analysis showed that most accessions with similar seed shattering degree tended to group together,indicating that these genespecific marker have potential application in the molecular marker-assisted selection of seed shattering in the future.