Identification And Functional Analysis Of Candidate Genes For Seed Shattering In Elymus Sibiricus Based On Transcriptome Sequencing

Elymus sibiricus is a perennial forage grass belonging to the genus Elymus,it has great cold and drought resistance,high yield,good quality and strong adaptability to the environment.E.sibiricus is often used as excellent forage for ecological restoration and livestock industry in Northwest China,especially the Qinghai-Tibet Plateau.However,the severe seed shattering often leads to a great seed yield losses,which limits the development and promotion of E.sibiricus new cultivars.At present,only 8 bred E.sibiricus cultivars have been registered in China,and the breeding goals of these cultivars are mostly focused on yield,and there were no reports on the breeding of shattering resistant cultivars.In this study,based on the evaluation of genetic diversity in E.sibiricus,we studied the mechanism of E.sibiricus seed shattering from histology,physiology,transcriptomics,etc.,and screened a group of candidate genes related to grain shattering.Meanwhile,the gene function verification was carried out for the hub genes related to seed shattering.The main results were as follows:1.According to the previous studies,53 E.sibiricus germplasms with different seed shattering were screened.The genetic diversity and relationships of these germplasms were studied using SCoT markers.The results showed that Start Codon Targeted?SCoT?markers were used for studying the genetic diversity and relationships among 53 E.sibiricus accessions from its primary distribution area in China.A total of173 bands were generated from 16 SCoT primers,159 bands of which were polymorphic with the percentage of polymorphic bands of 91.91%.Based upon population structure analysis five groups were formed.The cluster analysis separated the accessions into two major clusters and three sub-clusters,similar to the results of principal coordinate analysis.The molecular variance analysisshowed that genetic variation was greater within geographical regions than between them.It is interesting that the E.sibiricus germplasm from the Qinghai-Tibet Plateau possessed the highest genetic diversity among all geographical populations,these materials are important for future breeding program.2.Based on previous evaluations of seed shattering and genetic diversity analysis,6 wild E.sibiricus accessions from the northeastern margin of the Qinghai-Tibet Plateau were selected,including 2 low seed shattering accessions and 4 high seed shattering accessions.Histological and physiological studies on the seed abscission zone of these materials were performed by scanning electron microscopy,paraffin sections and ELISA.The results showed that the abscission layer of seeds was present before seed shattering and it was composed of 2 to 3 layers of small volume cells with an oval shape and compact arrangement.Meanwhile,the degree of lignification of these cells was higher than the surrounding cells.The lignified cell number of low seed shattering accessions was also larger than high seed shattering materials;Low seed shattering accessions hadn't fracture abscission layer at seed maturity stage.These accessions had a rough fracture surface and integrated abscission layer structure.While high seed shattering materials occurred clear fracture of the abscission layer during seed maturation.These materials had smooth fracture surface and incomplete abscission layer structure;The changes of cellulose?CE?and polygalacturonase?PG?activities of six accessions were different at different seed developmental stages.The activities of the two cell wall hydrolases were both closely related to seed shattering;The CE and PG activity of six accessions at high seed shattering stage had same order,and this was similar with electron microscopy observation;High seed shattering material XH09possessed the highest enzyme activity(XH09_CE=479.52 IU·L^-1,XH09_PG=188.87pg·mL^-1)in the critical period of seed shattering,but low seed shattering material ZHN03 had the lowest enzyme activity.3.Transcriptome analysis of the seed abscission zone at three different developmental stages of two genotypes?XH09 and ZhN03?with different seed shattering degree was performed.cDNA libraries from abscission zone tissue at 7 days,21 days and 28 days after heading?DAH?were constructed and sequenced.A total of86,634 unigenes were annotated and 7,110 differentially expressed transcripts?DETs?were predicted from“XH09-7 vs ZhN03-7”,“XH09-21 vs ZhN03-21”and“XH09-28vs ZhN03-28”,corresponding to 2,058 up-regulated and 5,052 down-regulated unigenes.The expression profiles of 10 candidate transcripts involved in cell wall-degrading enzymes,lignin biosynthesis and phytohormone activity were validated using quantitative real-time PCR?qRT-PCR?,8 of which were up-regulated in low seed shattering genotype ZhN03,suggesting these genes may be associated with the reduction of seed shattering.In addition,we also performed the transcriptome analysis of seed abscission zone at four different developmental stages in high seed shattering genotype XH09.The abscission zone tissues at four different developmental stages of XH09 were used to construct cDNA libraries and sequenced.The results showed that:a total of 92,832 Unigenes were annotated and 29,089 differentially expressed genes were predicted from"DAH7 vs DAH14","DAH7 vs DAH21"and"DAH7 vs DAH28"gene sets.Multiple analysis methods have suggested that a large number of differentially expressed genes in"cell wall component metabolism","lignin component metabolism"and"plant hormone signaling pathway"may participate in the seed shattering process.The analysis of the weighted gene co-expression network screened out 38 hub genes encoding transcription factors,these genes included RAP2,eRF1,BEL1,HOX4/16,SRF,AGL14,AS1,WRKY11.We predicted that these genes may control seed shattering of E.sibiricus by regulating differentiation,formation,development of abscission zone,and cell lignification.4.The transgenic technology was used to overexpress the hub gene Es-BEL1related to seed shattering in Arabidopsis thaliana to verify the gene function.The results showed that:three overexpressed lines were screened in this study.The overexpressed lines showed stunted growth,dwarfed plants,shortened silique spacing,delayed flower organ shedding,and reduced pods shattering when compared with wild-type A.thaliana.Scanning electron microscopy showed that the transgenic plants had delayed cell development and reduced volume.The results of physiological detection showed that an increasing trend of auxin content and polygalacturonase activity in overexpressed lines pods,while the abscisic acid and lignin contents were decreased.The qPCR results showed that the genes involved in the abscission zone development of Arabidopsis fruit pods,lignification of abscission layer cells,and pod?flower?shedding were all down-regulated in overexpressed lines.These results indicated that the Es-BEL1 gene may be involved in the abscission zone development of E.sibiricus.The high expression of this gene can regulate and reduce seed shattering through multiple pathways.