Whole Genome Wide Analysis And Development Of Molecular Markers Based On LTR Retrotransposon In Cleistogenes Songorica

The innovation and utilization of germplasm resources and plant breeding are inseparable from the analysis of genetic diversity at the molecular level.As an extremely drought-resistant grass species,Cleistogenes songorica also has excellent characteristics such as high seed viability and good nutritional value,and can be used as purpose of ecological grass,forage grass and lawn grass.However,due to the lack of genomic data,the current research on this grass is concentrated on the morphological level,and the research on the molecular level is still lagging behind.Long terminal repeat retrotransposons(LTR-RTs)are major components of the plant genome and play significant roles in functional genome diversity and phenotypic variations.LTR-RTs are abundantly distributed in plant genomes,facilitating the development of markers based on LTR-RTs for germplasm identification,genetic diversity and phylogenetic analysis,and marker assisted breeding.The whole genome sequencing of C.songorica has provided the basis for the analysis and development of molecular markers for LTR-RTs.Based on whole-genome data,this study identified and analyzed LTR-RTs,screened potentially active full-length candidate LTR-RTs,and developed four types of molecular markers based on LTR-RTs.It is expected to provide the basis for the mining of LTR-RTs data and molecular-assisted breeding.The main findings are as follows:1.Whole genome analysis of LTR-RTs was performed in C.songorica.A total of 299,079 LTR-RTs was identified and classified as Gypsy type(199,460),Copia type(81,865),or others type(17,754).LTR-RTs were widely distributed in the genome,enriched in the centromere region of the chromosome,and negatively correlated with gene distribution.A comparative analysis of homologous LTR-RTs was performed with Oropetium thomaeum,Oryza sativa,Brachypodium distachyon,and Arabidopsis thaliana,and the homologous LTR-RTs were searched.The results showed that the number of homologous LTR-RTs and genetic distance between the two species were negatively correlated.In addition,845 full-length LTR-RTs with potential activity were screened,of which 410 belonged to the Gypsy superfamily and 435 belonged to the Copia superfamily.The average ratio of Copia and Gypsy elements was 1.17.The phylogenetic tree construction and insertion time analysis of these sequences were performed.The members of the Copia superfamily were classified into the four lineages of Sire,Oryco,Retrofit,and Tork.The members of the Gypsy superfamily were classified into Athila,Tat,Reina,CRM,and Del/Tekay five lineages.A large number of transpositions of LTR-RTs occurred within 4 million years.The insertion time of full-length LTR-RTs was relatively short,and the peak insertion time was between 1 and 1.5 million years.2.Based on the genome-wide LTR-RTs data,350 pairs of primers were developed,including retrotransposon-based insertion polymorphisms(280),inter-retrotransposon amplified polymorphisms(54),insertion site-based polymorphisms(11),and retrotransposon-microsatellite amplified polymorphisms(5).Polymorphism was evaluated in 23 accessions of the genus Cleistogenes,of which 80 primers showed polymorphism,collectively yielding 332 alleles with an average of 4.2 alleles per locus.The polymorphic information content(PIC)values extended from 0.08 to 0.87 with a mean of 0.49.Furthermore we tested the cross-species transferability of 99 newly developed primers in 5 Gramineae and 6 non-Gramineae species,collectively yielding 325 alleles with an average transfer rate of 42.70%,which is the highest in O.sativa.