Genome-Wide Analysis Of Repetitive Sequences And Identification Of Chromosome Structural Variation In Lolium Species

Grass crops are important crops in animal husbandry,and their planting area is about twice that of grain crops.Among them,the turf grass,and ornamental grass,are the main component of livestock feed.They can also perform functions such as soil restoration and biodiversity improvement.It has good social benefits,economic value and ornamental value.Lolium belongs to the genus Lolium L.of Poaceae.It is an excellent forage grass with the characteristics of high yield,fast growth and good palatability.It is also considered as the most worldwide major forage grass in the plant system and grows in many agricultural and non-agricultural areas.Elymus L.belongs to the Triticeae family of Poaceae and is an allopolyploid species.Most species of Elymus are fine forage grasses and are resistant to some diseases and insect pests of barley and cultivated wheat.It can be used as an excellent gene resource bank for improving other crops.There are relatively few cytological studies on Lolium and Elymus in China.In this study,precise chromosome identification and molecular marker analysis were carried out on diploid,tetraploid Lolium multiflorum Lam.and hexaploid Elymus dahuricus.The main results are as follows:1.The homology groups of tetraploid Lolium chromosomes were determined by Oligo-FISH Painting system,and Lolium 2L chromosome was successfully identified by the homology group probe Synt2.Then,the polymorphism analysis of rDNA was carried out,and it was found that there was genetic linkage between 5S rDNA and 18S rDNA on chromosome 2L.However,the evolution patterns of 5S rDNA and 18S rDNA were different,the 5S rDNA region was relatively stable,and its quantity and distribution polymorphism were low.5S rDNA was located only on the short arm of chromosome 2L,and had 2 signal sites in diploid Lolium and 4 signal sites in tetraploid Lolium.The number and distribution position of 18S rDNA were highly polymorphic.Among the diploid Lolium materials analyzed in total,there were 4,5,6,7,and 8 chromosomes containing Oligo-18SrDNA hybridization signals,in tetraploid Lolium materials,there were 9,10,11,12,13,14 and 15 chromosomes contain Oligo-18SrDNA hybridization signals.At the same time,it was observed through karyotype analysis that the ryegrass species had a large number of chromosome breaks,and the breakage points were mainly enriched in the 18S rDNA region,indicating that the 18S rDNA site was a fragile site.2.Through non-denaturing fluorescence in situ hybridization(ND-FISH),some Lolium specific probes were screened out.A total of seven probes consistent with the signal site of Oligo-5SrDNA were found:Oligo-1RS-2,Oligo-Ai-15,Oligo-3D1,Oligo5A1,Oligo-Ai-9,Oligo-Y308,Oligo-Stc12.Six probes consistent with Oligo-18SrDNA signal site:Oligo-1RS-1,Oligo-1RS-9,Oligo-5D-156,Oligo-3H-1,Oligo-2H-1-100,Oligo-E3900.Thirteen probes with signal at both the centromere and at the telomere:Oligo-Thp3961,Oligo-6H-1-50,Oligo-4B-6,Oligo-oat-cen2,Oligo-Lp61-5,OligoY358,Oligo-4H-9-50,Oligo-Dv235,Oligo-Dvl18,Oligo-Lp-Telo-1,Oligo-Lp-Telo-2,Oligo-Ai-6,Oligo-Y372.Two telomeric probes:Oligo-Y20,Oligo-Y29.One centromere probe:Oligo-(GT)10 and one genomic probe:Oligo-Y322.These probes can be used for genetic diversity analysis of Lolium materials.The hybridization signals of some probe sequences were significantly different in the diploid Lolium reference genome.It indicated that the current Lolium reference genome sequence assembly needs to be further revised.3.The genetic diversity of 24 diploid Lolium(2n=14),including 12 Oregon annual diploid Lolium,6 Hongcao and 6 Tang X So 865 Lolium,was analyzed by IT molecular marker.40 pairs of primers were selected,and total 485 bands were detected,427 of which were polymorphic.The rate of polymorphism was 88.04%.The cluster analysis was conducted according to UPGMA.The genetic similarity coefficient was 0.72,the 24 individuals clustered into 5 branches,and the genetic differences among the groups were significant.It was conducive to indepth conservation of species diversity and selection of new materials for grass breeding.4.By genome in situ hybridization,Oligo-FISH Painting system and ND-FISH for precise karyotype identification of the hexaploid Elymus dahuricus(2n=6x=42),and the FISH map of E.dahuricus was constructed.Using the probes Oligo-HvCSR and OligopTa535 to distinguish the H genome of E.dahuricus,using GISH with the whole genome of Pseudoroegneria as a probe to distinguish the St genome,and the remaining 14 chromosomes were the Y genome.The chromosomes of the seven homologous groups of E.dahuricus were accurately analyzed by the homology group probes Syntl-Synt7.Then,continuous ND-FISH was carried out to accurately identify the karyotype of 42 chromosomes and construct a FISH map.Through the rearrangement identification of the homology group probes Synt2 and Synt5,Robertson translocation was found in the short arm of chromosomes 2H and 5Y.The present study identified novel translocation karyotypes of 5YS.2HL and 2HS.5YL in E.dahuricus.In summary,in this study,a combination of molecular and cytogenetic methods was used to analyze the genetic diversity of Lolium species.And some specific probes of Lolium species were screened out,which provided important reference value for the correction of Lolium sequencing and assembly.Using the new technology of Oligo-FISH Painting system developed by our laboratory,we have completed the accurate identification of seven homologous group chromosomes of E.dahuricus,and the FISH map of the whole genome was constructed,which laid the foundation for the utilization and mining of E.dahuricus echinatus genetic resources.