Comparative Analysis And Chromosome Location Of Spinach Repetitive Sequence Based On Whole Genome Re-sequencing

Repetitive sequences mainly refer to transposon sequences(DNA transposon sequence,retrotransposon sequence)and tandem repeat sequences(satellite sequences,small satellite sequences,microsatellite sequences).They not only play an important role in the evolution of the genome,but also play a role in the evolution of the sex chromosome of dioecious plants.Spinacia is a dioecious plant.It is a model material for studying plant sex determination and sex differentiation.Because it has cultivars of homosexual chromosomes(Spinacia oleracea L.),a wild species of heteromorphic chromosome(S.tetrandra Stev.),and related species(Beta vulgaris L.),these procide very good materials for studying the role of repeated sequences in the evolution of sex chromosomes.The purpose of this study was to analyze the distribution characteristics of repeat sequences in the genomes of different species of Spinach by using Next-Generation Sequencing(NGS)data and fluorescence in situ hybridization(FISH)technology to explore its relationship with the evolution of sex chromosomes.The main findings are as follows:1.Using the Repeat Explorer software platform and genomic resequencing NGS(Next-Generation Sequencing)data,the differences between the male and female genomes of spinacia plants were elucidated in different species S.turkestanica,S.oleracea and S.tetrandra.Differences between the tetrandra genome,and between the spinach and its related species(B.vulgaris)genome.Angela,Bianca,SIRE,Tork,Athila,Ogre,CRM,Helitron and En Spm＿CACTA have significant differences in spinach and B.vulgaris genomes.But there was no significant difference between S.turkestanica,S.oleracea and S.tetrandra genome;Galadriel has significant differences among the three different species of B.vulgaris and spinach,but there is no significant difference between the male and female genomes of each species of spinach.The content of SIRE,TAR,Ogre,CRM,Tekay,Helitron,En Spm＿CACTA,Mu DR＿Mutator and Satellite in male spinach genome is higher than that in female,but the difference is not significant;the proportion of Galadriel in female genome is higher than that in male,but the difference is not significant;With the proportion of repeated sequences in the spinach genera increases,the proportion of Ale,Angela,Athila,Ogre and Retand in the male genome is also rising.The average proportion of repeats in the S.tetrandra genome is 53.41%,which is higher than that of S.oleracea(average 50.5%)and S.turkestanica(average 49.56%).Theproportion of repeats in the spinach genome is much higher than that of sugar beets.In the spinach genome,the proportion of repeat genomes in the Ty3/Gypsy superfamily is higher than that of Ty1/Copia.However,in the beet genome,the proportion of repeat genomes in Ty1/Copia is the most abundant.The LTR retroposons of Ty3/Gypsy and Ty1/Copia are the main components of repeat sequences.In Ty1/Copia,Angela predominates,accounting for more than 94.97% of Ty1/Copia in spinach species.In the genome of B.vulgaris,SIRE occupies the largest proportion,accounting for 71.44% of Ty1/Copia.Analysis of Ty3/Gypsy shows that Ogre is the most common type in spinach species,accounting for 69.28% of the genome.For beets,Retand and Tekai account for a relatively large proportion of Ty3/Gypsy,accounting for28.5 and 38.6%,respectively.2.Analysis was performed using the TAndem REpeat ANalyzer(TAREAN)software in the Repeat Explorer software platform.The results showed that the proportion of satellite DNA in the genome of spinach and beet genomes was about 0.3%.There were 15 types of satellite DNA.The size is variable between these four species,ranging from 21 bp to 358 bp.Bvu Sat01-59-s,Bvu Sat02-141-s,Bvu Sat03-141-s,Bvu Sat04-159-s,Bvu Sat05-329-s and Bvu Sat06-358-s are only found in the genome of B.vulgaris.Sp Sat01-21,Sp Sat02-52,Sp Sat03-179,Sp Sat04-324 and Sp Sat05-141 were found in the genomes of three spinach species,and Stu Sat01-158 was found only in the S.turkestanica’s genome,Sol Sat01-171-s was found only in the S.oleracea’s genome,Ste Sat01-99-s was found only in the S.tetrandra’s genome,and Sp Sat02-52 accounted for 3.500% of the S.tetrandra’s genome,much higher that in S.turkestanica’s and S.oleracea’s genomes.3.Using fluorescence in situ hybridization technology and the 15 satellite DNAs identified above as probes,the results show: Bvu Sat01-59-s,Bvu Sat02-141-s,Bvu Sat03-141-s,Bvu Sat04-159-s,Bvu Sat05-329-s and Bvu Sat06-358-s and Bvu Sat07-358-s only have signals on beet metaphase chromosomes,but no signals are found on spinach chromosomes.Sp Sat01-21,Sp Sat02-52,Sp Sat03-179,Sp Sat04-324,Sp Sat05-141,Stu Sat01-158-s,Sol Sat01-171-s and Ste Sat01-99-s are only located on the spinach metaphase chromosome,and there is no signal distribution on the beet chromosome.We also found that the signals of Sp Sat03-179,Sp Sat04-324,Sp Sat05-141,Stu Sat01-158-s and Sol Sat01-171-s are located on chromosome 1.Therefore,we speculate that the accumulation of satellite DNA on chromosome1 may play an important role in the evolution of sexual chromosomes of spinach plants.4.Using Sp Sat01-21,Sp Sat02-52,Sp Sat03-179,Sp Sat04-324,Sp Sat05-141,Stu Sat01-158-s,Sol Sat01-171-s and Ste Sat01-99-s as probes,fluorescence in situ hybridization was peformed to hybrid to S.turkestanica and S.oleracea mitotic metaphase chromosome at the mitotic phase and pachytene,telophase and metaphase I chromosome at the first meiotic prophase to observe whether the fluorescent probe signals differed between male and female metaphase of spinach metaphase chromosome,between different stages of meiosis and between different varieties.According to the evolutionary level of different species and the signals distribution of probes,it is speculated that repetitive sequences may play a role in the evolution of sexual staining in spinach.The results showed that the every probe had same signals on the chromosomes of two spinach species.In addition,no significant difference was found in the signals between that on X and Y chromosome.The reason for this may be related to the early stage of spinach sex chromosome evolution.The differentiation of X and Y chromosome are relatively recent,and the genes on MSY were different from their homologous genes on the X chromosome in about 0.40 + 0.08 Mya.5.Four satellite DNA sequences,Sp Sat01-21,Sp Sat02-52,Sp Sat03-179 and Sp Sat04-324,were identified by TAREAN software.Clusters were characterized by a star or circular topology.Based on the fluorescence signal characteristics of these four satellite DNA probes,a more accurate karyotype of spinach was established.A series of FISH results showed that chromosome 1 pair had hybridization signals of Sp Sat01-21 and Sp Sat03-179 on the telomere region of both arms,Sp Sat04-324 on the telomere region of the short arm;Chromosome pair 2: Sp Sat01-21 and Sp Sat03-179 on the telomere region of the long arm,and 45 S on the telomere region of the short arm;chromosome pair 3: Sp Sat02-52 hybridization signal on two arm telomere region,Sp Sat01-21 and Sp Sat03-179 hybridization signal on short arm telomere region;chromosome pair 4: Sp Sat01-21 and Sp Sat02-52 hybridization signal on long arm telomere region;chromosome pair 5: Sp Sat01-21 hybridization signal on long arm telomere region,45 S r DNA hybridization signal on the short arm telomere region;chromosome pair 6: hybridization signal of Sp Sat01-21 and Sp Sat03-179 on long arm telomere region and 45 S r DNA on short arm telomere region.This result can better distinguish the characteristics of each chromosome in spinach,which provides sufficient evidence for the future research and differentiation of spinach chromosomes.