Separation And Chromosome Localization Of Male-Bias NUPT In Spinach

Spinacia oleracea L.(2n=2x=12)is a one or two years old dioecious herbaceous plant and has a high economic value.Spinach is determined XY type sex chromosomes,2n = 12,usually dioecious.Spinach’s sex chromosome is 1 chromosome being in the early stage of the evolution of sex chromosomes.The chromosome of spinach is small,about 989 Mbp,It is a favorable experiment system for research on sex differentiation in dioecious plants due to short growth cycle,being easy to cultivate.it is an ideal material for the study of plant sex differentiation and chromosome evolution.The phenomenon of organelle DNA fragments transferred to the nuclear genome often occurs in eukaryotes.In higher plants,the transfer of plasmid DNA and integrated into the nuclear genome will form DNA(nuclear plastid DNA karyoplasms,NUPTs).The latest study found NUPTs in dioecious Silene Y chromosome specific accumulation,indicating that the NUPTs may and sex chromosome origin and evolution has certain relation.However,whether the accumulation of NUPTs is a commen phenomenon,if it is,and how to participate in the origin and evolution of sex chromosomes.These issues are still not clear.Based on the problems above,this study with spinach as experimental material,using genomic subtractive hybridization screening and separating the difference of spinach male and female genome sequence of NUPTs.According to the spinach chloroplast genome data,we obtained long fragment NUPTs sequence and investigated chromosomal localization analysis using chromosome fluorescence in situ hybridization,the study to investigate the spinach chloroplast genome is transferred to the nuclear genome research and chromosome evolution provides data.The main results are as follows:(1)A subtractive hybridization library of spinach was constructed.4706 single colonies were selected from the library for PCR,and then the positive clones were screened by dot blot hybridization.A total of 39 male specific sequences were obtained.The average length of the sequences was about 154 bp,the longest was 308 bp,and the shortest was 75 bp.Analysis of the sequence obtained by Blastn homology analysis showed that there were 23 homologous alignment results,12 of which were related to chloroplast,and the similarity of chloroplast genome was more than 98%.Another 16 are new sequences.(2)primers and PCR were designed to screen out the sequence of the chloroplast.Finally,two maleand female genomes were named sp5 and sp8,respectively,and their length was 146 bp and 199 bp,respectively.The sp5 and sp8 in the NCBI database by blastx for similarity comparison,find out these sequences where long sequences,with the long sequence primers were designed sp5 and sp8,expanding a conducive to the chromosomal localization of the long fragment length,respectively 3706 bp and 2391 bp.Semi quantitative PCR amplification analysis revealed that the two NUPTs sequences had obvious differences in the male and female genomes of spinach,showing the male preference,indicating that the NUPTs sequence had more accumulation in the male genome.(3)the chromosome location of two NUPTs sequences obtained by the technique of chromosome in situ hybridization.Structure found that NUPTs has a stable fluorescence signal on all chromosomes of spinach.Which chromosome 1,namely the chromosome with the strongest signal,because after the verification of the two NUPTs sequence sp5 and sp8 in the male genome accumulation than in the female genome,referring to Deng et method verify the chromosome Y chromosomes of spinach,namely the two ordered NUPTs columns in the spinach on the Y chromosome fluorescence signal than on the X chromosome.By karyotype analysis and band pattern analysis,the fluorescence signal of NUPTs was mainly distributed in the vicinity of the chromosome.