Isolation And Chromosomal Localization Of Repetitive Sequence On Y Chromosome In Spinach

Spinach(Spinacia oleracea)is generaly an annual or biennial herbaceous plant,belonging to spiancia genus in subfamily Chenopodioideae of family Amaranthaceae.Its sex-determining mechanism is XY genotype.The genome of spinach is very small,about 989 Mbp.So spinach is the ideal material for studying early stage sex chromosome evolution.Cytological researches have demonstrated that the biggest chromosomes of spinach are the sex chromosomes,which have been microdissected and painted successfully in our lab.The amplified products of the microdissected Y chromosome lay a foundation for study of the structure and evolution of spinach sex chromosomes.The role of repetitive DNA sequences on sex chromosome evolution in other dioecious plant model,such as Carica papaya,Silene latifolia and Rumex acetosa,has been studied.However,its role on spinach sex chromosome evolution has not been reported.Previous research results showed that the DOP-PCR amplification products of microdissected single chromosome will gather a lot of repetitive DNA sequences.Using DOP-PCR amplification products of microdissected spinach Y chromosome as material,Subtractive Hybridization and Dot Hybridization techniques were used to screen sex biased repetitive DNA sequences.Then,using the repetitive DNA sequences as fluorescence probes.The root mitosis metaphase chromosomes of female or male spinach plants were hybridized.According to the fluorescence signals distribution on sex chromosomes and autosomes,the role of repetitive DNA sequences on sex chromosome evolution of spinach was discussed.The main results were as follows:1.The subtractive hybridization library of spinach Y chromosome was successfully constructed and recombinant plasmids were also successfully screened;2.A spinach male-specific repeat DNA sequence SP74-1 was successfully obtained;3.Using the male specific or biasedrepetitive DNA sequences as probes,such as SP73,SP74-1,SP74-3,SP75,SP76,SP77,SP78 and SP79,FISH results showed that the fluorescence signals of SP73 and SP75 on sex chromosomes were stronger than that on autosome and the fuorescence signals on Y chromosome and X chromosome have no significant difference.Male specific molecular markers SP74-1 and sex biased DNA sequences of SP73,SP74-3,SP75,SP76,SP77,SP78 and SP79 all contained Ty1-copia and Ty3-gypsy retrotransposon sequences.The fluorescence signals of class Ty1-copia retrotransposons and Ty3-gypsy reverse transcriptase sequences were distributed on the sex chromosomes.It can be inferred that there were the accumulation of retrotransposons in the process of the sex chromosome evolution in spinach.The results of this study lay the foundation for studying the molecular mechanism of repetitive sequence on the evolution of sex chromosomes in spinach.