The Construction And Screening Of Male Fosmid Library And FISH Location Of Sex-biased DNA Fragments In Spinach

Spinach(Spinacia oleracea),belonging to spiancia of chenopodioideae in amaranthaceae,which generaly is the annual or biennial herbaceous plant.Spinach is significant dioecious plants.And its sex-determining mechanism is XY type,which is in the second stage of the sex chromosome evolution level.The genome of spinach is very small,about 989 Mbp.So spinach is the important experimental material to study sex chromosome evolution of dioecian plant.At present,the cytological researches have demonstrated that the biggest chromosomes are the sex chromosomes,which have been microdissected and painted successfully in our lab.The sequencing of spinach transcriptome has also been completed.Some sex-linked molecular markers were obtained too.But the cloning of sex-controlled regions has not been reported at present.Up to now,the mechanisms of molecular sex determination and the sex chromosome evolution are also unclear.In this study,we constructed the spinach male-genome Fosmid library firstly,then we used the spinach male-specific molecular marker that was reported in document and we have developed in this study to screen the Fosmid library and obtain the Fosmid monoclone then hybrided to spinach root mitosis metaphase chromosomes using FISH technique.In addition,some DNA fragments that have difference between the male genome and female genome were obtained,sequenced and analyzed using semi-quantitative technique,The results of the study were as follows:1.The spinach male-genome Fosmid library was successfully constructed and the capacity,fraction of coverage and the quality were detected;2.A new technical method which combine spinach male-genome Fosmid library PCR screening with FISH technique for cloning spinach male sex-determinating region was set up successfully;3.In this study,a spinach male-specific molecular marker SP1168 was created,which is very stable and can be used to identify the sex of spinach at seeding stage.Some DNA fragments that have difference between the male and the female were also got in this paper;4.When the sex different DNA fragments SP1168,SP464 ex,scEST and SpYscs were located on the spinach root mitosis metaphase chromosomes,the FISH signals of these DNA fragments were showndifferent on X and Y chromosomes,of which the FISH signals were more brighter on Y chromosome than that on X chromosome.In this paper,the results showed that not only the male-specific DNA marker SP1168 but also sex-different DNA markers SP464 ex,scEST and SpYscs all contains certain Ty1-copia transposon DNA sequences.Although the FISH signals of these DNA fragments were shown on all the spinach root mitosis metaphase chromosomes,the difference of the FISH signals on X and Y chromosomes were obvious,which indicated that the transposons were accumulated in the process of the evolution of sex chromosomes and the the amount of the accumulation on X chorosome is more than the Y chorosome.So we infer that the transposons play an important role in the evolution of the spinach sex chromosomes.In this paper,the result of the study can provide provide a foundation to abtain the Sex determination region of spainch the research of the sex determination and differentiation mechanism,while can provide a theoretical basis to explorate the mechanism of spinach sex chromosome evolution.