Cloning And Identification Of SRAP Molecular Marker In Spinacia Oleracea L. And Humulus Scandens L.

Spinacia oleracea L.(2n=2x=12) is a one or two years old dioecious herbaceous plant and has a higheconomic value. It is a favorable experiment system for research on sex differentiation in dioecious plantsdue to short growth cycle, being easy to cultivate, unexpressed flower primordia heterotype and so on. Upto now, the studies of S. oleracea sex differentiation focus on its cytology, morphology, physiology andbiochemistry, but the researches in molecular level are few. What is more, the report of sex-determinationgene cloning has not been reported.Humulus scandens L. is an important perennialdioecious plant. Female plants of this species containthe set of chromosomes2n=16=14+XX, whereas male plants have2n=17=14+ХY₁Y₂. So it is asuitable model material for studying sex determination and sex chromosomes evolution in plants.Compared with Rumex acetosa L., the research on molecular mechanism of Humulus sex determination isstill at the primary stage.Therefore, SRAP (Sequence Related Amplified Polymorphism) DNA marker was used to screen maleand female genome pools of S. oleracea L. and H. scandens L. Then sex-related fragements were clonedand sequenced. At last, by using of FISH and Dot blotting technique, these fragements were identified. Themain results were as follows:⑴Sequence-related amplifid polymorphism (SRAP) was used to amplify the male and female genepools of S. oleracea L., so as to find and clone the sex-linked framents. Among the256primer pairs tested,20primers could amplify stable and remarkable specific bands and the primer polymorphism is7.813%.The amplified products were checked by PAGE electrophoresis, a total number of382bands weregenerated, among which76bands were polymorphic, and the percentage of polymorphic bands was19.9%.Then, the sex-specific fragments were excised from agarose gel and colonies containing targetfragments were sequenced. Of these special fragments, we found a male-associated fragment with a lengthof about391bp, which has a higher homology with18s rRNA (homology reached99%).By using fluorescence in situ hybridization (FISH), the DNA marker was physically located on threepair chromosomes of S. oleracea L. In the future, the differences of fluorescence signals of the DNAmarker between male and female spinach chromosomes will be investigated. ⑵SRAP was also used to amplify the male and female gene pools of H. scandens. A total number of671bands were got by using50primer-pairs of256primers with the optimized SRAP reaction condition.Primer polymorphism is19.53%and the percentage of polymorphic bands was36.81%, which werechecked by polyacrylamide gel electrophoresis (PAGE). Then, the specific bands were cloned to besequenced for comparing their DNA sequences. One special fragment we got was a male-linked fragmentwith a length of666bp. The analysis showed that it might be a part of transposons. Dot blot result showedall sex linked fragements had the positive signals. It indicated that the difference between male and femalegenome was small. The other methods should be used to analyse the fragements.