黄瓜（Cucumis sativus L.）性别分化相关的遗传标记研究
摘要
本文以多代自交的全雌性黄瓜栽多星(EC1)和多代自交的弱雌性黄瓜北京截头(CC3)为亲本，进行杂交和自交获得F_2分离群体，采用同工酶标记、RAPD分子标记和AFLP分子标记等方法对黄瓜性别分化相关遗传标记进行研究。主要结果如下：
在与黄瓜性别分化有关的同工酶标记研究中，对全雌性黄瓜EC1和弱雌性黄瓜CC3及其自交后在F_2代分离出的全雌株、强雌株和弱雌株进行了MDH、AAT与POD三种同工酶分析。结果表明，MDH在EC1和CC3之间可找到2个多态性的同工酶位点，但不能在F_2群体间不同性别单株区分开；AAT在EC1和CC3之间和F_2群体间均无明显差异；POD在EC1和CC3之间和F_2群体间均能找到有1～2个差异的同工酶位点。
与黄瓜性别分化有关的RAPD分子标记研究包括DNA提取方法的筛选、优化RAPD反应体系、黄瓜亲本RAPD分子标记多态性随机引物的筛选和黄瓜性别相关的RAPD分子标记筛选四个方面。在DNA提取方法筛选的研究中，2％浓度的CTAB法提取的黄瓜叶片DNA具有典型的天然DNA分子的标准紫外吸收光谱特点，其A_(260/280)值在1.6-1.8之间，DNA的产量为870.63μg/g。此方法在黄瓜上相对优于其他DNA提取方法，提出的DNA适合进一步RAPD分析。
在进行优化RAPD反应体系研究中，以黄瓜基因组总DNA为模板，通过对聚合酶链式反应(PCR)体系中Mg~(2+)浓度、dNTP浓度、Mg~(2+)浓度与dNTP浓度互作、模板DNA的浓度、随机引物的浓度和Taq酶浓度梯度进行试验，结果表明，反应体系为：超纯水12.2μl，Buffer(10x)2μ1，MgCl_2(25mM)_2μl，dNTP(2mM)1.5μl，Primer(8pmol/rec)1μl，Taq酶(1.5U/rec)0.3μl，TemplateDNA(50ng/μl)1μl时筛选出的PCR反应体系谱带清晰、条带丰富、可重复性好。
在黄瓜亲本RAPD分子标记多态性随机引物的筛选研究中，通过全雌性与弱雌性黄瓜亲本基因组DNA的RAPD-PCR扩增对620条随机引物进行筛选，共有61条随机引物有明显差异。筛选出的引物可进一步用于黄瓜性别相关的分子标记研究。在对黄瓜性别分化相关的RAPD分子标记筛选的研究中，通过对黄瓜亲本显示多态性的61条随机引物对F_2分离群体进行扩增，随机引物AK04在1000bp、AK10在200bp、AK20在400bp处产生与性别相关的特异性条带。
黄瓜(cucumis sativoL)性别分化相关的遗传标记研究
在与黄瓜性别分化有关的AFLP分子标记研究中，对全雌性黄瓜Ecl和弱雌
性黄瓜CC3及其自交后在F:代分离出的全雌株、强雌株和弱雌株进行了AFLP分
析.结果表明，全雌性黄瓜ECI和弱雌性黄瓜CC3的人FLP图谱每个泳道有55-
64条带，在ECI与Cc3之间至少有6个明显的多态性位点;在FZ代中全雌群体、
强雌群体和弱雌群体之间显示出7个多态性条带;在黄瓜不同性型的单株之间每
个AFLP引物至少可以发现5个多态性条带。
This paper, using a gynoecious cucumber Deltastar (EC1) and monoecious cucumber Beijing Jietou (CC3) as parents to make F1 to get the F2 segregation population, studied the genetic markers related to the sex differentiation of cucumber with isozyme, RAPD and AFLP methods. The main results of this paper are as follows:
In the study on the isozyme markers related to sex differentiation of cucumber, the research is about the analysis in three isozyme systems (MDH, AAT, POD) of gynoecious cucumber EC1, monoecious cucumber CC3 and their F2 segregation population. The results showed that 2 polymorphic loci were observed in MDH between EC1 and CC3, but no variation among F2 segregation population was observed. No variation was observed in AAT both between EC1 and CC3 and among F2 segregation population. 1 -2 polymorphic loci were observed in POD both between EC 1 and CC3 and among F2 segregation population.
Studies on the RAPD molecular markers related to sex differentiation of cucumber included the DNA extraction methods screening, the optimization of RAPD reaction system, the random primers screening of polymorphism for the RAPD molecular markers of parents of cucumber and sex-related RAPD molecular markers screening of cucumber. In the study on the DNA extraction methods screening, the method of CTAB with 2% concentration possessed the standard ultraviolet absorbance spectrum of the pure natural DNA and the A260/280 was between 1.6-1.8, the yield of DNA was 870.63ug/g young leaves. This method adapted to the further analysis of RAPD.
In the study on the optimization of RAPD reaction system for cucumber, the research, using genomic DNA of cucumber as the template, tested the concentrations of Mg2+, dNTP, DNA, random primers and Taq enzyme. The results indicated that the optimized reaction system including DDH2O 12.2 ul, Buffer (10x) 2 ul, MgCl2(25mM) 2 ul, dNTP (2mM) 1.5 ul, Primer (8pmol/rec) 1 ul, Taq enzyme (1.5U/rec) 0.3 ul and Template DNA (50ng/ ul) 1 ul, had the traits of clear, good polymorphic and well repeated electrophoresis patterns.
In the study on the random primers screening of polymorphism for the RAPD molecular markers of parents of cucumber, this research is about the analysis in screening 620 random primers by RAPD-PCR amplification of the gynoecious and monoecious genomic DNA of parents
of cucumber. The results indicated that 61 random primers showed the apparent difference. The primers screened can be used for further study of molecular markers linked to sex of cucumber. In the study on the sex-related RAPD molecular markers screening of cucumber, This study is about the analysis in amplifying p2 segregation population with 61 random primers that showed the polymorphism among the parents of cucumber. The results indicated that random primers AK04, AK10, and AK.20 showed sex-specific bands at 1000, 200 and 400bp respectively.
In the study on the AFLP molecular markers related to sex differentiation of cucumber, the research is about the analysis of gynoecious cucumber EC1, monoecious cucumber CC3 and their Fj segregation population using AFLP molecular markers. The results showed that the difference of AFLP markers related to the sex differentiation of cucumber is abundant. There are 55-64 bands per lane and 6 polymorphic loci between EC1 and CC3. There are 7 polymorphic loci among the gynoecious, subgynoecious and monoecious population in F2 segregation population There are at least 5 polymorphic loci among individuals of different sex types of cucumber per primer.
在与黄瓜性别分化有关的同工酶标记研究中，对全雌性黄瓜EC1和弱雌性黄瓜CC3及其自交后在F_2代分离出的全雌株、强雌株和弱雌株进行了MDH、AAT与POD三种同工酶分析。结果表明，MDH在EC1和CC3之间可找到2个多态性的同工酶位点，但不能在F_2群体间不同性别单株区分开；AAT在EC1和CC3之间和F_2群体间均无明显差异；POD在EC1和CC3之间和F_2群体间均能找到有1～2个差异的同工酶位点。
与黄瓜性别分化有关的RAPD分子标记研究包括DNA提取方法的筛选、优化RAPD反应体系、黄瓜亲本RAPD分子标记多态性随机引物的筛选和黄瓜性别相关的RAPD分子标记筛选四个方面。在DNA提取方法筛选的研究中，2％浓度的CTAB法提取的黄瓜叶片DNA具有典型的天然DNA分子的标准紫外吸收光谱特点，其A_(260/280)值在1.6-1.8之间，DNA的产量为870.63μg/g。此方法在黄瓜上相对优于其他DNA提取方法，提出的DNA适合进一步RAPD分析。
在进行优化RAPD反应体系研究中，以黄瓜基因组总DNA为模板，通过对聚合酶链式反应(PCR)体系中Mg~(2+)浓度、dNTP浓度、Mg~(2+)浓度与dNTP浓度互作、模板DNA的浓度、随机引物的浓度和Taq酶浓度梯度进行试验，结果表明，反应体系为：超纯水12.2μl，Buffer(10x)2μ1，MgCl_2(25mM)_2μl，dNTP(2mM)1.5μl，Primer(8pmol/rec)1μl，Taq酶(1.5U/rec)0.3μl，TemplateDNA(50ng/μl)1μl时筛选出的PCR反应体系谱带清晰、条带丰富、可重复性好。
在黄瓜亲本RAPD分子标记多态性随机引物的筛选研究中，通过全雌性与弱雌性黄瓜亲本基因组DNA的RAPD-PCR扩增对620条随机引物进行筛选，共有61条随机引物有明显差异。筛选出的引物可进一步用于黄瓜性别相关的分子标记研究。在对黄瓜性别分化相关的RAPD分子标记筛选的研究中，通过对黄瓜亲本显示多态性的61条随机引物对F_2分离群体进行扩增，随机引物AK04在1000bp、AK10在200bp、AK20在400bp处产生与性别相关的特异性条带。
黄瓜(cucumis sativoL)性别分化相关的遗传标记研究
在与黄瓜性别分化有关的AFLP分子标记研究中，对全雌性黄瓜Ecl和弱雌
性黄瓜CC3及其自交后在F:代分离出的全雌株、强雌株和弱雌株进行了AFLP分
析.结果表明，全雌性黄瓜ECI和弱雌性黄瓜CC3的人FLP图谱每个泳道有55-
64条带，在ECI与Cc3之间至少有6个明显的多态性位点;在FZ代中全雌群体、
强雌群体和弱雌群体之间显示出7个多态性条带;在黄瓜不同性型的单株之间每
个AFLP引物至少可以发现5个多态性条带。
This paper, using a gynoecious cucumber Deltastar (EC1) and monoecious cucumber Beijing Jietou (CC3) as parents to make F1 to get the F2 segregation population, studied the genetic markers related to the sex differentiation of cucumber with isozyme, RAPD and AFLP methods. The main results of this paper are as follows:
In the study on the isozyme markers related to sex differentiation of cucumber, the research is about the analysis in three isozyme systems (MDH, AAT, POD) of gynoecious cucumber EC1, monoecious cucumber CC3 and their F2 segregation population. The results showed that 2 polymorphic loci were observed in MDH between EC1 and CC3, but no variation among F2 segregation population was observed. No variation was observed in AAT both between EC1 and CC3 and among F2 segregation population. 1 -2 polymorphic loci were observed in POD both between EC 1 and CC3 and among F2 segregation population.
Studies on the RAPD molecular markers related to sex differentiation of cucumber included the DNA extraction methods screening, the optimization of RAPD reaction system, the random primers screening of polymorphism for the RAPD molecular markers of parents of cucumber and sex-related RAPD molecular markers screening of cucumber. In the study on the DNA extraction methods screening, the method of CTAB with 2% concentration possessed the standard ultraviolet absorbance spectrum of the pure natural DNA and the A260/280 was between 1.6-1.8, the yield of DNA was 870.63ug/g young leaves. This method adapted to the further analysis of RAPD.
In the study on the optimization of RAPD reaction system for cucumber, the research, using genomic DNA of cucumber as the template, tested the concentrations of Mg2+, dNTP, DNA, random primers and Taq enzyme. The results indicated that the optimized reaction system including DDH2O 12.2 ul, Buffer (10x) 2 ul, MgCl2(25mM) 2 ul, dNTP (2mM) 1.5 ul, Primer (8pmol/rec) 1 ul, Taq enzyme (1.5U/rec) 0.3 ul and Template DNA (50ng/ ul) 1 ul, had the traits of clear, good polymorphic and well repeated electrophoresis patterns.
In the study on the random primers screening of polymorphism for the RAPD molecular markers of parents of cucumber, this research is about the analysis in screening 620 random primers by RAPD-PCR amplification of the gynoecious and monoecious genomic DNA of parents
of cucumber. The results indicated that 61 random primers showed the apparent difference. The primers screened can be used for further study of molecular markers linked to sex of cucumber. In the study on the sex-related RAPD molecular markers screening of cucumber, This study is about the analysis in amplifying p2 segregation population with 61 random primers that showed the polymorphism among the parents of cucumber. The results indicated that random primers AK04, AK10, and AK.20 showed sex-specific bands at 1000, 200 and 400bp respectively.
In the study on the AFLP molecular markers related to sex differentiation of cucumber, the research is about the analysis of gynoecious cucumber EC1, monoecious cucumber CC3 and their Fj segregation population using AFLP molecular markers. The results showed that the difference of AFLP markers related to the sex differentiation of cucumber is abundant. There are 55-64 bands per lane and 6 polymorphic loci between EC1 and CC3. There are 7 polymorphic loci among the gynoecious, subgynoecious and monoecious population in F2 segregation population There are at least 5 polymorphic loci among individuals of different sex types of cucumber per primer.
引文
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