与黄瓜全雌性基因连锁的SSR分子标记

Identification of SSR marker linked to gynoecious loci in cucumber(Cucumis sativus L.)

黄瓜(Cucumis sativus L.)是重要的蔬菜栽培作物,其雌花率的高低直接影响着黄瓜产量。目前优良的黄瓜品种都具备全雌性或强雌性特征,全雌性也是黄瓜优势育种的重要途径。但由于黄瓜性别表现受到遗传和环境等多种因素的影响,传统的从表型上进行全雌性基因的选择效率不高。然而,借助与目的基因相连锁的分子标记进行辅助育种能直接从基因型上对后代单株进行选择,准确率高,能够在苗期进行性型鉴定,从而大大地提高育种效率。以全雌品系240-1-2-2-3-1自交系和弱雌品系3-5-1-3-2-1-1-1-1-2及其F1、F2、BC1P1和BC1P2世代为试验材料,进行田间鉴定和遗传规律分析。结果表明:黄瓜性别表达由寡基因控制,并受到一些背景基因的修饰;黄瓜全雌性相关基因遗传模型符合加性-显性-上位性遗传模型。利用PCR技术和SSR分子标记方法,通过亲本、F2全雌和全雄基因池筛选,从699对SSR引物组合中获得稳定的多态性引物组合2对,即CSWCT25和SSR18956;经回收、测序,特异片段全长分别为331bp和145bp,与黄瓜全雌性基因的连锁距离分别为7.7cM和6.8cM,均可用于黄瓜全雌系品种的辅助选育。

Summary Gynoecy plays an important role in cucumber （Cucumis sativus L. ） heterosis breeding and identification of the markers linked to this character will facilitate selection of gynoecious cucumber line in breeding program. Traditional selection for cucumber cultivars with gynoecious line has required evaluation in complicated environments over several years, which is long period, time and labor consuming. Molecular markers offer a faster and more accurate way for breeding, as selection can be based on genotype rather than phenotype. The use of molecular markers for indirect selection of important agronomic characters, or marker-assisted selection （MAS） can improve the efficiency of traditional breeding. Many studies developed a lot of SSR markers, which had greatly facilitated MAS in cucumber breeding. Now some studies showed that some markers were connected with gynoecious gene but the distances were not compact, so few were availably applied to breeding. The aim of this study was genetic analysis of gynoecy and identification of molecular marker associated with gynoecious gene using gynoecious line, monoecious line, and SSR marker. The genetic analysis of cucumber gynoecious was evaluated with a gynoecious line 240- 1-2- 2- 3- 1,monoecious line 3 - 5 - 1 - 3 - 2 - 1 - i - 1 - 1 - 2 and their F1 , F2, BC1 P1, BC1 P1 populations in the present study. Total DNA of parents and F2 were isolated from freeze-dried leaf tissue by the CTAB method. SSR markers were analyzed withgynoeciousline240-1-2-2-3-1, monoecious line3-5-1-3-2-1-1-1-1-2, F2, and 699 pairs of SSR primers. SSR analysis was performed with the primers. PCR was performed in 20 μL reaction containing 2 μL genomic DNA （20 ng）, 10 pmol/μL primers 0.4 μL, respectively, 2.5 mmol/L Mg2＋ 2 gl, 2 mmol/L dNTP 1 /μL, 5 U/uL Taq DNA polymerase enzyme 0.1 μL, 10N buffer 2 μL and double distilled water. The amplification profiles were 5 min at 94℃, followed by 35 cycles of 30 s at 94 ℃ ; 1 min at 55℃, 1 Min at 72 ℃ ; then 10 min at 72 ℃. After amplification, the PCR products were mixed with loading buHer （2.5 mg/mL bromophenol blue, 2.5 mg/mL diphenylamine blue, 10 mmol/L EDTA, 950//oo （V/V） formamide）, denatured for 5 min at 94% and put on ice for 5 min. The denatured PCR products were separated on 6 （W/V） denaturing polyacrylamide gel at 100 W power and visualized by silver straining. Polymorphic fragments of primers were cloned and sequehced. Linkage analysis used the software of Mapmaker V3.0. During analysing the separated rate of F and F2, the results showed that the gynoeey in 240 - 1 - 2 - 2 - 3 - 1 was controlled by oligogene with some background genes modified. Inheritance of gynoecy was accord with the additive dominant-epistatic model. From 699 pairs of SSR primer, two pair of stable SSR markers （CSWCT25 and SSR18956）, 331 bp and 145 bp in bands size were obtained respectively during PCR products of two SSL markers cloned and sequenced, and linkage analysis indicated that its genetic distance to the gynoecious loci was 7.7 cM and 6.8 cM, respectively. Two SSR markers are tightly linked to gynoecious loci on the chromosome 6. In sum, knowledge of location of gynoecious gene in cucumber and its related traits in crosses will be helpful to the design of more effective selection schemes to develop gynoecious cucumber genotypes. Progress in breeding gynoecious lines is still slow because of the complex inheritance of this character. However, gynoecious cultivars and the markers identified in this study can contribute to improving gynoecious line. Two SSR markers could be used effectively for molecular markerassisted selection in breeding programs to develop cucumber gynoecious line breeding.