西瓜白化致死基因的分子标记和遗传分析
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摘要
西瓜是一种重要的经济作物,我国西瓜常年栽培面积100万公顷,年西瓜总产3000万吨以上。西瓜的遗传多样性较为丰富,不同的种质资源之间存在着较多的性状差异,然而西瓜的遗传多样性研究起步较晚,特别是现代分子生物技术的应用尚不充分。为了给西瓜遗传基础研究提供新方法,本试验以课题组发现的西瓜白化致死近等基因系和F2代群体及亲本为试材,分别利用SRAP和EST-SSR进行分子标记,并结合主要的种质性状进行遗传分析。同时,试验还探索了利用柱头涂抹法对西瓜进行T-DNA导入标记的方法研究。主要的试验结果如下:
(1)对公共数据中的8619条西瓜EST序列进行处理和分析,获得202个EST-SSR,分布在191条EST序列中,出现频率为2.31%,分布密度为1/19.1kb。其优势重复基序为二核苷酸和三核苷酸(68.8%),在二核苷酸基序中以AG/TG类型为最多(46%);在三核苷酸基序中,以AAG类型为最多(43%)。基序重复次数以4次(17.8%)、7次(15.3%)和11次(14.9%)为最多。长度分布主要在20~30bp之间(76%)。利用上述EST-SSR序列设计获得24对引物在西瓜白化致死近等基因系中进行验证,结果获得了16对有效的扩增引物,占总设计引物对数的66.7%。结果表明:利用公共数据库中的EST序列开发西瓜EST-SSR标记是一种有效的方法。
(2)对独家发现的西瓜白化致死近等基因系和F2代群体进行SRAP标记和遗传分析,结果在400对引物中未找到与西瓜白化致死基因连锁的分子标记,但获得了9对SRAP引物组合的10个呈3:1分离的标记。遗传分析表明,除种皮颜色外,果皮颜色、果肉颜色、果实形状、种子大小、果柄顶部形状和果皮厚度等7个种质性状与西瓜白化致死基因之间没有连锁关系。但上述10个SRAP标记与7个性状之间有一定的连锁关系,其中标记E6-M2-170和E14-M2-240之间相似性特强,连锁距离为0cM;种子大小与果皮厚度两个性状之间的连锁最紧密,连锁距离为5.6cM;果柄顶部形状与果实形状和熟性与果皮厚度之间的连锁较紧密,连锁距离分别为8.0cM和8.1cM。
(3)借鉴拟南芥蘸花进行外源DNA导入的方法,通过柱头涂抹进行西瓜T-DNA导入标记的方法研究。结果表明:品种ZXG01078坐瓜率最高,且个别单瓜有变异苗产生;卡那霉素筛选处理西瓜种子的最佳浓度500~700 mg/L,不同品种间有差异;卡那霉素筛选处理西瓜叶片的最佳部位为嫩叶或幼叶,最佳浓度4000~8000 mg/L,不同品种间差异不明显。连续两年的试验中个别单瓜后代能稳定的产生无心苗和连体苗。
Watermenlon is an important economic crops. In China, Watermelon cultivation area is maintained at 1,000,000 hectares per year. Annual output of watermelon more than 30 million tons. Watermelon has a relatively rich genetic diversity. Different germolasm resources exist large differences of trarts. However, the research in watermelon on genetic diversities started too late, especially in the application of modern molecular techniques are inadequate. In order to explore a new method to study the molecular genetic in watermelon. We utilized albino lethal near-isogenic lines of watermelon, F2 generation and their parents for the experimental material. Then using SRAP and EST-SSR molecular markers combined with the main germplasm traits for genetic analysis. In addition, the experiment also explored stigma smear to transfer T-DNA and molecular marker research in watermelon. The main results as follows:
We analysed a total of 8619 watermelon ESTs from GenBank and sought out 202 SSR contained in 191 EST sequences. The frequency of the SSRs was 2.31% and mean the distribution density was 1/19.1kb in watermelon. The dominant repeat motifs were dinucleotide and trinucleotide. AG/TG was the most repeat type in dinucleotide(46%) and AAG was the most repeat type in trinucleotide(43%).The most motif repeat times were 4 (17.8%), 7 (15.3%) and 11 (14.9%). The length was mainly distributed between 20 to 30bp.We obtained 24 primer pairs from 202 EST-SSRs in watermelon.Then utilized albino lethal near-isogenic lines of watermelon for verify these primers.We obtained 16 effective amplified primer pairs,accouting for 66.7% of the total number of designed primer pairs. The results suggest that use of public EST databases to develop EST-SSR primers of watermelon are effective method.
We utilized albino lethal near-isogenic lines of watermelon and F2 generation for the experimental material. Then using SRAP technique and genetic analysis to research, the result is that we found no molecular marker tightly linked to albino lethal gene among 400 primer pairs.On the other hand,we got 9 SRAP primer pairs which amplified out of 10 difference bands that showed 3:1 separation. Genetic analysis expressed that the rind color,flesh color, fruit shape, seed size, fruit peduncle top shape and thickness of rind et.al. 7 major germplasm traits were not linked to albino lethal gene in watermelon excepted the seed colour. There was certain linkage between the 10 SRAP molecular marker and the 7 major germplasm traits. Among them, markers E6-M2-170 and E14-M2-240 were completely linkage with a distance of 0cM. Seed size was closely linked to thickness of rind at 5.6cM. Fruit peduncle top shape and development period were linked to fruit shape and thickness of rind at 8.0cM and 8.1cM, respectively.
From the method of foreign DNA introduced to Arabidopsis thaliana via flowers dip, we used stigma smear to transfer T-DNA into watermelon and its molecular marker research. The results showed the highest fruit setting rate specie is ZXG01078 and its individual single fruit have deviant seedlings. The best concentration of kanamycin treating watermelon seeds is 500~700mg/L with differences among the species. The best position is spire leaf or youngleaf and the best concentration of kanamycin treating the watermelon leaf is 4000~8000mg/ L with no difference among the species. The offspring of individual single fruit can steadily produce no pointing and twinborn seedlings in two years experiment.
(1)对公共数据中的8619条西瓜EST序列进行处理和分析,获得202个EST-SSR,分布在191条EST序列中,出现频率为2.31%,分布密度为1/19.1kb。其优势重复基序为二核苷酸和三核苷酸(68.8%),在二核苷酸基序中以AG/TG类型为最多(46%);在三核苷酸基序中,以AAG类型为最多(43%)。基序重复次数以4次(17.8%)、7次(15.3%)和11次(14.9%)为最多。长度分布主要在20~30bp之间(76%)。利用上述EST-SSR序列设计获得24对引物在西瓜白化致死近等基因系中进行验证,结果获得了16对有效的扩增引物,占总设计引物对数的66.7%。结果表明:利用公共数据库中的EST序列开发西瓜EST-SSR标记是一种有效的方法。
(2)对独家发现的西瓜白化致死近等基因系和F2代群体进行SRAP标记和遗传分析,结果在400对引物中未找到与西瓜白化致死基因连锁的分子标记,但获得了9对SRAP引物组合的10个呈3:1分离的标记。遗传分析表明,除种皮颜色外,果皮颜色、果肉颜色、果实形状、种子大小、果柄顶部形状和果皮厚度等7个种质性状与西瓜白化致死基因之间没有连锁关系。但上述10个SRAP标记与7个性状之间有一定的连锁关系,其中标记E6-M2-170和E14-M2-240之间相似性特强,连锁距离为0cM;种子大小与果皮厚度两个性状之间的连锁最紧密,连锁距离为5.6cM;果柄顶部形状与果实形状和熟性与果皮厚度之间的连锁较紧密,连锁距离分别为8.0cM和8.1cM。
(3)借鉴拟南芥蘸花进行外源DNA导入的方法,通过柱头涂抹进行西瓜T-DNA导入标记的方法研究。结果表明:品种ZXG01078坐瓜率最高,且个别单瓜有变异苗产生;卡那霉素筛选处理西瓜种子的最佳浓度500~700 mg/L,不同品种间有差异;卡那霉素筛选处理西瓜叶片的最佳部位为嫩叶或幼叶,最佳浓度4000~8000 mg/L,不同品种间差异不明显。连续两年的试验中个别单瓜后代能稳定的产生无心苗和连体苗。
Watermenlon is an important economic crops. In China, Watermelon cultivation area is maintained at 1,000,000 hectares per year. Annual output of watermelon more than 30 million tons. Watermelon has a relatively rich genetic diversity. Different germolasm resources exist large differences of trarts. However, the research in watermelon on genetic diversities started too late, especially in the application of modern molecular techniques are inadequate. In order to explore a new method to study the molecular genetic in watermelon. We utilized albino lethal near-isogenic lines of watermelon, F2 generation and their parents for the experimental material. Then using SRAP and EST-SSR molecular markers combined with the main germplasm traits for genetic analysis. In addition, the experiment also explored stigma smear to transfer T-DNA and molecular marker research in watermelon. The main results as follows:
We analysed a total of 8619 watermelon ESTs from GenBank and sought out 202 SSR contained in 191 EST sequences. The frequency of the SSRs was 2.31% and mean the distribution density was 1/19.1kb in watermelon. The dominant repeat motifs were dinucleotide and trinucleotide. AG/TG was the most repeat type in dinucleotide(46%) and AAG was the most repeat type in trinucleotide(43%).The most motif repeat times were 4 (17.8%), 7 (15.3%) and 11 (14.9%). The length was mainly distributed between 20 to 30bp.We obtained 24 primer pairs from 202 EST-SSRs in watermelon.Then utilized albino lethal near-isogenic lines of watermelon for verify these primers.We obtained 16 effective amplified primer pairs,accouting for 66.7% of the total number of designed primer pairs. The results suggest that use of public EST databases to develop EST-SSR primers of watermelon are effective method.
We utilized albino lethal near-isogenic lines of watermelon and F2 generation for the experimental material. Then using SRAP technique and genetic analysis to research, the result is that we found no molecular marker tightly linked to albino lethal gene among 400 primer pairs.On the other hand,we got 9 SRAP primer pairs which amplified out of 10 difference bands that showed 3:1 separation. Genetic analysis expressed that the rind color,flesh color, fruit shape, seed size, fruit peduncle top shape and thickness of rind et.al. 7 major germplasm traits were not linked to albino lethal gene in watermelon excepted the seed colour. There was certain linkage between the 10 SRAP molecular marker and the 7 major germplasm traits. Among them, markers E6-M2-170 and E14-M2-240 were completely linkage with a distance of 0cM. Seed size was closely linked to thickness of rind at 5.6cM. Fruit peduncle top shape and development period were linked to fruit shape and thickness of rind at 8.0cM and 8.1cM, respectively.
From the method of foreign DNA introduced to Arabidopsis thaliana via flowers dip, we used stigma smear to transfer T-DNA into watermelon and its molecular marker research. The results showed the highest fruit setting rate specie is ZXG01078 and its individual single fruit have deviant seedlings. The best concentration of kanamycin treating watermelon seeds is 500~700mg/L with differences among the species. The best position is spire leaf or youngleaf and the best concentration of kanamycin treating the watermelon leaf is 4000~8000mg/ L with no difference among the species. The offspring of individual single fruit can steadily produce no pointing and twinborn seedlings in two years experiment.
引文
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