Kayesh E, Zhang Y Y, Liu G S, Bilkish N, Sun X, Leng X P, Fang J G
College of Horticulture, Nanjing Agricultural University, Nanjing, China.
Genet Mol Res. 2013 Sep 23;12(3):3871-8. doi: 10.4238/2013.September.23.5.
The objectives of this investigation were to develop and validate the expressed sequence tag (EST)-simple sequence repeat (SSR) markers from large EST sequences, and to study the segregation and distribution of SSRs within two grapevine parental lines. In total, 94 F₁ lines crossed between "Early Rose" and "Red Globe" were studied. Approximately 2100 EST-SSR sequences of Vitis vinifera L. were searched for SSRs and analyzed for the design of polymerase chain reaction (PCR) primers amplifying the SSR-rich regions. Trinucleotide repeats were found to be the most abundant, followed by other nucleotide repeats. A total of 182 SSR primer pairs were first developed for the study on the parental polymorphism. Among the 182 SSR primers, 142 primer pairs (78%) could amplify the anticipated PCR products, among which only 52 primer pairs (36.62%) showed polymorphism between the two parents. These polymorphic bands were further surveyed among the 94 F₁ lines, and the results showed that a total of 162 bands were amplified, and 98 of them were polymorphic in both parents (60.86% polymorphism), with an average of 1.88 polymorphic DNA bands for each primer pair. After testing with the chi-square test, 33 of the clearly amplified polymorphic bands followed a 3:1 ratio, and 37 followed a 1:1 ratio. The rest showed distorted segregation ratios.
本研究的目的是从大量表达序列标签（EST）中开发并验证EST-简单序列重复（SSR）标记，并研究SSR在两个葡萄亲本系中的分离和分布情况。总共研究了“早玫瑰”和“红地球”杂交产生的94个F₁代株系。在葡萄（Vitis vinifera L.）的约2100个EST-SSR序列中搜索SSR，并分析用于扩增富含SSR区域的聚合酶链反应（PCR）引物的设计。发现三核苷酸重复最为丰富，其次是其他核苷酸重复。总共首次开发了182对SSR引物用于亲本多态性研究。在这182对SSR引物中，有142对引物（78%）能扩增出预期的PCR产物，其中只有52对引物（36.62%）在两个亲本之间表现出多态性。在94个F₁代株系中进一步检测这些多态性条带，结果显示总共扩增出162条带，其中98条在两个亲本中均为多态性（多态性为60.86%），每个引物对平均有1.88条多态性DNA条带。经卡方检验，33条清晰扩增的多态性条带符合3:1的比例，37条符合1:1的比例。其余的显示出偏离的分离比例。
