Ritschel Patricia Silva, Lins Tulio Cesar de Lima, Tristan Rodrigo Lourenço, Buso Gláucia Salles Cortopassi, Buso José Amauri, Ferreira Márcio Elias
Cellular Biology Department, IB - University of Brasília (UnB) - Campus Universitário, Asa Norte, CEP 70.910-900, Brasilia, DF, Brazil.
BMC Plant Biol. 2004 May 18;4:9. doi: 10.1186/1471-2229-4-9.
Despite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species.
Seven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups.
Genomic library microsatellite enrichment is an efficient procedure for marker development in melon. One-hundred and forty-four new markers were developed from Tsp-AG/TC genomic library. This is the first reported attempt of successfully using enriched library for microsatellite marker development in the species. A sample of the microsatellite markers tested proved efficient for genetic analysis of melon, including genetic distance estimates and identity tests. Linkage analysis indicated that the markers developed are dispersed throughout the genome and should be very useful for genetic analysis of melon.
尽管在几种作物物种中基因组技术取得了巨大进展，但诸如微卫星标记等分子工具在甜瓜（Cucumis melo L.）和葫芦科物种中的可用性一直有限。微卫星标记的开发将对甜瓜的遗传分析和育种产生重大影响，特别是对标记饱和遗传图谱的构建以及标记辅助育种计划的实施。基因组微卫星富集文库可能是此类物种中标记开发的一种有效替代方法。
从一个Tsp - AG/TC微卫星富集文库中鉴定出700个含有微卫星序列的克隆，并设计合成了144对引物。当在一组甜瓜和其他葫芦科种质上测试67个微卫星标记时，65个标记在甜瓜种质中显示出DNA多态性。对于一些葫芦科物种，如黄瓜（Cucumis sativus），高达50%的甜瓜微卫星标记可直接用于DNA多态性评估，这显著降低了标记开发成本。从新的微卫星标记集中随机抽取25个微卫星标记，并在40份甜瓜种质上进行特征分析，生成了该物种的等位基因频率数据库。平均期望杂合度为0.52，范围从0.45到0.70，表明一小部分选定的标记应该足以解决关于基因型鉴定和品种保护的问题。基于微卫星多态性的遗传距离与从RAPD标记分析获得的数据一致。用55个新开发的标记开始进行图谱分析，大多数引物显示出符合孟德尔预期的分离。连锁分析检测到56%的标记之间存在连锁，分布在9个连锁群中。
基因组文库微卫星富集是甜瓜标记开发的一种有效方法。从Tsp - AG/TC基因组文库中开发了144个新标记。这是首次报道成功使用富集文库进行该物种微卫星标记开发的尝试。测试的微卫星标记样本被证明对甜瓜的遗传分析有效，包括遗传距离估计和同一性测试。连锁分析表明开发的标记分散在整个基因组中，对甜瓜的遗传分析应该非常有用。
