Characterization and identification of (CT)n microsatellites in soybean using sheared genomic libraries
Characterization and identification of (CT)n microsatellites in soybean using sheared genomic libraries
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DOI：
10.1093/dnares/7.2.103
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发表时间：
2000-04-28
期刊：
影响因子：
4.1
通讯作者：
Harada, K
中科院分区：
文献类型：
--
作者：
Hossain, KG;Kawai, H;Harada, K
通过移液和DNase I处理大豆DNA，构建了三个小插入片段（300 bp类似于600 bp）剪切的基因组文库。对来自每个文库的约15，000个克隆进行CT-简单序列重复（CT-SSR）筛选。CT-SSR在大豆基因组中含量丰富，估计频率约为每110 kb基因组DNA一个SSR。对129个阳性克隆测序后，对阳性克隆中CT重复序列的重复类型和频率进行了表征。设计了49对引物，并初步评价了它们扩增一组6个品种基因组DNA的能力，包括一个作图家族的父母。扩增产物经10%PAGE分析。在53 ℃退火温度下，88%的引物能够扩增出所有这些基因组DNA，其中22个（45%）在6个品种中具有多态性，其中14个在作图家系的亲本中具有多态性。利用16个大豆品种的DNA对多态性引物进行了进一步的等位基因信息分析。平均等位基因数为4个，最高多态信息含量为0.84。利用现有的大豆RFLP图谱，对其中14个SSR进行了作图。这些发现将促进我们对大豆基因组的理解，并有助于绘制基因组图谱和区分该物种的密切相关品种。
Three small insert (300 similar to 600 bp) sheared genomic libraries were constructed by pipetting and DNase I treatment of soybean DNA. About 15,000 clones from each library were screened for CT- simple sequence repeats (CT-SSRs). The CT-SSRs were abundant in the soybean genome at an estimated frequency of approximately one SSR per 110 kb of genomic DNA. Following the sequencing of 129 positive clones, the repeat types and frequency of CT repeats among the positive clones were characterized. Forty-nine primer pairs were designed and preliminarily evaluated for their ability to amplify genomic DNA from a set of six varieties, including parents of a mapping family. Amplified products were analyzed by 10% PAGE. Eighty-eight percent of the designed primers were able to amplify all these genomic DNAs using a single PCR profile of 53 degrees C annealing temperature, of which 22 (45%) were polymorphic in the six varieties, and 14 of them were polymorphic in the parents of the mapping family. The polymorphic primer sets were further assessed for allelic information using DNA from 16 soybean cultivars. The average number of alleles was 4, ranging from 2 to 7 with the highest polymorphism information content value 0.84. Fourteen of these SSRs were mapped, using an existing soybean RFLP map. The findings presented here will advance our understanding of the soybean genome, and assist in the mapping genome and discrimination of closely related varieties of this species.