Hu Wei, Zhou Tianhao, Wang Pengfei, Wang Bo, Song Jiaming, Han Zhongmin, Chen Lingling, Liu Kede, Xing Yongzhong
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China.
College of Agriculture, Yangtze University, Jingzhou, 434000, China.
Rice (N Y). 2020 Jan 6;13(1):1. doi: 10.1186/s12284-019-0361-3.
The level of difficulty involved in separating marker genotypes greatly determines the utilization of such marker-aided selection (MAS) by breeders. Genotyping by use of agarose gel electrophoresis is easily accepted by breeders due to its simple requirements and easy operation in the lab. Here, we extracted 19,937 large fragment insertions/deletions (LInDels) that were 30-55 bp based on two indica rice and one japonica rice reference genome sequences. Thousands of primer pairs were designed by the Primer 3 program to amplify the corresponding LInDels, and 6582 LInDel markers with unique genome loci were reserved after being tested by e-PCR; 346 of these markers were validated in a panel of 22 cultivars by running on a 1.5% agarose gel. Most LInDel markers had a considerable number of polymorphisms. The LInDel markers have an equivalent efficiency to that of the SSR and SNP markers in identifying hybrids, estimating genetic distance and developing genetic linkage maps. The hybrid genotypes of the LInDel markers exhibited three bands, which were the result of heteroduplex formation between the insertion allele and the deletion allele. Fifty-five breeding markers, including 9 intragenic markers and 46 closely linked LInDel markers, were developed for 55 known genes that are related to yield, biotic and abiotic stress tolerance. These agarose-resolvable LInDel markers will be welcomed by breeders and will play an important role in MAS.
分离标记基因型所涉及的难度水平在很大程度上决定了育种者对这种标记辅助选择（MAS）的利用情况。利用琼脂糖凝胶电泳进行基因分型因其要求简单且在实验室操作容易而容易被育种者接受。在此，我们基于两个籼稻和一个粳稻参考基因组序列提取了19937个30 - 55 bp的大片段插入/缺失（LInDels）。通过Primer 3程序设计了数千对引物来扩增相应的LInDels，经电子PCR（e-PCR）测试后保留了6582个具有独特基因组位点的LInDel标记；其中346个标记在22个品种的样本中通过在1.5%琼脂糖凝胶上电泳进行了验证。大多数LInDel标记具有相当数量的多态性。LInDel标记在鉴定杂种、估计遗传距离和构建遗传连锁图谱方面与SSR和SNP标记具有同等效率。LInDel标记的杂种基因型呈现出三条带，这是插入等位基因和缺失等位基因之间形成异源双链的结果。针对55个与产量、生物和非生物胁迫耐受性相关的已知基因开发了55个育种标记，包括9个基因内标记和46个紧密连锁的LInDel标记。这些可通过琼脂糖分辨的LInDel标记将受到育种者的欢迎，并将在标记辅助选择中发挥重要作用。
