Abstract
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.
Highlights
DNA markers have been widely used for genetic improvement of rice due to their abundant number and even distribution across the genome
10,361 large fragment insertions/ deletions (LInDels) had large insertion/deletion sizes ranging from 20 bp to 90 bp, which could be separated by agarose gel electrophoresis
Universality of Agarose-Resolvable LInDel Markers in Rice Currently, the reference genome sequences for five rice varieties have been released to the public, including sequences of the four following indica rice subspecies: 9311, Zhenshan 97 (ZS97), Minghui 63 (MH63), R498 (Du et al 2017; Yu et al 2002; Zhang et al 2016a) and one japonica rice subspecies sequence of Nipponbare (Sasaki 2005)
Summary
DNA markers have been widely used for genetic improvement of rice due to their abundant number and even distribution across the genome. Since the 1980s, three generations of DNA markers have been developed and widely utilized for the genetic improvement of plants. With the development of next-generation sequencing, SNPs and InDel have been more popular in plant studies due to their codominant, genome-wide distribution and low cost. Agarose gel electrophoresis equipment is easy to obtain; LInDel markers are expected to be used for quickly genotyping the plants that are produced in the breeding program. This makes the breeding process more autonomous and enhances the work efficiency. With the release of the genome reference sequence, InDel markers have been confirmed to be evenly spread throughout the whole genomes of many crop plants, such as cotton, rice, maize, oilseed rape and so on (Li et al 2014; Liu et al 2015a; Liu et al 2015b; Mahmood et al 2016; Moghaddam et al 2014; Shen et al 2004; Song et al 2015)
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