Development of Whole-Genome Agarose-Resolvable LInDel Markers in Rice

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.