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.

The Extent of Migration of the Holliday Junction Is a Crucial Factor for Gene Conversion in Rhizobium etli

ABSTRACT Gene conversion, defined as the nonreciprocal transfer of DNA, is one result of homologous recombination. Three steps in recombination could give rise to gene conversion: (i) DNA synthesis for repair of the degraded segment, (ii) Holliday junction migration, leading to heteroduplex formation, and (iii) repair of mismatches in the heteroduplex. There are at least three proteins (RuvAB, RecG, and RadA) that participate in the second step. Their roles have been studied for homologous recombination, but evidence of their relative role in gene conversion is lacking. In this work, we showed the effect on gene conversion of mutations in ruvB, recG, and radA in Rhizobium etli, either alone or in combination, using a cointegration strategy previously developed in our laboratory. The results indicate that the RuvAB system is highly efficient for gene conversion, since its absence provokes smaller gene conversion segments than those in the wild type as well as a shift in the preferred position of conversion tracts. The RecG system possesses a dual role for gene conversion. Inactivation of recG leads to longer gene conversion tracts than those in the wild type, indicating that its activity may hinder heteroduplex extension. However, under circumstances where it is the only migration activity present (as in the ruvB radA double mutant), conversion segments can still be seen, indicating that RecG can also promote gene conversion. RadA is the least efficient system in R. etli but is still needed for the production of detectable gene conversion tracts.