The Sequencing-Based Mapping Method for Effectively Cloning Plant Mutated Genes

Abstract

A forward genetic approach is a powerful tool for identifying the genes underlying the phenotypes of interest. However, the conventional map-based cloning method is lengthy, requires a large mapping population and confirmation of many candidate genes in a broad genetic region to clone the causal variant. The whole-genome sequencing method clones the variants with a certain failure probability for multiple reasons, especially for heterozygotes, and could not be used to clone the mutation of epigenetic modifications. Here, we applied the highly complementary characteristics of these two methods and developed a sequencing-based mapping method (SBM) for identifying the location of plant variants effectively with a small population and low cost, which is very user-friendly for most popular laboratories. This method used the whole-genome sequencing data of two pooled populations to screen out enough markers. These markers were used to identify and narrow the candidate region by analyzing the marker-indexes and recombinants. Finally, the possible mutational sites were identified using the whole-genome sequencing data and verified in individual mutants. To elaborate the new method, we displayed the cloned processes in one Arabidopsis heterozygous mutant and two rice homozygous mutants. Thus, the sequencing-based mapping method could clone effectively different types of plant mutations and was a powerful tool for studying the functions of plant genes in the species with known genomic sequences.

Vitamin B1 THIAMIN REQUIRING1 synthase mediates the maintenance of chloroplast function by regulating sugar and fatty acid metabolism in rice

“…To identify the cles locus, we applied a sequencing‐based mapping method reported previously (Yu et al, 2021). The mapping population was generated by crossing cles (“Hwayoung” background) with “Zhonghua 11”, leading to an F 2 segregation ratio of 1:3.54 (50:177, χ 2 = 1.07, P > 0.05) (Table S1).…”

“…We obtained a serial of mutants from Rice T‐DNA Insertion Sequence Database but found a cles mutant without T‐DNA insertion. For cloning the cles mutant, we used a sequencing‐based mapping method (Yu et al, 2021). The chlorotic plants from the F2 population crossed between cles and Zhonghua 11 ( Oryza sativa ssp.…”

“…To identify the cles locus, we applied a sequencing-based mapping method reported previously (Yu et al, 2021). The…”

The Genetic Homogeneity of Uganda’s East African Highland Bananas (Mutika/Lujugira) Does Not Match the Extensive Morphological Variation Identified in this Subgroup

“…Recently, cultivar cultivar-specific markers, mutations, and chimerism were reported in Cavendish banana somaclonal variants [72]. Segregation tests, cloning, and sequencing of relevant genes are necessary to prove that any character is due to mutations or epigenetic variation [73].…”

Rice DSP controls stigma, panicle and tiller primordium initiation

“…SSR markers (http://www. gramene.org/) and detailed methods were illustrated in Yu's article (Yu et al, 2021).…”