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TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea).

ABSTRACT: Targeting Induced Local Lesions in Genomes (TILLING) is a powerful reverse genetics approach for functional genomics studies. We used high-throughput sequencing, combined with a two-dimensional pooling strategy, with either minimum read percentage with non-reference nucleotide or minimum variance multiplier as mutation prediction parameters, to detect genes related to abiotic and biotic stress resistances. In peanut, lipoxygenase genes were reported to be highly induced in mature seeds infected with Aspergillus spp., indicating their importance in plant-fungus interactions. Recent studies showed that phospholipase D (PLD) expression was elevated more quickly in drought sensitive lines than in drought tolerant lines of peanut. A newly discovered lipoxygenase (LOX) gene in peanut, along with two peanut PLD genes from previous publications were selected for TILLING. Additionally, two major allergen genes Ara h 1 and Ara h 2, and fatty acid desaturase AhFAD2, a gene which controls the ratio of oleic to linoleic acid in the seed, were also used in our study. The objectives of this research were to develop a suitable TILLING by sequencing method for this allotetraploid, and use this method to identify mutations induced in stress related genes.We screened a peanut root cDNA library and identified three candidate LOX genes. The gene AhLOX7 was selected for TILLING due to its high expression in seeds and roots. By screening 768 M2 lines from the TILLING population, four missense mutations were identified for AhLOX7, three missense mutations were identified for AhPLD, one missense and two silent mutations were identified for Ara h 1.01, three silent and five missense mutations were identified for Ara h 1.02, one missense mutation was identified for AhFAD2B, and one silent mutation was identified for Ara h 2.02. The overall mutation frequency was 1 SNP/1,066 kb. The SNP detection frequency for single copy genes was 1 SNP/344 kb and 1 SNP/3,028 kb for multiple copy genes.Our TILLING by sequencing approach is efficient to identify mutations in single and multi-copy genes. The mutations identified in our study can be used to further study gene function and have potential usefulness in breeding programs.

SUBMITTER: Guo Y

PROVIDER: S-EPMC4369367 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea).

Guo Yufang Y Abernathy Brian B Zeng Yajuan Y Ozias-Akins Peggy P

BMC genomics 20150307

<h4>Background</h4>Targeting Induced Local Lesions in Genomes (TILLING) is a powerful reverse genetics approach for functional genomics studies. We used high-throughput sequencing, combined with a two-dimensional pooling strategy, with either minimum read percentage with non-reference nucleotide or minimum variance multiplier as mutation prediction parameters, to detect genes related to abiotic and biotic stress resistances. In peanut, lipoxygenase genes were reported to be highly induced in mat ...[more]

PMID: 25881128

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Project description:BackgroundAllergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal, but avoidance is difficult due to the prevalence of peanut-derived products in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins through mutagenesis. Other seed quality traits could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach, was used to identify mutations affecting seed traits in peanut.ResultsTwo similar copies of a major allergen gene, Ara h 1, have been identified in tetraploid peanut, one in each subgenome. The same situation has been shown for major allergen Ara h 2. Due to the challenge of discriminating between homeologous genes in allotetraploid peanut, nested PCR was employed, in which both gene copies were amplified using unlabeled primers. This was followed by a second PCR using gene-specific labeled primers, heteroduplex formation, CEL1 nuclease digestion, and electrophoretic detection of labeled fragments. Using ethyl methanesulfonate (EMS) as a mutagen, a mutation frequency of 1 SNP/967 kb (3,420 M2 individuals screened) was observed. The most significant mutations identified were a disrupted start codon in Ara h 2.02 and a premature stop codon in Ara h 1.02. Homozygous individuals were recovered in succeeding generations for each of these mutations, and elimination of Ara h 2.02 protein was confirmed. Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses. TILLING also was used to identify mutations in fatty acid desaturase AhFAD2 (also present in two copies), a gene which controls the ratio of oleic to linoleic acid in the seed. A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein. A mutation in AhFAD2A was predicted to restore function to the normally inactive enzyme.ConclusionsThis work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity. TILLING in peanut can be extended to virtually any gene, and could be used to modify other traits such as nutritional properties of the seed, as shown in this study.

Project description:Bacterial wilt (BW) caused by Ralstonia solanacearum is a serious, global, disease of peanut (Arachis hypogaea L.), but it is especially destructive in China. Identification of DNA markers linked to the resistance to this disease will help peanut breeders efficiently develop resistant cultivars through molecular breeding. A F2 population, from a cross between disease-resistant and disease-susceptible cultivars, was used to detect quantitative trait loci (QTL) associated with the resistance to this disease in the cultivated peanut. Genome-wide SNPs were identified from restriction-site-associated DNA sequencing tags using next-generation DNA sequencing technology. SNPs linked to disease resistance were determined in two bulks of 30 resistant and 30 susceptible plants along with two parental plants using bulk segregant analysis. Polymorphic SSR and SNP markers were utilized for construction of a linkage map and for performing the QTL analysis, and a moderately dense linkage map was constructed in the F2 population. Two QTL (qBW-1 and qBW-2) detected for resistance to BW disease were located in the linkage groups LG1 and LG10 and account for 21 and 12 % of the bacterial wilt phenotypic variance. To confirm these QTL, the F8 RIL population with 223 plants was utilized for genotyping and phenotyping plants by year and location as compared to the F2 population. The QTL qBW-1 was consistent in the location of LG1 in the F8 population though the QTL qBW-2 could not be clarified due to fewer markers used and mapped in LG10. The QTL qBW-1, including four linked SNP markers and one SSR marker within 14.4-cM interval in the F8, was closely related to a disease resistance gene homolog and was considered as a candidate gene for resistance to BW. QTL identified in this study would be useful to conduct marker-assisted selection and may permit cloning of resistance genes. Our study shows that bulk segregant analysis of genome-wide SNPs is a useful approach to expedite the identification of genetic markers linked to disease resistance traits in the allotetraploidy species peanut.

Dataset Information

TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea).

Publications

TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea).

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