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Selection criteria for SNP loci to maximize robustness of high-resolution melting analysis for plant breeding.

ABSTRACT: DNA markers are useful for identifying genes and developing new genetic materials for breeding and genetic research. High-resolution melting (HRM) analysis can detect a single nucleotide polymorphism (SNP) in two polymerase chain reaction (PCR) fragments as a melting temperature (Tm) difference without additional experimental steps, such as gel electrophoresis. To design a method for developing reliable HRM markers that discriminate between homozygous alleles containing SNPs, we tested new evaluation indexes related to the thermodynamics of double-stranded DNA to find one that maximizes the difference in Tm values between PCR fragments. We found that differences in the change in Gibbs free energy (?G°) correlated with actual differences in Tm values. Optimization of the nearest neighboring nucleotide (NNN) of a SNP by nucleotide substitution in the primer and reducing the size of the PCR fragment both enlarged the actual differences in Tm. The genetic DNA markers we developed by NNN substitution, termed NNNs-HRM markers, could be precisely mapped within soybean chromosomes by linkage analysis. We developed a Perl script pipeline to enable the automatic design of a massive number of NNNs-HRM markers; these scripts are freely available and would be useful for practical breeding programs for other plant species.

SUBMITTER: Yamagata Y

PROVIDER: S-EPMC6198901 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Selection criteria for SNP loci to maximize robustness of high-resolution melting analysis for plant breeding.

Yamagata Yoshiyuki Y Yoshimura Atsushi A Anai Toyoaki T Watanabe Satoshi S

Breeding science 20180904 4

DNA markers are useful for identifying genes and developing new genetic materials for breeding and genetic research. High-resolution melting (HRM) analysis can detect a single nucleotide polymorphism (SNP) in two polymerase chain reaction (PCR) fragments as a melting temperature (Tm) difference without additional experimental steps, such as gel electrophoresis. To design a method for developing reliable HRM markers that discriminate between homozygous alleles containing SNPs, we tested new evalu ...[more]

PMID: 30369824

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Project description:The chief aim of plant breeding is to improve varieties so as to increase their yield and breeding traits. One of the first stages of breeding is the selection of parental forms from the available gene pool of existing varieties. To date, costly and laborious methods based on multiple crossbreeding and phenotypic selection have been necessary to properly assess genetic resources in terms of productivity, quality parameters, and susceptibility to biotic and abiotic stressors. The often long and complicated breeding cycle can be significantly shortened through selection using DNA markers. To this end, use is made of close couplings between the marker and the locus responsible for the inheritance of the functional trait. The aim of this study was to identify single nucleotide polymorphism (SNP) and SilicoDArT markers associated with yield traits and to predict the heterosis effect for yield traits in maize (Zea mays L.). The plant material used in the research consisted of 19 inbred maize lines derived from different starting materials, and 13 hybrids resulting from crossing them. A two-year field experiment with inbred lines and hybrids was established at two Polish breeding stations on 10 m2 plots in a randomized block design with three replicates. The biometric measurements included cob length, cob diameter, core length, core diameter, number of rows of grain, number of grains in a row, mass of grain from the cob, weight of one thousand grains, and yield. The isolated DNA was subjected to DArTseq genotyping. Association mapping was performed in this study using a method based on the mixed linear model with the population structure estimated by eigenanalysis (principal component analysis of all markers) and modeled by random effects. Narew, Popis, Kozak, M Glejt, and Grom were the hybrids used in the study that showed the highest significant heterosis effect in 2013 and 2014. The similarity between parental components determined on the basis of SNP and SilicoDArT marker analysis did not exceed 33%. It was found that the genetic similarity between parental components, determined on the basis of SNP and SilicoDArT markers, reflected their degree of relationship, and correlated significantly with the effect of heterosis. As the results indicate, the parental components for heterosis crosses can be selected based on genetic similarity between parental components evaluated using SNP and SilicoDArT markers, supported with information on the origin of parental forms. Of the markers we analyzed, 76 were selected as being significantly associated with at least six traits observed in 2013 and 2014 at both the Łagiewniki and Smolice stations.

Project description:BackgroundTogether single nucleotide substitutions and small insertion/deletion variants are the most common form of sequence variation in the human gene pool.High-resolution SNP profile and/or haplotype analyses enable the identification of modest-risk susceptibility genes to common diseases, genes that may modulate responses to pharmaceutical agents, and SNPs that can affect either their expression or function. In addition, sensitive techniques for germline or somatic mutation detection are important tools for characterizing sequence variations in genes responsible for tumor predisposition. Cost-effective methods are highly desirable. Many of the recently developed high-throughput technologies are geared toward industrial scale genetic studies and arguably do not provide useful solutions for small laboratory investigator-initiated projects. Recently, the use of new fluorescent dyes allowed the high-resolution analysis of DNA melting curves (HRM).ResultsHere, we compared the capacity of HRM, applicable to both genotyping and mutation scanning, to detect genetic variations in the tumor suppressor gene TP53 with that of mutation screening by full resequencing. We also assessed the performance of a variety of available HRM-based genotyping assays by genotyping 30 TP53 SNPs. We describe a series of solutions to handle the difficulties that may arise in large-scale application of HRM to mutation screening and genotyping at the TP53 locus. In particular, we developed specific HRM assays that render possible genotyping of 2 or more, sometimes closely spaced, polymorphisms within the same amplicon. We also show that simultaneous genotyping of 2 SNPs from 2 different amplicons using a multiplex PCR reaction is feasible; the data can be analyzed in a single HRM run, potentially improving the efficiency of HRM genotyping workflows.ConclusionThe HRM technique showed high sensitivity and specificity (1.0, and 0.8, respectively, for amplicons of <400 bp) for mutation screening and provided useful genotyping assays as assessed by comparing the results with those obtained with Sanger sequencing. Thus, HRM is particularly suitable for either performing mutation scanning of a large number of samples, even in the situation where the amplicon(s) of interest harbor a common variant that may disturb the analysis, or in a context where gathering common SNP genotypes is of interest.

Dataset Information

Selection criteria for SNP loci to maximize robustness of high-resolution melting analysis for plant breeding.

Publications

Selection criteria for SNP loci to maximize robustness of high-resolution melting analysis for plant breeding.

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