Marker discovery by RNA sequencing
Single nucleotide polymorphisms (SNP) are important molecular markers in plant genetics and breeding. SNP is widely employed in molecular breeding programs nowadays due to its abundance, biallelic nature, and low mutation rate. Moreover, the amenability of SNP markers for high-throughput assays makes it a marker of choice for plant breeders.
Next-generation sequencing (NGS) is a versatile tool for SNP discovery. However, the development of markers in species with a limited genomic resource like genome and marker database is challenging as the method has to identify the marker among the lines and also be able to fetch the border sequences to the marker to create an essay.
In recent days, NGS approaches such as transcriptome sequencing with marker mining analysis resulted in the generation of comprehensive transcriptome sequence data and SNP markers resources in several horticultural plants such as okra and eggplant. This approach is very useful for
- Discovery of markers for hybrid testing in crops.
- Bulked RNA sequencing between wild type, mutant and F2 lines to identify genic mutations associated with the trait.
Bulked segregant analysis (BSA) using RNA-seq. Mutant and non-mutant parent plants are processed independently and the BSA analysis allows visualization of the probability of each SNP marker being in complete linkage disequilibrium with the mutated gene
SNP discovery for the hybrid testing marker. Each parent and F1 line are processed independently. In this example, parent-1 is denovo assembled and used as a reference to align parent 2 and F1 to identify the marker
RNAseq is an affordable and accurate method for plant species with limited genomic resources as RNA-seq data sets themselves can be used to create sequence assemblies for subsequent mapping of RNA-seq reads. It can be used for comparisons of samples not necessarily generated together, or even as part of the same experiment allowing the RNAseq data to be a useful resource for current experiments and future studies.
If you are interested in SNP marker development for (1) diversity study among different lines, (2) Hybrid testing markers for your hybrid QC or (3) bulk segregant analysis(BSA) for mapping mutant genes you can choose the RNA seq approach.