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Experimental Pipeline for SNP and SSR Discovery and Genotyping Analysis of Mango (Mangifera indica L.)   

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Original research article

A brief version of this protocol appeared in:
BMC Plant Biology
Dec 2015

Abstract

Establishing a reservoir of polymorphic markers is an important key for marker-assisted breeding. Many crops are still lack of such genomic infrastructure. Single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) are useful as markers because they are widespread over the genome and many technologies were developed for high throughput genotyping. We present here a pipeline for developing a reservoir of SNP and SSR markers for Mangifera indica L. as an example for fruit tree crops having no genomic information available. Our pipeline includes de novo assembly of reference transcriptome with MIRA and CAP3 based on reads produced by 454-GS FLX technology; Polymorphic loci discovery by alignment of Illumina resequencing to the transcriptome reference; Identifying a subset of loci that are polymorphic in the entire germplasm collection for downstream diversity analysis by genotyping with Fluidigm technology.

Keywords: SNP discovery, Diversity, Marker-assisted selection, SSR

Background

Considerations of high-throughput sequencing: This pipeline does not include RNA/DNA extraction and other molecular biology lab protocols for next generation sequencing (NGS). It is common to outsourcing NGS. Therefore, it includes DNA preparation for genotyping only. Before describing the pipeline below, we would like to comment about the considerations regarding the sequencing.

Assumption: In this pipeline, we assume a non-model organism which has no genomic infrastructure at all. For marker discovery, one will need a reference and resequencing to discover the polymorphism. The ultimate reference is a genome. However, due to the fact that having a good draft or a complete reference genome is still expensive task our recommendation is to sequence a reference transcriptome from a pool of tissues. The pool of tissues should compensate the unequal gene representation as a result of tissue-specific expression.

Technology: For the purpose of a reference transcriptome sequencing, 454-GS Flx Titanium or any long reads NGS technology is preferred. For marker discovery by resequencing, a pool of genomic DNA (gDNA) from the population under study is a cost-effective solution. Polymorphic loci in such pool are representative sample of the polymorphic loci in the population. Here the important factor is the reads’ depth which should strive to an average coverage of 50x and no less than 20x. In a case of large genomes the choice of gDNA resequencing might be too expensive to get coverage of 50x. Alternatively, mRNA extraction of a pool of tissues and population individuals would be a cheaper option.

The aim of this protocol is to provide a pipeline (Figure 1) for the bioinformatics and genomics support unit that assist the breeder of a crop which has no genomic information to establish a set of polymorphic SNP and SSR markers. This set can be used for marker-assisted breeding studies as well as for exploring the diversity in the crop’s germplasm collection diversity.


Figure 1. Flowchart of a pipeline for marker discovery. The reference transcriptome here (represented as a database shape) is the link connecting function annotation with genetic variation.

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Sharabi-Schwager, M., Rubinstein, M., Ish shalom, M., Eshed, R., Rozen, A., Sherman, A., Cohen, Y. and Ophir, R. (2016). Experimental Pipeline for SNP and SSR Discovery and Genotyping Analysis of Mango (Mangifera indica L.). Bio-protocol 6(16): e1910. DOI: 10.21769/BioProtoc.1910.
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