Genetic mapping of Pinus flexilis major gene (Cr4) for resistance to white pine blister rust using transcriptome-based SNP genotyping

Abstract

BackgroundLinkage of DNA markers with phenotypic traits provides essential information to dissect clustered genes with potential phenotypic contributions in a target genome region. Pinus flexilis E. James (limber pine) is a keystone five-needle pine species in mountain-top ecosystems of North America. White pine blister rust (WPBR), caused by a non-native fungal pathogen Cronartium ribicola (J.C. Fisch.), has resulted in mortality in this conifer species and is still spreading through the distribution. The objective of this research was to develop P. flexilis transcriptome-wide single nucleotide polymorphism (SNP) markers using RNA-seq analysis for genetic mapping of the major gene (Cr4) that confers complete resistance to C. ribicola.ResultsNeedle tissues of one resistant and two susceptible seedling families were subjected to RNA-seq analysis. In silico SNP markers were uncovered by mapping the RNA-seq reads back to the de novo assembled transcriptomes. A total of 110,573 in silico SNPs and 2,870 indels were identified with an average of 3.7 SNPs per Kb. These SNPs were distributed in 17,041 unigenes. Of these polymorphic P. flexilis unigenes, 6,584 were highly conserved as compared to the genome sequence of P. taeda L (loblolly pine). High-throughput genotyping arrays were designed and were used to search for Cr4-linked genic SNPs in megagametophyte populations of four maternal trees by haploid-segregation analysis. A total of 32 SNP markers in 25 genes were localized on the Cr4 linkage group (LG). Syntenic relationships of this Cr4-LG map with the model conifer species P. taeda anchored Cr4 on Pinus consensus LG8, indicating that R genes against C. ribicola have evolved independently in different five-needle pines. Functional genes close to Cr4 were annotated and their potential roles in Cr4-mediated resistance were further discussed.ConclusionsWe demonstrated a very effective, low-cost strategy for developing a SNP genetic map of a phenotypic trait of interest. SNP discovery through transcriptome comparison was integrated with high-throughput genotyping of a small set of in silico SNPs. This strategy may be applied to mapping any trait in non-model plant species that have complex genomes. Whole transcriptome sequencing provides a powerful tool for SNP discovery in conifers and other species with complex genomes, for which sequencing and annotation of complex genomes is still challenging. The genic SNP map for the consensus Cr4-LG may help future molecular breeding efforts by enabling both Cr4 positional characterization and selection of this gene against WPBR.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3079-2) contains supplementary material, which is available to authorized users.

Limber pine (Pinus flexilis James) genetic map constructed by exome‐seq provides insight into the evolution of disease resistance and a genomic resource for genomics‐based breeding

“…The accuracy of a genetic map is affected by several factors, including the distribution of targeted genomic regions, the quality of genotypic data, the number of mapped markers and the mapping population size. In this study, a limber pine transcriptome (Liu et al ., ) was used for exome capture because a limber pine genome sequence is not yet available. A total of 14 706 unique protein‐coding transcripts were selected as exome‐enrichment targets.…”

“…The first comprehensive genomic resource for limber pine, a newly assembled transcriptome, has enabled the in silico mining of SNPs in a large number of expressed genes across the genome through comparison of transcriptomes among different genotypes. This work anchored a limber pine R gene ( Cr4 ) on the Pinus consensus linkage group 8 (LG8) by synteny‐based comparative mapping (Liu et al ., ). Nonetheless, genetic and genomic research on the limber pine has lagged behind the need and potential utility for its breeding and conservation.…”

Genetic Diversity and Population Structure of Whitebark Pine (Pinus albicaulis Engelm.) in Western North America

“…[40]. RNA-seq analysis using Illumia HiSeq platforms has been applied to the development of genomic resources and molecular tools for breeding programs of western white pine and limber pine [26,30,32,41]. However, limitations are still present for application of cutting-edge technologies to five-needle pine species due to the huge size (20~30 GB) of their highly repetitive genomes [25,29,42].…”

“…To design genotyping arrays, SNPs within the WBP transcriptome were evaluated as previously reported [26,32]. We selected SNPs based on their variant types (non-coding vs .…”

“…Genomic DNA was extracted and purified from needle tissues individually using a QIAGEN DNeasy plant mini kit (Qiagene, CA, USA). SNP genotyping was performed as previously described [26,32] using a Sequenom iPlex MASSARRAY platform at Laval University (Quebec City, Canada).…”