Development of single nucleotide polymorphism markers in the large and complex rubber tree genome using next-generation sequence data

Abstract

The development of single nucleotide polymorphism (SNP) markers provides the opportunity to improve many areas of plant breeding and population genetics. Unfortunately, for species such as the rubber tree (Hevea brasiliensis), the use of next-generation sequencing for genomic SNP discovery is very difficult because of the large genome size and the abundance of repeated sequences. Access to a set of validated SNP markers is a significant advantage for rubber researchers who wish to apply SNPs in scientific research. Here, we performed genomic sequencing of H. brasiliensis and generated 10,993,648 short reads, which were assembled into 10,071 contigs (N50 = 3078) by a de novo assembly strategy. A total of 2446 contigs presented no hits in the current H. brasiliensis genome assembly and may therefore be considered novel genomic sequences of rubber tree. A total of 143 putative polymorphic positions were selected, gene annotations were available for 58.7 % of the markers, and all of the sequences could be anchored to the released H. brasiliensis genome. These SNPs were validated in eight genotypes of H. brasiliensis and 15 F1 plants from a mapping population, resulting in 30 (20.9 %) positions correctly classified. The analysis revealed key candidate genes responsible for defence mechanisms and provided markers for further genetic improvement of Hevea in breeding programmes. (Résumé d'auteur

High-Resolution Genetic Map and QTL Analysis of Growth-Related Traits of Hevea brasiliensis Cultivated Under Suboptimal Temperature and Humidity Conditions

“…Compared with other species from Malpighiales, Hevea species contain the largest genomes but might also have the largest repeat content ( Tang et al, 2016 ). Seventy percent of the H. brasiliensis genome is composed of repeat elements, which, together with the lack of information at the chromosomal level, leads to difficulties with assembly ( Rahman et al, 2013 ) and challenges in the identification and development of SNP assays ( Souza et al, 2016 ). Moreover, differences in the estimated rubber tree genome size have been observed by microdensitometry, flow cytometry ( Bennett and Smith, 1997 ), and 17-mer sequence estimation ( Tang et al, 2016 ) approaches.…”

“…Ninety-six other polymorphic SNPs obtained from genomic ( Souza et al, 2016 ) and transcriptomic ( Salgado et al, 2014 ) data were also selected for genotyping using the BioMark TM Real-Time PCR system with the 96.96 Dynamic Array TM IFC (Fluidigm Corporation, San Francisco, CA, United States). For kompetitive allele-specific PCR (KASP) assays, 1.4 μL of KASP assay reagent, 5 μL of 2X assay loading reagent, 3.6 μL of ultrapure water, and 2.8 μL of KASP assay mix (12 μM allele-specific forward primer and 30 μM reverse primer) were used.…”

Genetic Evaluation of Natural Populations of the Endangered Conifer Thuja koraiensis Using Microsatellite Markers by Restriction-Associated DNA Sequencing

“…koraiensis has been hindered by a lack of genomic information, which are usually not available for rare and endangered species. The rapid developments of next-generation sequencing (NGS) technologies offer the possibility to generate large amounts of DNA sequences and develop molecular marker from nonmodel plants such as Ziziphus jujube [ 23 ], Hevea brasiliensis [ 24 ], and Pistacia vera [ 25 ]. Restriction-associated DNA (RAD) sequencing is one such NGS-based method which uses restriction enzymes on genomic DNA for complexity reduction [ 26 , 27 ].…”

Biosynthesis of Natural Rubber: Current State and Perspectives

“…H. brasiliensis has the highest number of disease resistance-related genes compared with other Euphorbiaceae genomes, indicating that these genes may play import roles in response to stresses and diseases caused by fungal pathogens [22]. Through genome or RNA sequencing, molecular markers such as simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) have been identified, and a high-density genetic linkage map was constructed for H. brasiliensis [94,95,96,97,98,99,100,101,102]. These data would be a valuable information resource for future genetic analyses such as quantitative trait loci (QTL) identification, linkage disequilibrium investigations and phylogenetic studies and a powerful tool for breeding elite rubber tree cultivars.…”