De Novo Assembly and Characterization of Bud, Leaf and Flowers Transcriptome from Juglans Regia L. for the Identification and Characterization of New EST-SSRs

Abstract

Persian walnut (Juglans regia L.), valued for both its nut and wood, is an ecologically important temperate tree species native to the mountainous regions of central Asia. Despite its importance, there are still few transcriptomic resources in public databases for J. regia, limiting gene discovery and breeding. Here, more than 49.9 million sequencing reads were generated using Illumina sequencing technology in the characterization of the transcriptome of four J. regia organs (bud, leaf, female flowers, and male flowers). De novo assembly yielded 117,229 unigenes with an N50 of 1955 bp. Based on sequence similarity searches against known proteins, a total of 20,413 (17.41%) genes were identified and annotated. A set of 27,584 unigenes with SSR (simple sequence repeats) motifs were identified as potential molecular markers, and a sample of 77 of these EST-SSRs (express sequence tags) were further evaluated to validate their amplification and assess their polymorphism. Next, we developed 39 polymorphic microsatellite markers to screen 88 Persian walnut individuals collected from 11 populations. These markers and transcriptomic resources will be useful for future studies of population genetic structure, evolutionary ecology, and breeding of Persian walnut and other Juglans species.

Population genetic structure and adaptive differentiation of iron walnut Juglans regia subsp. sigillata in southwestern China

“…Total genomic DNA was isolated from the dried leaf tissue using a Plant Genomic DNA extraction kit from TIANGEN (TIANGEN, Beijing, China). Genotypes of all DNA samples were detected using 22 pairs of microsatellite primers developed for Juglans (Dang et al, , ; Hu et al, , ). Each forward primer was marked with fluorescent dye (FAM, TAMRM, HEX, and ROX).…”

“…Transcriptome data generated by high‐throughput sequencing have been an excellent resource for SSR marker development (Dang et al, ; Hu et al, ). EST‐SSRs have a variety of uses; some sequences contain polymorphisms that are transferable to related taxa (Feng et al, ).…”

“…Here, we use a multidisciplinary approach including molecular phylogeographic, ecological niche modeling, and phylogenetic approaches to investigate the biogeography, genetic structure, and demographic history of iron walnut in China. Specifically, we used 22 simple sequence repeat (EST-SSR) markers and sequence variation at five chloroplast fragments (cpDNAs) to (a) determine the genetic diversity and population structure of J. sigillata at chloroplast DNA sequences and Genotypes of all DNA samples were detected using 22 pairs of microsatellite primers developed for Juglans (Dang et al, 2015(Dang et al, , 2016Hu et al, 2015Hu et al, , 2016. Each forward primer was marked with fluorescent dye (FAM, TAMRM, HEX, and ROX).…”

Walnut: past and future of genetic improvement

“…The following sections depict an overview of these molecular studies and their application (Table 2). Woeste et al 2002;Dangl et al 2005;Hoban et al 2008;Zhang et al 2010;Qi et al 2011;Yi et al 2011;Wu et al 2012;Zhang et al 2013;Chen et al 2013;Najafi et al 2014;Topçu et al 2015;Ikhsan et al 2016;Dang et al 2015;Hu et al 2015;Dang et al 2016 Interspecific hybrids identification Pollegioni et al 2008Pollegioni et al , 2009 Genetic diversity/relationships determination Foroni et al 2005;Dangl et al 2005;Victory et al 2006;Robichaud et al 2006;Wang et al 2008;Aradhya et al 2009;Gunn et al 2010;Karimi et al 2010;Bai et al 2010;Mohsenipoor et al 2010;Ebrahimi et al 2011;Ruiz-Garcia et al 2011;Pollegioni et al 2011;Qi et al 2011;Grauke et al 2012;Mahmoodi et al 2013;Zhang et al 2013;Pop et al 2013;Najafi et al 2014;Dang et al 2015;Hu et al 2015;Han et al 2016;Noor Shah et al 2016;Ćelepirović et al 2016;Wang et al 2016;…”

Management of a Walnut Germplasm Collection: Which of SSR or SNP Markers Are Most Suitable to Preserve Biodiversity?

“…regia, by reviewing the literature [37][38][39]. When working with biallelic markers such as SNPs, it is known that the genetic distances can be equivalent to those calculated with SSRs, using this formula: n(k-1), where k is the average number of alleles per locus, and n is the number of loci [40].…”