Development and Characterization of Novel EST-SSRs from Larix gmelinii and Their Cross-Species Transferability.

Guojun Zhang,Di Zhou,Zhenzhen Sun,Zunzheng Wei,Min Xiong,Xian Wang,Junming Yang

doi:10.3390/molecules200712469

Abstract

A set of 899 L. gmelinii expression sequence tags (ESTs), available at the National Center of Biotechnology Information (NCBI), was employed to address the feasibility on development of simple sequence repeat (SSR) markers for Larch species. Totally, 634 non-redundant unigenes including 145 contigs and 489 singletons were finally identified and mainly involved in biosynthetic, metabolic processes and response to stress according to BLASTX results, gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) maps. Approximately 11.7% (74) unigenes contained 90 candidate SSRs, which were mainly trinucleotides (29, 32.2%) and dinucleotides (26, 28.9%). A relatively high frequency of SSRs was respectively found in the Open Reading Frame (ORF, about 54.4%) and 5′-untranslated region (5′-UTR, 31.2%), while a low frequency was observed in the 3′-untranslated region (3′-UTR, about 14.4%). Of the 45 novel EST-SSRs markers, nine were found to be polymorphic at two L. gmelinii populations. The number of alleles per locus (Na) ranged from two to four, and the observed (Ho) and expected (He) heterozygosity values were 0.200–0.733 and 0.408–0.604, respectively. The inbreeding coefficients (FIS) for all loci were more than zero except Lg41. Most of these 9EST-SSR markers were transferable to its related species L. kaempferi, L. principis-rupprechtii and L. olgensis. These novel EST-SSRs will be useful for further research on comparative genomics, genetic resources conservation and molecular breeding in larch trees.

Highlights

Larix spp., a deciduous coniferous species with a widespread distribution in the world, play vital role in maintaining the structure and functional stability of the ecosystems, and serving wood source and timber industry
The expression sequence tags (ESTs)-simple sequence repeat (SSR) transferable results in the Larix genus are in agreement with report by Yang et al [16,20], who verifiedall 6 EST-SSR developed from L. kaempferi could be successfully amplified over four species including L. gmelinii, L. principis-rupprechtii, L. olgensis and L. olgensis var Koreana
The development of functional molecular markers like EST-SSRs is becoming a valuable objective for plants, especially in marker-assisted breeding programs

Summary

Introduction

Larix spp., a deciduous coniferous species with a widespread distribution in the world, play vital role in maintaining the structure and functional stability of the ecosystems, and serving wood source and timber industry. L. leptolepis, and 1147 UniESTs from somatic embryos and a stem Suppression Subtractive Hybridization library (SSH) of larch hybrids (L. leptolepis × L. principis-rupprechtii and L. eptolepis × L. olgensis) were first employed to develop six polymorphism EST-SSRs for L. leptolepis, and all of them showed high cross-species transferability in the Larix genus [16]. Only 125 EST-SSRs produced amplification products in the parents (L. leptolepis and L. gmelinii) and its hybrids Such a small proportion of successful primer pairs may be attributed to the high levels of complexity and differences among coniferous species genomes. The objective in present study was to: (1) assemble and annotate these non-redundant sequences; (2) characterize the SSRs motif type and frequency; (3) develop an EST-SSR primer and evaluate its polymorphismin in L. gmelinii; (4) test its cross-species transferability in other related larch species

EST Data Assemble and Annotation

Plant Materials

SSR Identification and Primer Designs

Conclusions