Development and application of microsatellites in candidate genes related to wood properties in the Chinese white poplar (Populus tomentosa Carr.)

Abstract

Gene-derived simple sequence repeats (genic SSRs), also known as functional markers, are often preferred over random genomic markers because they represent variation in gene coding and/or regulatory regions. We characterized 544 genic SSR loci derived from 138 candidate genes involved in wood formation, distributed throughout the genome of Populus tomentosa, a key ecological and cultivated wood production species. Of these SSRs, three-quarters were located in the promoter or intron regions, and dinucleotide (59.7%) and trinucleotide repeat motifs (26.5%) predominated. By screening 15 wild P. tomentosa ecotypes, we identified 188 polymorphic genic SSRs with 861 alleles, 2-7 alleles for each marker. Transferability analysis of 30 random genic SSRs, testing whether these SSRs work in 26 genotypes of five genus Populus sections (outgroup, Salix matsudana), showed that 72% of the SSRs could be amplified in Turanga and 100% could be amplified in Leuce. Based on genotyping of these 26 genotypes, a neighbour-joining analysis showed the expected six phylogenetic groupings. In silico analysis of SSR variation in 220 sequences that are homologous between P. tomentosa and Populus trichocarpa suggested that genic SSR variations between relatives were predominantly affected by repeat motif variations or flanking sequence mutations. Inheritance tests and single-marker associations demonstrated the power of genic SSRs in family-based linkage mapping and candidate gene-based association studies, as well as marker-assisted selection and comparative genomic studies of P. tomentosa and related species.

Figure 1.

Flow diagram of P. tomentosa genic SSR marker development and applications in this study.

Figure 2.

(A) Distribution of repeat motifs in 544 genic SSRs identified in 138 P. tomentosa genes; (B) Distribution of dinucleotide repeat motifs detected in 138 P. tomentosa genes; (C) Distribution of repeat numbers in di- and trinucleotide SSRs. Bars of different colours show the number of genic SSRs from dinucleotide and trinucleotide.

Figure 3.

Phylogenic relationship among 26 genotypes belonging to 5 sections of the genus Populus (S. matsudana as outgroup) based on 30 polymorphic genic SSR markers. The different colour branches denote the divergent clusters.

Figure 4.

Alignment and comparison of variations and mutations in genic SSRs with sequences homologous between P. tomentosa (01) and P. trichocarpa (02), based on a number of genic SSRs. Ten types of mutations and variations for genic SSR loci with their flanking regions were identified (See Table 2 for details).