High-density genetic linkage mapping reveals low stability of QTLs across environments for economic traits in Eucalyptus.

Introduction: Eucalyptus urophylla, E. tereticornis and their hybrids are the most important commercial forest tree species in South China where they are grown for pulpwood and solid wood production. Construction of a fine-scale genetic linkage map and detecting quantitative trait loci (QTL) for economically important traits linked to these end-uses will facilitate identification of the main candidate genes and elucidate the regulatory mechanisms. Method: A high-density consensus map (a total of 2754 SNPs with 1359.18 cM) was constructed using genotyping by sequencing (GBS) on clonal progenies of E. urophylla × tereticornis hybrids. QTL mapping of growth and wood property traits were conducted in three common garden experiments, resulting in a total of 108 QTLs. A total of 1052 candidate genes were screened by the efficient combination of QTL mapping and transcriptome analysis. Results: Only ten QTLs were found to be stable across two environments, and only one (qSG10Stable mapped on chromosome 10, and associated with lignin syringyl-to-guaiacyl ratio) was stable across all three environments. Compared to other QTLs, qSG10Stable explained a very high level of phenotypic variation (18.4-23.6%), perhaps suggesting that QTLs with strong effects may be more stably inherited across multiple environments. Screened candidate genes were associated with some transcription factor families, such as TALE, which play an important role in the secondary growth of plant cell walls and the regulation of wood formation. Discussion: While QTLs such as qSG10Stable, found to be stable across three sites, appear to be comparatively uncommon, their identification is likely to be a key to practical QTL-based breeding. Further research involving clonally-replicated populations, deployed across multiple target planting sites, will be required to further elucidate QTL-by-environment interactions.

Transcriptome-derived microsatellite markers for population diversity analysis in Archidendron clypearia (Jack) I.C. Nielsen.

BACKGROUND: The medicinal woody leguminous genus Archidendron F. Mueller serves as important herbal resources for curing upper respiratory tract infection, acute pharyngitis, tonsillitis, and gastroenteritis. However, genomic resources including transcriptomic sequences and molecular markers remain scarce in the genus. METHODS AND RESULTS: Transcriptome sequencing, genic microsatellite marker development, and population diversity analysis were conducted in Archidendron clypearia (Jack) I.C. Nielsen. Flower and flower bud transcriptomes were de novo assembled into 173,172 transcripts, with an average transcript length of 1597.3 bp and an N50 length of 2427 bp. A total of 34,701 microsatellite loci were identified from 26,716 (15.4 %) transcripts. Primer pairs were designed for 718 microsatellite loci, of which 456 (63.5 %) were polymorphic. Of the 456 polymorphic markers, 391 (85.7 %) and 402 (88.1 %) were transferable to A. lucidum (Benth.) I.C. Nielsen and A. multifoliolatum (H.Q. Wen) T.L. Wu, respectively. Using a subset of 15 microsatellite markers, relatively high genetic diversity was detected over two A. clypearia populations, with overall mean expected heterozygosity (He) being 0.707 and demonstrating the necessity of conservation. Relatively low differentiation between the two populations was revealed despite the distant separation (about 700 km), with overall inbreeding coefficient of sub-population to the total population (Fst) being 8.7 %. CONCLUSIONS: This study represents the first attempt to conduct transcriptome sequencing, SSR marker development, and population genetics analysis in the medicinally important genus Archidendron. Our results will offer valuable resources and information for further genetic studies and practical applications in Archidendron and the related taxa.

The first identification of genomic loci in plants associated with resistance to galling insects: a case study in Eucalyptus L'Hér. (Myrtaceae).

Genomic loci related with resistance to gall-inducing insects have not been identified in any plants. Here, association mapping was used to identify molecular markers for resistance to the gall wasp Leptocybe invasa in two Eucalyptus species. A total of 86 simple sequence repeats (SSR) markers were screened out from 839 SSRs and used for association mapping in E. grandis. By applying the mixed linear model, seven markers were identified to be associated significantly (P ≤ 0.05) with the gall wasp resistance in E. grandis, including two validated with a correction of permutation test (P ≤ 0.008). The proportion of the variance in resistance explained by a significant marker ranged from 3.3% to 37.8%. Four out of the seven significant associations in E. grandis were verified and also validated (P ≤ 0.073 in a permutation test) in E. tereticornis, with the variation explained ranging from 24.3% to 48.5%. Favourable alleles with positive effect were also mined from the significant markers in both species. These results provide insight into the genetic control of gall wasp resistance in plants and have great potential for marker-assisted selection for resistance to L. invasa in the important tree genus Eucalyptus.

Genome scans for divergent selection in natural populations of the widespread hardwood species Eucalyptus grandis (Myrtaceae) using microsatellites.

Identification of loci or genes under natural selection is important for both understanding the genetic basis of local adaptation and practical applications, and genome scans provide a powerful means for such identification purposes. In this study, genome-wide simple sequence repeats markers (SSRs) were used to scan for molecular footprints of divergent selection in Eucalyptus grandis, a hardwood species occurring widely in costal areas from 32° S to 16° S in Australia. High population diversity levels and weak population structure were detected with putatively neutral genomic SSRs. Using three FST outlier detection methods, a total of 58 outlying SSRs were collectively identified as loci under divergent selection against three non-correlated climatic variables, namely, mean annual temperature, isothermality and annual precipitation. Using a spatial analysis method, nine significant associations were revealed between FST outlier allele frequencies and climatic variables, involving seven alleles from five SSR loci. Of the five significant SSRs, two (EUCeSSR1044 and Embra394) contained alleles of putative genes with known functional importance for response to climatic factors. Our study presents critical information on the population diversity and structure of the important woody species E. grandis and provides insight into the adaptive responses of perennial trees to climatic variations.

Comparative Genomics Analyses Reveal Extensive Chromosome Colinearity and Novel Quantitative Trait Loci in Eucalyptus.

Dense genetic maps, along with quantitative trait loci (QTLs) detected on such maps, are powerful tools for genomics and molecular breeding studies. In the important woody genus Eucalyptus, the recent release of E. grandis genome sequence allows for sequence-based genomic comparison and searching for positional candidate genes within QTL regions. Here, dense genetic maps were constructed for E. urophylla and E. tereticornis using genomic simple sequence repeats (SSR), expressed sequence tag (EST) derived SSR, EST-derived cleaved amplified polymorphic sequence (EST-CAPS), and diversity arrays technology (DArT) markers. The E. urophylla and E. tereticornis maps comprised 700 and 585 markers across 11 linkage groups, totaling at 1,208.2 and 1,241.4 cM in length, respectively. Extensive synteny and colinearity were observed as compared to three earlier DArT-based eucalypt maps (two maps with E. grandis × E. urophylla and one map of E. globulus) and with the E. grandis genome sequence. Fifty-three QTLs for growth (10-56 months of age) and wood density (56 months) were identified in 22 discrete regions on both maps, in which only one colocalizaiton was found between growth and wood density. Novel QTLs were revealed as compared with those previously detected on DArT-based maps for similar ages in Eucalyptus. Eleven to 585 positional candidate genes were obained for a 56-month-old QTL through aligning QTL confidence interval with the E. grandis genome. These results will assist in comparative genomics studies, targeted gene characterization, and marker-assisted selection in Eucalyptus and the related taxa.

Development of 198 novel EST-derived microsatellites in Eucalyptus (Myrtaceae).

PREMISE OF THE STUDY: Expressed sequence tag (EST)-derived microsatellites were identified in Eucalyptus through screening the GenBank database. The loci were sequence-verified and explored for polymorphism among 20 genotypes. METHODS AND RESULTS: In total, 198 novel microsatellites were developed from 8262 unigenes, with the identity of 73.6-100% to the original sequences and presence of the expected repeat motifs. One hundred and eighty-four markers proved to be polymorphic among 10 E. urophylla and 10 E. tereticornis genotypes, with the number of alleles per locus, observed heterozygosity, and polymorphic information content being 2-17 (mean: 7.11), 0-1.0 (mean: 0.4511), and 0.0940-0.9131 (mean: 0.6571), respectively. CONCLUSIONS: These markers will be useful for germplasm characterization, genome mapping, and gene tagging for economic traits in the two species examined and may have potential for genetic applications in Eucalyptus.