Fagen Li

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

PREMISE OF THE STUDYnExpressed 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.nnnMETHODS AND RESULTSnIn 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.nnnCONCLUSIONSnThese 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.

Development of 240 novel EST-SSRs in Eucalyptus L’Hérit

Simple sequence repeat (SSR) markers derived from expressed sequence tag (EST) resources provide great potential for comparative mapping, direct gene tagging of quantitative trait loci and functional diversity studies. Here we report on the development of 240 novel EST-SSRs for the important tree genus Eucalyptus L’Hérit. Of the 240 EST-SSR loci, 218 (90.8xa0%) were polymorphic among 12 individuals of E. grandis Hill ex Maiden, with the number of alleles per locus (Na), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC) averaging at 5.0, 0.403, 0.598 and 0.529, respectively. High rates of cross-species/subgenus amplification were observed. The EST-SSRs developed herein would be a valuable addition of functional markers for genetics and breeding applications in a wide range of eucalypt species. The primer sequences for the 240 EST-SSRs have been deposited in the Probe database of GenBank (IDs Pr016588534–773).

A male-specific SCAR marker in Calamus simplicifolius, a dioecious rattan species endemic to China

A male-specific SCAR marker (CsMale1) was developed in the dioecious rattan species Calamus simplicifolius. The marker was 509xa0bp in size and had a GC content of 50.1%. The sequence contained two open reading frames, indicating that CsMale1 represented probably a coding genome region. No highly similar sequence was found within the GenBank database. The SCAR marker could be used for precise and quick identification of plant sex types in C. simplicifolius, especially at very early developmental stages.

Quantitative genetics of cold hardiness and growth in Eucalyptus as estimated from E.urophylla × E. tereticornis hybrids

Interspecific hybrids of Eucalyptus urophylla S. T. Blakexa0×xa0E. tereticornis Smith were used for quantitative genetic analysis of cold hardiness (CH) and growth traits height (H), diameter at breast height (D) and volume (V) at 0.5, 1.5, 2.5 and 4xa0years of age. The effect of E. tereticornis male variance was significant (Pxa0<xa00.05) for CH as well as 2.5- and 4-year-old growth except for H4, and E. urophylla femalexa0×xa0E. tereticornis male interaction was significant for 0.5- and 1.5-year-old growth and CH except for CH0.5, whereas E. urophylla female effect was not significant for the majority of traits analyzed. Both additive and dominance gene action were present in the genetics of all the traits measured at most ages, indicating that those traits may be improved by hybrid breeding. The σD2/σA2 ratios tended to decline after 1.5xa0years of age in CH and growth traits, implying that breeding value predictions derived from older performance might be more reliable. Estimates of narrow-sense heritability (h2) for CH and growth ranged from 0.03xa0±xa00.05 to 0.28xa0±xa00.13, depending on trait and age. Additive genetic correlations between CH and V were 0.28xa0±xa00.48 and 0.69xa0±xa00.30 at ages 1.5 and 2.5, respectively, suggesting that selection for growth would cause a favorable increase in cold hardiness in E. urophyllaxa0×xa0E. tereticornis hybrids.

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.

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.

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 (Pu2009≤u20090.05) with the gall wasp resistance in E. grandis, including two validated with a correction of permutation test (Pu2009≤u20090.008). 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 (Pu2009≤u20090.073 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.

DiSNPindel: improved intra-individual SNP and InDel detection in direct amplicon sequencing of a diploid

BackgroundAmplicon re-sequencing based on the automated Sanger method remains popular for detection of single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels) for a spectrum of genetics applications. However, existing software tools for detecting intra-individual SNPs and InDels in direct amplicon sequencing of diploid samples are insufficient in analyzing single traces and their accuracy is still limited.ResultsWe developed a novel computation tool, named DiSNPindel, to improve the detection of intra-individual SNPs and InDels in direct amplicon sequencing of a diploid. Neither reference sequence nor additional sample was required. Using two real datasets, we demonstrated the usefulness of DiSNPindel in its ability to improve largely the true SNP and InDel discovery rates and reduce largely the missed and false positive rates as compared with existing detection methods.ConclusionsThe software DiSNPindel presented here provides an efficient tool for intra-individual SNP and InDel detection in diploid amplicon sequencing. It will also be useful for identification of DNA variations in expressed sequence tag (EST) re-sequencing.

Genetic parameters for growth and wood chemical properties in Eucalyptus urophylla × E. tereticornis hybrids

Key messageGrowth and wood chemical properties are important pulpwood traits. Their narrow-sense heritability ranged from 0.03 to 0.49 inEucalyptus urophylla×E. tereticornishybrids, indicating low to moderate levels of genetic control. Genetic correlations were mostly favorable for simultaneous improvement on growth and wood traits. Additive and non-additive genetic effects should be considered in making a hybrid breeding strategy.ContextEucalypt hybrids are widely planted for pulpwood production purposes. Genetic variations and correlations for growth and wood chemical traits remain to be explored in Eucalyptus interspecific hybrids.AimsOur objectives were to clarify the heritability of growth and wood chemical traits and determine the genetic correlations between traits and between trials in E. urophylla × E. tereticornis hybrids.MethodsTwo trials of 59 E. urophylla × E. tereticornis hybrids derived from an incomplete factorial mating design were investigated at age 10 for growth (height and diameter) and wood chemical properties (basic density, cellulose content, hemi-cellulose content, lignin content, and syringyl-to-guaiacyl ratio). Mixed linear models were used to estimate genetic parameters.ResultsNarrow-sense heritability estimates were 0.13−0.22 in growth and 0.03−0.49 in wood traits, indicating low to moderate levels of additive genetic control. Genetic correlations were mostly positively significant for growth with basic density and cellulose content but negatively significant with hemi-cellulose and lignin contents, being favourablefavorable for pulpwood breeding purpose. Type-B correlations between sites were significant for all the traits except diameter and lignin content.ConclusionHybrid superiority warrants the breeding efforts. An appropriate breeding strategy should be able to capture both additive and non-additive genetic effects.

A novel set of EST-InDel markers in Eucalyptus L’Hérit.: polymorphisms, cross-species amplification, physical positions and genetic mapping

Insertion/deletion (InDel) markers are valuable for genetic applications in plant species, and the public databases of expressed sequence tags (ESTs) have facilitated the development of genic InDel markers. In this study, we developed a novel set of 144 InDel markers in an important tree genus Eucalyptus L’Hérit. using the ESTs of GenBank. Amplicon sequencing against two parents of a mapping population (Eucalyptusurophylla S. T. Blakexa0×xa0E. tereticornis Smith) revealed that the InDel size ranged from 2 to 44 bases, and the dinucleotide type was the most abundant (37.3xa0%). The cross-species/subgenus amplification rate ranged from 62.5xa0% in E. tessellaris F. Muell. (subgenus Blakella) to 99.3xa0% in E. grandis Hill ex Maiden (subgenus Symphyomyrtus) with an average of 85.4xa0%. There were 121 EST-InDels (84.0xa0%) polymorphic among 12 individuals of E. grandis, and the mean number of alleles per polymorphic locus (Na), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC) were 4.0, 0.278, 0.538 and 0.465, respectively. Physical positions of 143 EST-InDels were predicted on the E. grandis genome sequence. A total of 81 EST-InDels were incorporated into prior dense genetic maps of E. urophylla and E. tereticonis, and extensive synteny and colinearity were observed between E. grandis genome sequence and the mapped EST-InDel markers. These EST-InDels will provide a valuable resource of functional markers for genetic diversity evaluation, genome comparison, QTL mapping and marker-assisted breeding in Eucalyptus.