Effects of drought and rehydration on root gene expression in seedlings of Pinus massoniana Lamb.

The mechanisms underlying plant response to drought involve the expression of numerous functional and regulatory genes. Transcriptome sequencing based on the second- and/or third-generation high-throughput sequencing platforms has proven to be powerful for investigating the transcriptional landscape under drought stress. However, the full-length transcriptomes related to drought responses in the important conifer genus Pinus L. remained to be delineated using the third-generation sequencing technology. With the objectives of identifying the candidate genes responsible for drought and/or rehydration and clarifying the expression profile of key genes involved in drought regulation, we combined the third- and second-generation sequencing techniques to perform transcriptome analysis on seedling roots under drought stress and rewatering in the drought-tolerant conifer Pinus massoniana Lamb. A sum of 294,114 unique full-length transcripts were produced with a mean length of 3217 bp and N50 estimate of 5075 bp, including 279,560 and 124,438 unique full-length transcripts being functionally annotated and Gene Ontology enriched, respectively. A total of 4076, 6295 and 18,093 differentially expressed genes (DEGs) were identified in three pair-wise comparisons of drought-treatment versus control transcriptomes, including 2703, 3576 and 8273 upregulated and 1373, 2719 and 9820 downregulated DEGs, respectively. Moreover, 157, 196 and 691 DEGs were identified as transcription factors in the three transcriptome comparisons and grouped into 26, 34 and 44 transcription factor families, respectively. Gene Ontology enrichment analysis revealed that a remarkable number of DEGs were enriched in soluble sugar-related and cell wall-related processes. A subset of 75, 68 and 97 DEGs were annotated to be associated with starch, sucrose and raffinose metabolism, respectively, while 32 and 70 DEGs were associated with suberin and lignin biosynthesis, respectively. Weighted gene co-expression network analysis revealed modules and hub genes closely related to drought and rehydration. This study provides novel insights into root transcriptomic changes in response to drought dynamics in Masson pine and serves as a fundamental work for further molecular investigation on drought tolerance in conifers.

Effects of typical marine environmental factors on the bioluminescence intensity of individual Noctiluca scintillans.

Red Noctiluca scintillans (RNS) is one of the major red tide species and dominant bioluminescent plankton in the global offshore. Bioluminescence offers a number of applications for ocean environment assessments such as interval waves study, fish stocks evaluation and underwater target detection making it of significant interest in forecasting bioluminescence occurrence and intensity. RNS is susceptible to changes in marine environmental factors. However, the effects of marine environmental factors on the bioluminescent intensity (BLI, photon s-1) of individual RNS cells (IRNSC) is poorly known. In this study, the effects of temperature, salinity and nutrients on the BLI were studied by field and laboratory culture experiments. In the field experiments, bulk BLI was measured by an underwater bioluminescence assessment tool at various temperature, salinity and nutrient concentrations. To exclude the contribution by other bioluminescent planktons, an identification method of IRNSC was first developed using the features of the bioluminescence flash kinetics (BFK) curve of RNS to identify and extract BLI emitted by an individual RNS cell. To decouple the effects of each environmental factor, laboratory culture experiments were conducted to examine the effects of a single factor on the BLI of IRNSC. The field experiments showed that BLI of IRNSC negatively correlated with temperature (3-27°C) and salinity (30-35‰). The logarithmic BLI can be well fitted using a linear equation with temperature or salinity with Pearson correlation coefficients of -0.95 and -0.80, respectively. The fitting function with salinity was verified by the laboratory culture experiment. On the other hand, no significant correlation was observed between BLI of IRNSC and nutrients. These relationships could be used in the RNS bioluminescence prediction model to improve the prediction accuracy of bioluminescent intensity and spatial distribution.

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-wide analysis of the TPX2 family proteins in Eucalyptus grandis.

BACKGROUND: The Xklp2 (TPX2) proteins belong to the microtubule-associated (MAP) family of proteins. All members of the family contain the conserved TPX2 motif, which can interact with microtubules, regulate microtubule dynamics or assist with different microtubule functions, for example, maintenance of cell morphology or regulation of cell growth and development. However, the role of members of the TPX family have not been studied in the model tree species Eucalyptus to date. Here, we report the identification of the members of the TPX2 family in Eucalyptus grandis (Eg) and analyse the expression patterns and functions of these genes. RESULTS: In present study, a comprehensive analysis of the plant TPX2 family proteins was performed. Phylogenetic analyses indicated that the genes can be classified into 6 distinct subfamilies. A genome-wide survey identified 12 members of the TPX2 family in the sequenced genome of Eucalyptus grandis. The basic genetic properties of the TPX2 family in Eucalyptus were analysed. Our results suggest that the TPX2 family proteins within different sub-groups are relatively conserved but there are important differences between groups. Quantitative real-time PCR (qRT-PCR) was performed to confirm the expression levels of the genes in different tissues. The results showed that in the whole plant, the levels of EgWDL5 transcript are the highest, followed by those of EgWDL4. Compared with other tissues, the level of the EgMAP20 transcript is the highest in the root. Over-expression of EgMAP20 in Arabidopsis resulted in organ twisting. The cotyledon petioles showed left-handed twisting while the hypocotyl epidermal cells produced right-handed helical twisting. Finally, EgMAP20, EgWDL3 and EgWDL3L were all able to decorate microtubules. CONCLUSIONS: Plant TPX2 family proteins were systematically analysed using bioinformatics methods. There are 12 TPX2 family proteins in Eucalyptus. We have performed an initial characterization of the functions of several members of the TPX2 family. We found that the gene products are localized to the microtubule cytoskeleton. Our results lay the foundation for future efforts to reveal the biological significance of TPX2 family proteins in 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.

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

BACKGROUND: Amplicon 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. RESULTS: We 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. CONCLUSIONS: The 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.

Biochemical and molecular changes associated with heteroxylan biosynthesis in Neolamarckia cadamba (Rubiaceae) during xylogenesis.

Wood, derived from plant secondary growth, is a commercially important material. Both cellulose and lignin assembly have been well studied during wood formation (xylogenesis), but heteroxylan biosynthesis is less well defined. Elucidation of the heteroxylan biosynthetic pathway is crucial to understand the mechanism of wood formation. Here, we use Neolamarckia cadamba, a fast-growing tropical tree, as a sample to analyze heteroxylan formation at the biochemical and molecular levels during wood formation. Analysis of the non-cellulosic polysaccharides isolated from N. cadamba stems shows that heteroxylans dominate non-cellulosic polysaccharides and increase with xylogenesis. Microsomes isolated from stems of 1-year-old N. cadamba exhibited UDP-Xyl synthase and xylosyltransferase activities with the highest activity present in the middle and basal stem regions. To further understand the genetic basis of heteroxylan synthesis, RNA sequencing (RNA-seq) was used to generate transcriptomes of N. cadamba during xylogenesis. The RNA-seq results showed that genes related to heteroxylan synthesis had higher expression levels in the middle and basal part of the stem compared to the apical part. Our results describe the heteroxylan distribution and heteroxylan synthesis trait in N. cadamba and give a new example for understanding the mechanism of heteroxylan synthesis in tropical tree species in future.

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.

Gene expression in Eucalyptus branch wood with marked variation in cellulose microfibril orientation and lacking G-layers.

In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G-layer). Gene expression was studied in Eucalyptus nitens branches oriented at 45 degrees using microarrays containing 4900 xylem cDNAs, and wood fibre characteristics revealed by X-ray diffraction, chemical and histochemical methods. Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G-layers and had higher cellulose and decreased Klason lignin compared with lower branch wood. Expression of two closely related fasciclin-like arabinogalactan proteins and a beta-tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches. Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G-layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified.

beta-tubulin affects cellulose microfibril orientation in plant secondary fibre cell walls.

Cellulose microfibrils are the major structural component of plant secondary cell walls. Their arrangement in plant primary cell walls, and its consequent influence on cell expansion and cellular morphology, is directed by cortical microtubules; cylindrical protein filaments composed of heterodimers of alpha- and beta-tubulin. In secondary cell walls of woody plant stems the orientation of cellulose microfibrils influences the strength and flexibility of wood, providing the physical support that has been instrumental in vascular plant colonization of the troposphere. Here we show that a Eucalyptus grandisbeta-tubulin gene (EgrTUB1) is involved in determining the orientation of cellulose microfibrils in plant secondary fibre cell walls. This finding is based on RNA expression studies in mature trees, where we identified and isolated EgrTUB1 as a candidate for association with wood-fibre formation, and on the analysis of somatically derived transgenic wood sectors in Eucalyptus. We show that cellulose microfibril angle (MFA) is correlated with EgrTUB1 expression, and that MFA was significantly altered as a consequence of stable transformation with EgrTUB1. Our findings present an important step towards the production of fibres with altered tensile strength, stiffness and elastic properties, and shed light on one of the molecular mechanisms that has enabled trees to dominate terrestrial ecosystems.

[Progress in research on forest tree genomics].

This is a review on forest tree genomics. In structural genomics, genetic maps have been constructed for up to 40 forest tree species, more than 30 commercially important QTLs have been detected, comparative mapping has been done for a few of forest tree taxa, and whole genome sequencing was completed for Populus and is under way for Eucalyptus. For functional genomics, huge EST databases from multiple tissues of a number of tree species have been rapidly accumulated, and molecular analyses on secondary growth and wood formation, flowering, and cold hardiness have given some insights into the metabolic pathways of those tree-specific development processes. The prospects of development in tree genomics are discussed, which may be implicative for accelerating forest tree genomics studies in China.

Moderate-density molecular maps of Eucalyptus urophylla S. T. Blake and E. tereticornis Smith genomes based on RAPD markers.

Moderate-density molecular maps were constructed for the genomes of Eucalyptus urophylla S. T. Blake and E. tereticornis Smith using RAPD markers and an interspecific cross between the two species. One hundred and eighty-three primers were employed to generate 245 and 264 parent-specific markers in E. urophylla and E. tereticornis, respectively, as well as 49 parent-shared markers. The normally segregating markers, including 208 (84.9%) specific to maternal E. urophylla, 175 (66.3%) to paternal E. tereticornis, and 48 shared by both parents, were used for framework map construction for each parental species. For maternal E. urophylla, the linkage map consisted of 23 linkage groups, 160 framework markers, and 60 accessory markers, defining a total map distance of 1504.6cM and an average interval of 11.0 +/- 8.07 cM. For paternal E. tereticornis, the linkage map contained 23 linkage groups, 126 framework markers, and 92 accessory markers, defining a total map distance of 1035.7 cM and an average interval of 10.1 +/- 7.23 cM. Genome length was estimated at 1585.7 and 1507.5 cM for E. urophylla and E. tereticornis, respectively, indicating map coverage of 94.9 and 68.7% of the corresponding genomes. Construction of such maps will be valuable for quantitative trait loci (QTLs) detection, marker-assisted selection (MAS), comparative mapping, and whole genome based fingerprint characterization in Eucalyptus breeding programs.