Genetic variation of wood chemical traits and association with underlying genes in Eucalyptus urophylla

We developed a quantitative and association genetic study with Eucalyptus urophylla using a progeny trial. Based on a sample of 831 trees distributed in 84 half-sib families whose wood was phenotyped by near-infrared spectroscopy, the results showed that traits related to lignin, cellulose, and wood extractives presented significant additive genetic variability with moderate to high narrow sense heritability (h ²?=?0.28 to 0.93). Genetic correlations varied with high standard error and showed low to moderate values. Using three cellulose synthase genes (EuCesA1, EuCesA2, and EuCesA3) and three candidate genes involved in the lignin pathway (EuC4H1, EuC4H2, and EuCAD2), an association study was performed for each of the gene action models (co-dominant, recessive, and dominant) using two methods. Firstly, single-marker association tests were done and 539 tests (49 single nucleotide polymorphisms (SNPs)?×?11 traits) were analyzed. After Bonferroni correction with a significance level of P?=?0.00102, only four SNPs presented significant association with syringyl and syringyl-to-guaiacyl ratio with an adjusted coefficient of determination varying between 2.6 and 4.4 %. Secondly, a model selection method, the backward approach, was implemented. Similar SNPs were detected by both the backward selection and the individual marker approaches. However, the latter detected new associations with other traits, genes, and SNPs and improved the quality of the model as shown by the BIC criteria and the higher adjusted determination coefficient (1.5 to 8.3 %). Our results reveal that cellulose genes can be associated with lignin traits (syringyl-to-guaiacyl ratio) and stress the possible pleiotropic effect of some genes.     

Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.Suchita Bhandari and Takeshi Fujino contributed equally to this research.     

Single-nucleotide polymorphisms in PtoCesA7 and their association with growth and wood properties in Populus tomentosa

Cellulose synthase (CesA) genes encode the enzymes that synthesize cellulose; therefore, CesAs play central roles in plant development and affect the yield and quality of wood, essential properties for industrial applications of plant biomass. To effectively manipulate wood biosynthesis in trees and improve wood quality, we thus require a better understanding of the natural variation in CesAs. Association studies have emerged as a powerful tool for identification of variation associated with quantitative traits. Here, we used a candidate gene-based association mapping approach to identify PtoCesA7 allelic variants that associate with growth and wood quality traits in Populus tomentosa. We isolated a full-length PtoCesA7 cDNA and observed high PtoCesA7 expression in xylem, consistent with the xylem-specific expression of CesA7. Nucleotide diversity and linkage disequilibrium (LD) in PtoCesA7, sampled from the P. tomentosa natural distribution, revealed that PtoCesA7 harbors high nucleotide diversity (π _T = 0.0091) and low LD (r ² ≥ 0.1, within 800 bp). By association analysis, we identified seven single-nucleotide polymorphisms (SNPs) (false discovery rate Q < 0.10) and 12 haplotypes (Q < 0.10) that associated with growth and wood properties, explaining 3.62–10.59 % of the phenotypic variance. We also validated 9 of the 10 significant marker–trait associations in at least one of three smaller subsets (climatic regions) or in a linkage-mapping population. Thus, our study identified functional PtoCesA7 allelic variants associated with growth and wood quality traits, giving new insights into genes affecting wood quality and quantity. From an applied perspective, the SNPs revealed in this study have potential applications in marker-assisted breeding.     

Effects of Nitrogen Sources on Cellulose and Synthetic Lignin Degradation by Phanerochaete chrysosporium

Phanerochaete chrysosporium degraded cellulose faster with organic nitrogen sources than with NH₄Cl. Simple and complex nitrogen sources added at the time of inoculation to N-limited cultures of P. chrysosporium, with glucose as carbon/energy source, transiently stimulated degradation of synthetic [¹⁴C]lignin to ¹⁴CO₂. The same nitrogen sources added 5 days after inoculation, when the cultures were entering secondary metabolism, delayed ¹⁴CO₂ production. The various N sources affected synthetic lignin degradation in defined medium differently than lignin degradation in aspen wood.     

Mapping of PARK2 and PACRG Overlapping Regulatory Region Reveals LD Structure and Functional Variants in Association with Leprosy in Unrelated Indian Population Groups

Leprosy is a chronic infectious disease caused by Mycobacterium Leprae, where the host genetic background plays an important role toward the disease pathogenesis. Various studies have identified a number of human genes in association with leprosy or its clinical forms. However, non-replication of results has hinted at the heterogeneity among associations between different population groups, which could be due to differently evolved LD structures and differential frequencies of SNPs within the studied regions of the genome. A need for systematic and saturated mapping of the associated regions with the disease is warranted to unravel the observed heterogeneity in different populations. Mapping of the PARK2 and PACRG gene regulatory region with 96 SNPs, with a resolution of 1 SNP per 1 Kb for PARK2 gene regulatory region in a North Indian population, showed an involvement of 11 SNPs in determining the susceptibility towards leprosy. The association was replicated in a geographically distinct and unrelated population from Orissa in eastern India. In vitro reporter assays revealed that the two significantly associated SNPs, located 63.8 kb upstream of PARK2 gene and represented in a single BIN of 8 SNPs, influenced the gene expression. A comparison of BINs between Indian and Vietnamese populations revealed differences in the BIN structures, explaining the heterogeneity and also the reason for non-replication of the associated genomic region in different populations.     

Effects of Nitrogen Supplements on Degradation of Aspen Wood Lignin and Carbohydrate Components by Phanerochaete chrysosporium

A supplement of KH₂PO₄, MgSO₄, CaCl₂, trace elements, and thiamine accelerated the initial rate of aspen wood decay by Phanerochaete chrysosporium but did not increase the extent of lignin degradation. Asparagine, casein hydrolysate, and urea supplements (1% added N) strongly inhibited lignin degradation and weight loss. The complex nitrogen sources peptone and yeast extract stimulated lignin degradation and weight loss. Albumen and NH₄Cl had intermediate effects. Conversion of [¹⁴C]lignin to ¹⁴CO₂ and water-soluble materials underestimated lignin degradation in the presence of the complex N sources. The highest ratio of lignin degradation to total weight loss and the largest increase in cellulase digestibility occurred during the decay of unsupplemented wood. Rotting of aspen wood by P. chrysosporium gives smaller digestibility increases than have been found with some other white-rot fungi.     

Ultrastructural Aspects of Wood Delignification by Phlebia (Merulius) tremellosus

Wood from aspen and birch that had been decayed for 12 weeks by Phlebia tremellosus had averages of 30 and 31% weight loss, respectively, and 70% lignin loss. Digestibility increased from averages of 21 and 13% for sound aspen and birch to 54 and 51% for decayed aspen and birch. Individual wood sugar analyses of decayed birch blocks indicated an average loss of 10% glucose, 45% xylose, and 19% mannose. Micromorphological studies demonstrated the removal of middle lamellae and separation of cells. Vessels also separated at perforation plates. Electron microscopy with OsO₄-glutaraldehyde-fixed and KMnO₄-fixed wood showed that lignin was progressively removed first from the secondary cell wall layers, beginning at the lumen surface, and later from the compound middle lamella. Extensive degradation of lignin was found throughout the secondary wall and middle lamella region between cells. In cells with advanced decay, the middle lamella between cells was completely degraded, but cell corner regions remained.     

Cloning and characterization of cellulose synthase-like gene, PtrCSLD2 from developing xylem of aspen trees

Genetic improvement of cell wall polymer synthesis in forest trees is one of the major goals of forest biotechnology that could possibly impact their end product utilization. Identification of genes involved in cell wall polymer biogenesis is essential for achieving this goal. Among various candidate cell wall-related genes, cellulose synthase-like D (CSLD) genes are intriguing due to their hitherto unknown functions in cell wall polymer synthesis but strong structural similarity with cellulose synthases (CesAs) involved in cellulose deposition. Little is known about CSLD genes from trees. In the present article PtrCSLD2, a first CSLD gene from an economically important tree, aspen (Populus tremuloides) is reported. PtrCSLD2 cDNA was isolated from an aspen xylem cDNA library and encodes a protein that shares 90% similarity with Arabidopsis AtCSLD3 protein involved in root hair tip growth. It is possible that xylem fibers that also grow by intrusive tip growth may need expression of PtrCSLD2 for controlling the length of xylem fibers, a wood quality trait of great economical importance. PtrCSLD2 protein has a N-terminal cysteine-rich putative zinc-binding domain; eight transmembrane domains; alternating conserved and hypervariable domains; and a processive glycosyltransferases signature, D, D, D, QXXRW; all similar to aspen CesA proteins. However, PtrCSLD2 shares only 43-48% overall identity with the known aspen CesAs suggesting its distinct functional role in cell wall polymer synthesis perhaps other than cellulose biosynthesis. Based on Southern analysis, the aspen CSLD gene family consists of at least three genes and this gene copy estimate is supported by phylogenetic analysis of available CSLDs from plants. Moreover, gene expression studies using RT-PCR and in situ mRNA hybridization showed that PtrCSLD2 is expressed at a low level in all aspen tissues examined with a slightly higher expression level in secondary cell wall-enriched aspen xylem as compared to primary cell wall enriched tissues. Together, these observations suggest that PtrCSLD2 gene may be involved in the synthesis of matrix polysaccharides that are dominant in secondary cell walls of poplar xylem. Future molecular genetic analyses will clarify the functional significance of CSLD genes in the development of woody trees.     

Down-regulation of lignin biosynthesis in transgenic Leucaena leucocephala harboring O-methyltransferase gene

In the present study, a 0.47 kb OMT gene construct from aspen, encoding for an enzyme O-methyltransferase (OMT, EC 2.1.1.6), in antisense orientation was used to down-regulate lignin biosynthesis in Leucaena leucocephala. The plants were transformed with Agrobacterium tumefaciens strain harboring the antisense gene, and the transformation was confirmed by PCR amplification of the npt II gene. The integration of a heterologous antisense OMT gene construct in transformed plants led to a maximum of 60% reduction in OMT activity relative to control. The evaluation of total lignin content by the Klason method revealed a maximum of 28% reduction. Histochemical analyses of stem sections depicted a reduction in lignin content and normal xylem development. The results also suggested a probable increase in aldehyde levels and a decrease in syringyl units. Lignin down-regulation was accompanied by an increase in methanol soluble phenolics to an extent that had no impact on wood discoloration, and the plants displayed a normal phenotype. Concomitantly, an increase of up to 9% in cellulose content was also observed. Upon alkali extraction, modified lignin was more extractable as evident from reduced Klason lignin in saponified residue and increased alkali soluble phenolics. The results together suggested that the extent of down-regulation of OMT activity achieved may lead to quality amelioration of Leucaena with respect to its applicability in pulp and paper manufacture as well as nutritive and easily digestible forage production.     

Nucleotide sequence analysis of two lignin genes in Acacia auriculiformis × Acacia mangium hybrid for enhancement of wood pulp quality

There is large potential in the establishment of Acacia auriculiformis × Acacia mangium hybrid plantations through selection of high quality parents especially with characteristics favourable for some end products like pulp and fibre. Trees altered in their lignin profile with reduced amounts of lignin or increased extractable syringyl are desirable in the pulp and paper industry. Cinnamoyl-CoA reductase (CCR) and caffeic acid O-methyltransferase (COMT) are two enzymes likely to regulate lignin content and composition in the syringyl (S)- and guaiacyl (G)-related monolignols at specific branches of the lignin biosynthetic pathway. A goal of this study was to discover the genetic variation in CCR and COMT genes in Acacia species using single nucleotide polymorphisms (SNPs). In this study, two lignin genes, CCR (3,317 bp) and COMT (2,764 bp), were isolated from the Acacia hybrid, sequenced and analysed in silico. Southern blot analysis suggested that there are one to two copies of genes encoding CCR and COMT in the Acacia hybrid. Upon genotyping 480 individuals from natural populations of A. auriculiformis and A. mangium, six CCR SNPs were found in A. auriculiformis and five CCR SNPs in A. mangium. Three COMT SNPs were found in A. auriculiformis and one COMT SNP in A. mangium. A pair of CCR SNPs showed high linkage disequlibrium (LD) with r ² value of 0.870 in an A. mangium population from Papua New Guinea. The SNPs will be further exploited through the candidate gene-based LD mapping to identify QTLs for the Acacia marker-assisted breeding. This is the first documentation of the isolation, cloning and re-sequencing of CCR and COMT genes in the Acacia hybrid and the natural populations of A. auriculiformis and A. mangium.     

Biological Delignification of Aspen Wood by Solid-State Fermentation with the White-Rot Fungus Merulius tremellosus

Solid-state fermentation of aspen (Populus tremuloides) wood with Merulius tremellosus for 8 weeks removed 52% of the lignin but only 12% of the total wood weight, and increased the cellulase digestibility to 53% from 18%. Water-soluble and enzyme-solubilized lignin degradation products accumulated. Delignification was fastest at temperatures between 25 and 32.5°C and at a water-to-wood ratio of 2. Initial pH values between 4 and 6 were optimal; M. tremellosus acidified the wood to below pH 3.5 as it grew. The fungus tolerated CO₂ concentrations of at least 14% and O₂ concentrations down to 7% in the bulk gas phase. Both simple and complex nitrogen supplements inhibited delignification. Supplementary KH₂PO₄, MgSO₄, CaCl₂, thiamine, and trace elements had little effect on the fermentation. Four isolates of M. tremellosus had very similar abilities to delignify aspen wood. Biological delignification with M. tremellosus may be a useful pretreatment for enzymatic saccharification or ruminant feeding.     

Multilocus Patterns of Nucleotide Diversity, Population Structure and Linkage Disequilibrium in Boechera stricta, a Wild Relative of Arabidopsis

Information about polymorphism, population structure, and linkage disequilibrium (LD) is crucial for association studies of complex trait variation. However, most genomewide studies have focused on model systems, with very few analyses of undisturbed natural populations. Here, we sequenced 86 mapped nuclear loci for a sample of 46 genotypes of Boechera stricta and two individuals of B. holboellii, both wild relatives of Arabidopsis. Isolation by distance was significant across the species range of B. stricta, and three geographic groups were identified by structure analysis, principal coordinates analysis, and distance-based phylogeny analyses. The allele frequency spectrum indicated a genomewide deviation from an equilibrium neutral model, with silent nucleotide diversity averaging 0.004. LD decayed rapidly, declining to background levels in ~10 kb or less. For tightly linked SNPs separated by <1 kb, LD was dependent on the reference population. LD was lower in the specieswide sample than within populations, suggesting that low levels of LD found in inbreeding species such as B. stricta, Arabidopsis thaliana, and barley may result from broad geographic sampling that spans heterogeneous genetic groups. Finally, analyses also showed that inbreeding B. stricta and A. thaliana have ~45% higher recombination per kilobase than outcrossing A. lyrata.     

Quantitative trait locus (QTL) analysis of wood quality traits in <Emphasis Type="Italic">Eucalyptus nitens</Emphasis>

To identify the chromosomal regions affecting wood quality traits, we conducted a genome-wide quantitative trait locus (QTL) analysis of wood quality traits in Eucalyptus nitens. This information is important to exploit the full potential of the impending Eucalyptus genome sequence. A three generational mapping population consisting of 296 progeny trees was used to identify QTL associated with several wood quality traits in E. nitens. Thirty-six QTL positions for cellulose content, pulp yield, lignin content, density, and microfibril angle (MFA) were identified across different linkage groups. On linkage groups (LG)2 and 8, cellulose QTL cluster with pulp yield and extractives QTL while on LG4 and 10 cellulose and pulp yield QTLs cluster together. Similarly, on LG4, 5, and 6 QTL for lignin traits were clustered together. At two positions, QTL for MFA, a physical trait related to wood stiffness, were clustered with QTL for lignin traits. Several cell wall candidate genes were co-located to QTL positions affecting different traits. Comparative QTL analysis with Eucalyptus globulus revealed two common QTL regions for cellulose and pulp yield. The QTL positions identified in this study provide a resource for identifying wood quality genes using the impending Eucalyptus genome sequence. Candidate genes identified in this study through co-location to QTL regions may be useful in association studies.