Qingzhang Du

The actin multigene family in Populus: organization, expression and phylogenetic analysis

Despite the significance of actin in plant growth and development, little is known of the structure, expression and evolution of the actin gene family in woody plants. In this study, we systematically examined the diversification of the actin gene family in Populus by integrating genomic organization, expression, and phylogeny data. Genome-wide analysis of the Populus genome indicated that actin is a multigene family consisting of eight members, all predicted to encode 377-amino acid polypeptides that share high sequence homology ranging from 94.2 to 100% identity. Microarray and real-time PCR expression analysis showed that the PtrACT family members are differentially expressed in different tissues, exhibiting overlapping and unique expression patterns. Of particular interest, all PtrACT genes have been found to be preferentially expressed in the stem phloem and xylem, suggesting that poplar PtrACTs are involved in the wood formation. Gene structural and phylogenetic analyses revealed that the PtrACT family is composed of two main subgroups that share an ancient common ancestor. Extremely high intraspecies synonymous nucleotide diversity of πsynxa0=xa00.01205 was detected, and the πnon-syn/πsyn ratio was significantly less than 1; therefore, the PtACT1 appears to be evolving in Populus, primarily under purifying selection. We demonstrated that the actin gene family in Populus is divided into two distinct subgroups, suggesting functional divergence. The results reported here will be useful in conducting future functional genomics studies to understand the detailed function of actin genes in tree growth and development.

Genetic Diversity and Population Structure in Chinese Indigenous Poplar (Populus simonii) Populations Using Microsatellite Markers

Populus simonii Carr. is an important ecological and commercial breeding species in northern China; however, human interference during the last few centuries has led to the reduction and fragmentation of natural populations. To evaluate genetic diversity and differentiation within and among existing populations, we used 20 microsatellite markers to examine the genetic variation and structure of 16 natural populations. Our results indicated that the level of genetic diversity differed among populations, with average number of alleles per locus (AR) and expected heterozygosity (He) ranging from 3.7 to 6.11 and 0.589 to 0.731, respectively. A marginal population from Qilian in the Qinghai–Tibetan Plateau showed the highest values (ARu2009=u20096.11, Heu2009=u20090.731), and the Zhangjiakou and Yishui populations showed the lowest values (ARu2009=u20094.08, Heu2009=u20090.589 and ARu2009=u20093.7, Heu2009=u20090.604). The inbreeding coefficient (FIS) values for all populations were positive, which indicated an excess of homozygotes. The microsatellites allowed the identification of a significant subpopulation structure (Ku2009=u20093), consistent with an isolation by distance model for P. simonii populations. Additionally, molecular variance analysis revealed that 14.2xa0% of the variation resided among populations, and 85.8xa0% could be attributed to variation within populations. These data provide valuable information for natural resource conservation and for optimization of breeding programs in the immediate future.

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 (πTxa0=xa00.0091) and low LD (r2xa0≥xa00.1, within 800xa0bp). By association analysis, we identified seven single-nucleotide polymorphisms (SNPs) (false discovery rate Qxa0<xa00.10) and 12 haplotypes (Qxa0<xa00.10) that associated with growth and wood properties, explaining 3.62–10.59xa0% 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.

Association mapping in Populus reveals the interaction between Pto-miR530a and its target Pto-KNAT1

AbstractMain conclusionWe used transcript profiling and multi-SNP association to investigate the genetic regulatory relationship between miRNA Pto-miR530a and its target Pto-KNAT1, identifying additive, dominant, and epistatic effects.n MicroRNAs (miRNAs) play crucial roles in the post-transcriptional regulation of plant growth and development; indeed, many studies have described the importance of miRNA–target interactions in herbaceous species. However, elucidation of the miRNA–target interactions in trees may require novel strategies. In the present study, we describe a strategy combining expression profiling by reverse transcription quantitative PCR (RT-qPCR) and association mapping with multiple single nucleotide polymorphisms (SNPs) to evaluate the interaction between Pto-miR530a and its target Pto-KNAT1 in Populus tomentosa. RT-qPCR analysis showed a negative correlation (rxa0=xa0−0.62, Pxa0<xa00.05) between expression levels of Pto-miR530a and Pto-KNAT1 in eight tissues. We used a Bayesian hierarchical model to identify allelic variants of Pto-miR530a and Pto-KNAT1 that associated with eight traits related to growth and wood properties, in a population of 460 unrelated individuals of P. tomentosa. This analysis identified 27 associations, with the proportions of phenotypic variance (R2) contributed by each SNP ranging of 0.82–15.81xa0%, the additive effects of each SNP ranging of 0.16–18.09, and the dominant effects ranging from −14.09 to 19.00. Epistatic interaction models showed a strong interaction among SNPs in the miRNA target with R2 of 0.1–3.56xa0%, and information gain of significant SNP pairs of −3.09 to 0.93xa0%, representing the regulatory interactions between the miRNA and the mRNA. Thus, we used a new strategy that combines association genetics and expression profiling based on SNPs to study the regulatory relationship between this miRNA and its target mRNA, thereby providing novel advances in our understanding of the genetic architecture of important traits.

Single-nucleotide polymorphisms in the 5' UTR of UDP-glucose dehydrogenase (PtUGDH) associate with wood properties in Populus tomentosa

Association studies have emerged as a powerful tool for identification of markers associated with quantitative traits in forest trees. The cytosolic enzyme uridine 5′ diphosphate-glucose dehydrogenase (UGDH) converts UDP-glucose to UDP-glucuronate and affects cell wall formation in higher plants. Here, we used association genetics to identify UDP-glucose dehydrogenase (PtUGDH) allelic variation that associates with wood quality traits in Populus tomentosa. We isolated a 1,828xa0bp PtUGDH cDNA encoding a polypeptide of 481 amino acids. Expression analysis revealed that PtUGDH was expressed predominantly in young root, developing xylem from vascular tissues, and young leaves, suggesting that UGDH functions in cell wall formation. We identified 59 single-nucleotide polymorphisms (SNPs; πTu2009=u20090.00475) by resequencing the PtUGDH locus of 40 individuals and genotyped the 22 most common SNPs (minor allele frequency >10xa0%) in a discovery population (nu2009=u2009426). Linkage disequilibrium (LD) analysis showed that LD did not extend over the entire gene (r2u2009<u20090.1, within 300xa0bp). Association studies indicated that three SNPs (false discovery rate, Qu2009<u20090.05) and 12 haplotypes (Qu2009<u20090.05) were significantly associated with wood properties. The three significant SNPs are all in the 5′ untranslated regions of PtUGDH, and the phenotypic variance explained by each SNP ranged from 5.37 to 11.97xa0%. We validated one association in a validation population (nu2009=u20091,200) and validated another association by examining its effect on gene expression. The present study provided molecular markers associated with fiber length and holocellulose content, markers that have potential applications in marker-assisted breeding.

Populus endo-β-1,4-glucanases gene family: genomic organization, phylogenetic analysis, expression profiles and association mapping.

Main conclusionExtensive characterization of the poplarGH9 gene family provides new insights intoGH9function and evolution in woody species, and may drive novel progress for molecular breeding in trees.AbstractIn higher plants, endo-β-1,4-glucanases (cellulases) belonging to the glycosyl hydrolase family 9 (GH9) have roles in cell wall synthesis, remodeling and degradation. To increase the understanding of the GH9 family in perennial woody species, we conducted an extensive characterization of the GH9 family in the model tree species, Populus. We characterized 25 putative GH9 members in Populus with three subclasses (A, B, and C), using structures and bioinformatic analysis. Phylogenetic analyses of 114 GH9s from plant (dicot, monocot, and conifer) and bacterial species (outgroup) demonstrated that plant GH9s are monophyletic with respect to bacteria GH9s. Three subclasses, A, B, and C, of plant GH9 are formed before the divergence of angiosperms and gymnosperms. Chromosomal localization and duplications of GH9s in the Populus genome showed that eight paralogous pairs remained in conserved positions on segmental duplicated blocks, suggesting duplication of chromosomal segments has contributed to the family expansion. By examining tissue-specific expression profiles for all 25 members, we found that GH9 members exhibited distinct but partially overlapping expression patterns, while certain members have higher transcript abundance in mature or developing xylem. Based on our understanding of intraspecific variation and linkage disequilibrium of two KORRIGANs (PtoKOR1 and PtoKOR2) in natural population of Populus tomentosa, two non-synonymous SNPs in PtoKOR1 associated with fiber width and holocellulose content were obtained. Characterizations of the poplar GH9 family provide new insights into GH9 function and evolution in woody species, and may drive novel progress for molecular breeding in trees.

Transcript abundance patterns of Populus C-repeat binding factor2 orthologs and genetic association of PsCBF2 allelic variation with physiological and biochemical traits in response to abiotic stress

AbstractMain conclusionWe conducted a candidate gene-based approach to search for genetic associations between 10 SNPs in PsCBF2 and 10 abiotic stress-related traits.n The increasing incidence of abiotic stresses and the limitations of available treatments, particularly in trees, highlight the need to improve our understanding of the mechanisms of stress responses. In Arabidopsis, C-repeat binding factor 2 (CBF2) plays an important role in freezing tolerance and cold acclimation. Here, we isolated orthologs of CBF2 from five Populus species. Expression profiling revealed that the PopulusCBF2s were preferentially induced in response to cold, with CBF2 transcript abundances ranging from 5.4- to 62-fold higher than in unstressed controls of the corresponding species. In addition, we used a candidate gene-based approach in Populussimonii Carr. to identify single nucleotide polymorphisms (SNPs) in PsCBF2 associated with physiological and biochemical traits. PsCBF2 showed high nucleotide diversity (πTxa0=xa00.00549, θwxa0=xa00.01406) and low average linkage disequilibrium (r2xa0=xa00.061). Association studies in 528 individuals of an association population showed that nine SNPs (false discovery rate Qxa0<xa00.10) and one haplotype (Qxa0<xa00.10) were significantly associated with differences in four physiological and biochemical traits (Pxa0<xa00.005), with each marker explaining 1.31–5.87xa0% of the total variance in the corresponding trait. PsCBF2 transcript levels differed significantly in abundance among genotypic classes for most of the significant SNPs. Identification of these significant associations will help reveal the molecular basis of physiological differences and provide a starting point for marker-assisted selection for traits involved in stress tolerance in P. simonii.

Association studies reveal the effect of genetic variation in lncRNA UGTRL and its putative target PtoUGT88A1 on wood formation in Populus tomentosa

Long noncoding RNAs (lncRNAs) play significant roles in the growth and development of herbaceous plants by regulating target genes; however, the significance of lncRNA-messenger RNA (mRNA) interactions needs to be investigated in perennial trees. Here, we combined transcript profiling and multi-single-nucleotide polymorphism (SNP) association mapping to analyze the genetic variation and putative interactions of the lncRNA UDP-glucosyltransferase-related lncRNA (UGTRL) and its predicted target PtoUGT88A1 in a natural population of 435 unrelated Populus tomentosa individuals. We detected 41 and 67 common SNPs (minor allele frequency >0.05) in UGTRL and PtoUGT88A1, respectively, in the association population. Using additive and dominant association models, we identified 86 associations with 12 traits measuring tree growth, wood properties, and photosynthetic parameters. These associations represent 36 significant SNPs (Pu2009<u20090.01) from UGTRL and its putative target and explained 0.06 to 7.28xa0% of the phenotypic variance, indicating that UGTRL and its putative target affect wood formation. An epistasis model uncovered 84 SNP-SNP association pairs representing 38.89xa0% of the significant SNPs in UGTRL and PtoUGT88A1 with information gain of −8.01 to 5.57xa0%, revealing the strong interactions between UGTRL and its putative target. Tissue-specific expression analysis in eight tissues, including xylem and cambium, showed that UGTRL and PtoUGT88A1 displayed similar expression patterns (ru2009=u20090.77), which implied the putative lncRNA-mRNA interaction and the potential roles of the lncRNA and its target in wood formation. Our study provides a novel method integrating association studies and expression profiling for functional annotation of lncRNAs and dissection of lncRNA-mRNA interactions in trees.

Association genetics and expression patterns of a CBF4 homolog in Populus under abiotic stress.

New strategies for prevention and treatment of abiotic stress require an improved understanding of stress responses. Here, we examined response differences of a C-repeat binding factor gene (PsCBF4) between five species in the genus Populus. We also used a candidate gene-based approach to identify single nucleotide polymorphisms (SNPs) within PsCBF4 that were associated with physiological and biochemical traits in a natural population (528 unrelated individuals) of Populussimonii. We first isolated a 1,044-bp PsCBF4 cDNA encoding a polypeptide of 256 amino acids. Expression profiling revealed that CBF4 is differentially expressed under cold, heat, drought, and salt conditions among five Populus species. Cold stress is the most significant interspecific difference, and PsCBF4 transcript levels ranged from 6.5 to 379.5 times higher than in unstressed controls. A natural population of P. simonii showed high nucleotide diversity (πTxa0=xa00.00880, θwxa0=xa00.01192) and low linkage disequilibrium (r2xa0≥xa00.1, within 700xa0bp) across PsCBF4. Association analysis showed that nine SNPs (false discovery rate Qxa0<xa00.10) and two haplotypes (Qxa0<xa00.10) were significantly associated with six physiological and biochemical traits, with each marker explaining 3.36–6.12xa0% of the phenotypic variance in the corresponding trait. Transcript analysis further detected significant differences among genotypic classes for all significant SNPs. Identification of these significant associations will help reveal the molecular bases of physiological and biochemical differences and provide a starting point for marker-assisted selection for traits involved in stress tolerance in P. simonii.

Single nucleotide polymorphisms in a cellulose synthase gene (PtoCesA3) are associated with growth and wood properties in Populus tomentosa

In plants, the composition and organization of the cell wall determine cell shape, enable cell expansion, and affect the properties of woody tissues. Cellulose synthase (CesA) genes encode the enzymes involved in the synthesis of cellulose which is the major component of plant primary and secondary cell walls. Here, we isolated a full-length PtoCesA3 cDNA from the stem cambium tissue of Populus tomentosa. Tissue-specific expression profiling showed that PtoCesA3 is highly expressed during primary cell wall formation. Estimation of single nucleotide polymorphism (SNP) diversity and linkage disequilibrium (LD) revealed that PtoCesA3 harbors high SNP diversity (πTxa0=xa00.00995 and θwxa0=xa00.0102) and low LD (r2xa0≥xa00.1, within 1,280xa0bp). Association analysis in a P. tomentosa association population (460 individuals) showed that seven SNPs (false discovery rate Qxa0<xa00.10) and five haplotypes (Qxa0<xa00.10) were significantly associated with growth and wood properties, explaining 4.09–7.02xa0% of the phenotypic variance. All significant marker-trait associations were validated in at least one of the three smaller subsets (climatic regions) while five associations were repeated in the linkage population. Variation in RNA transcript abundance among genotypic classes of significant loci was also confirmed in the association or linkage populations. Identification of PtoCesA3 and examining its allelic polymorphisms using association studies open an avenue to understand the mechanism of cellulose synthesis in the primary cell wall and its effects on the properties of woody tissues.