, Volume 241, Issue 6, pp 1417–1434 | Cite as

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

  • Qingzhang Du
  • Lu Wang
  • Xiaohui Yang
  • Chenrui Gong
  • Deqiang ZhangEmail author
Original Article


Main conclusion

Extensive characterization of the poplar GH9 gene family provides new insights into GH9 function and evolution in woody species, and may drive novel progress for molecular breeding in trees.


In 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.


Association mapping Chromosomal localization Glycosyl hydrolase family 9 KORRIGAN Populus Wood properties 



Carbohydrate-binding module


Cellulose synthase complex


Diameter at the breast height


Expressed sequence tags


False discovery rate


Glycosyl hydrolase family 9


General linear model






Linkage disequilibrium


Linkage groups


Minor allele frequencies


Mixed linear model


Million years






Reverse transcription PCR


Real-time quantitative PCR


The squared correlation of allele frequencies


Southern subset


Single-nucleotide polymorphisms


Divergence time


Transmembrane domain



This work was supported by grants from the Forestry Public Benefit Research Program (No. 201304102), the State Key Basic Research Program of China (No. 2012CB114506), and the Project of the National Natural Science Foundation of China (No. 31170622, 30872042).

Supplementary material

425_2015_2271_MOESM1_ESM.doc (184 kb)
Fig. S1 Molecular characterization of PtoKOR1 and PtoKOR2. a Nucleotide and deduced amino acid sequences of PtoKOR1 and PtoKOR2. Numbers on the left refer to the positions of nucleotides or amino acid residues. b Gene structures of PtoKOR1 and PtoKOR2 (DOC 183 kb)
425_2015_2271_MOESM2_ESM.doc (112 kb)
Fig. S2 Protein sequence alignment of PtoKOR1 and PtoKOR2 with other plant PtoGH9s. Numbers on the left are the positions of the amino acids in each protein, with gaps (dashes) included to maximize alignments. Identical and similar amino acids are shaded in red and blue, respectively. represents the residues essential for catalytic activity identified in other plant PtoGH9s which are also conserved in PtoKOR1 and PtoKOR2 (D163/165, H504 and E561); * indicates the eight predicted glycosylation sites. The predicted transmembrane domain is overlined; the conserved polarized targeting signals are boxed. At: Arabidopsis thaliana, Os: Oryza sativa, Sl: Solanum lycopersicum (DOC 111 kb)
425_2015_2271_MOESM3_ESM.doc (756 kb)
Fig. S3 The decay of linkage disequilibrium within PtoKOR1 (a) and PtoKOR2 (b) in the natural population. Pairwise correlations between SNPs are plotted against the physical distance between the SNPs in base pairs. The curves describe the nonlinear regressions of r 2 (Er2) onto the physical distance in base pairs (DOC 756 kb)
425_2015_2271_MOESM4_ESM.doc (52 kb)
Fig. S4 Significant pairwise linkage disequilibrium (r 2 > 0.75, P < 0.001) between SNP markers in PtoKOR1. Four significant common SNP blocks are shown on a schematic of PtoKOR1 (DOC 52 kb)
425_2015_2271_MOESM5_ESM.doc (459 kb)
Fig. S5 The haplotype effect and protein structures for PtoKOR1 containing two significant haplotypes (T-C-T-A and A-T-A-G). a Haplotype effects on haplotype 1 (T-C-T-A) and haplotype 2 (A-T-A-G) found in PtoKOR1 controlling fiber width in Populus tomentosa natural populations. b Three-dimensional (3D) protein structures for PtoKOR1 containing two significant haplotypes (T-C-T-A and A-T-A-G) were predicted using SWISS-MODEL ( Four significant fold architecture changes between these two protein structures are indicated with an arrow (DOC 459 kb)
425_2015_2271_MOESM6_ESM.xlsx (257 kb)
Sequence data from this article has been deposited in the GenBank Data Library under the accession Nos. HQ331247–HQ331273, HQ331276–HQ331300, and HQ380298–HQ380376. The phenotypic and genotypic data for SNP-trait association studies were provided as additional files (See Supplementary Information files S1 and S2). File S1 The phenotype data used in the SNP-traits association analysis in Populus tomentosa association population. File S2 The genotype data for SNPs in PtoKOR1 used in the SNP-traits association analysis in Populus tomentosa association population (XLSX 257 kb)
425_2015_2271_MOESM7_ESM.doc (46 kb)
Table S1 Geographical and meteorological parameters of three climatic distributional regions of Populus tomentosa in this study (DOC 46 kb)
425_2015_2271_MOESM8_ESM.doc (46 kb)
Table S2 Primers used for real-time PCR analysis (DOC 46 kb)
425_2015_2271_MOESM9_ESM.doc (36 kb)
Table S3 The substitution rate ratios of nonsynonymous (dN or Ka) versus synonymous (dS or Ks) mutations estimated for paralogous PtrGH9 proteins. Synonymous (dS and Ks) and non-synonymous (dN and Ka) were used as parameters of substitution rates (DOC 35 kb)
425_2015_2271_MOESM10_ESM.doc (30 kb)
Table S4 Summary of transitions and transversions patterns for these SNPs identified in PtoKOR1 and PtoKOR2 (DOC 29 kb)
425_2015_2271_MOESM11_ESM.doc (83 kb)
Table S5 Functional domains and motifs of GH9 proteins in poplar (DOC 83 kb)
425_2015_2271_MOESM12_ESM.doc (107 kb)
Table S6 Coding region nucleotide1 and amino acid2 sequence pairwise comparisons (% similarity) between poplar GH9 genes (DOC 107 kb)
425_2015_2271_MOESM13_ESM.doc (133 kb)
Table S7 List of GH9 protein sequences from different species used in this study (DOC 133 kb)
425_2015_2271_MOESM14_ESM.doc (80 kb)
Table S8 Summary of nucleotide polymorphisms at the PtoKOR1 and PtoKOR2 loci, respectively. Regions containing indels are excluded from the calculation 14 (DOC 80 kb)
425_2015_2271_MOESM15_ESM.doc (66 kb)
Table S9 Summary of significant associations identified in PtoKOR1 using two association models (DOC 66 kb)
425_2015_2271_MOESM16_ESM.doc (33 kb)
Table S10 Validation of significant associations in PtoKOR1 using three climatic regions in Populus tomentosa natural populations (DOC 33 kb)


  1. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefPubMedCentralPubMedGoogle Scholar
  2. Bhandari S, Fujino T, Thammanagowda S, Zhang D, Xu F, Joshi CP (2006) Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees. Planta 224:828–837CrossRefPubMedGoogle Scholar
  3. Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler ES (2007) TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23:2633–2635CrossRefPubMedGoogle Scholar
  4. Brummell DA, Catala C, Lashbrook CC, Bennett AB (1997) A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants. Proc Natl Acad Sci USA 94:4794–4799CrossRefPubMedCentralPubMedGoogle Scholar
  5. Buchanan M, Burton RA, Dhugga KS, Rafalski AJ, Tingey SV, Shirley NJ, Fincher GB (2012) Endo-(1,4)-beta-glucanase gene families in the grasses: temporal and spatial co-transcription of orthologous genes. BMC Plant Biol 12:235. doi: 10.1186/1471-2229-12-235 CrossRefPubMedCentralPubMedGoogle Scholar
  6. Collinson ME (1992) The early fossil history of Salicaceae—a brief review. Proc R Soc Edinb Sect B Biol Sci 98:155–167CrossRefGoogle Scholar
  7. Cosgrove DJ (1999) Enzymes and other agents that enhance cell wall extensibility. Annu Rev Plant Biol 50:391–417CrossRefGoogle Scholar
  8. Crowell EF, Gonneau M, Stierhof YD, Hofte H, Vernhettes S (2010) Regulated trafficking of cellulose synthases. Curr Opin Plant Biol 13:700–705CrossRefPubMedGoogle Scholar
  9. del Campillo E (1999) Multiple endo-1,4-β-d-glucanase (cellulase) genes in Arabidopsis. Curr Top Dev Biol 46:39–61CrossRefPubMedGoogle Scholar
  10. Du Q, Wang B, Wei Z, Zhang D, Li B (2012) Genetic diversity and population structure of Chinese white poplar (Populus tomentosa) revealed by SSR markers. J Hered 103:853–862CrossRefPubMedGoogle Scholar
  11. Du QZ, Pan W, Xu BH, Li BL, Zhang DQ (2013) Polymorphic SSR loci within PtoCesA genes are associated with growth and wood properties in Populus tomentosa. New Phytol 197:763–776CrossRefPubMedGoogle Scholar
  12. Du Q, Xu B, Gong C, Yang X, Pan W, Tian J, Li B, Zhang D (2014) Variation in growth, leaf and wood-property traits of Chinese white poplar (Populus tomentosa Carr.), a major industrial tree species in Northern China. Can J For Res 44:326–339CrossRefGoogle Scholar
  13. Fu YX, Li WH (1993) Statistical tests of neutrality of mutations. Genetics 133:693–709PubMedCentralPubMedGoogle Scholar
  14. Hardy OJ, Vekemans X (2002) SPAGEDi: a versatile computer program to analyze spatial genetic structure at the individual or population levels. Mol Ecol Notes 2:618–620CrossRefGoogle Scholar
  15. Hill WG, Robertson A (1968) Linkage disequilibrium in finite populations. Theor Appl Genet 38:226–231CrossRefPubMedGoogle Scholar
  16. Huang ZH (1992) The study on the climatic regionalization of the distributional region of Populus tomentosa. J Beijing For Univ 14:26–32Google Scholar
  17. Irimia M, Roy SW (2008) Spliceosomal introns as tools for genomic and evolutionary analysis. Nucleic Acids Res 36:1703–1712CrossRefPubMedCentralPubMedGoogle Scholar
  18. Kalluri UC, Difazio SP, Brunner AM, Tuskan GA (2007) Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa. BMC Plant Biol 7:59CrossRefPubMedCentralPubMedGoogle Scholar
  19. Lane DR, Wiedemeier A, Peng LC, Hofte H, Vernhettes S, Desprez T, Hocart CH, Birch RJ, Baskin TI, Burn JE, Arioli T, Betzner AS, Williamson RE (2001) Temperature-sensitive alleles of RSW2 link the KORRIGAN endo-1,4-β- glucanase to cellulose synthesis and cytokinesis in Arabidopsis. Plant Physiol 126:278–288CrossRefPubMedCentralPubMedGoogle Scholar
  20. Li L, Lu S, Chiang VL (2006) A genomic and molecular view of wood formation. Crit Rev Plant Sci 25:213–233CrossRefGoogle Scholar
  21. Li X, Wu H, Dillon S, Southerton SG (2009) Generation and analysis of expressed sequence tags from six developing xylem libraries in Pinus radiata D.Don. BMC Genom 10:1–18CrossRefGoogle Scholar
  22. Libertini E, Li Y, McQueen-Mason SJ (2004) Phylogenetic analysis of the plant endo-β-1,4-glucanase gene family. J Mol Evol 58:506–515CrossRefPubMedGoogle Scholar
  23. Liebminger E, Grass J, Altmann F, Mach L, Strasser R (2013) Characterizing the link between glycosylation state and enzymatic activity of the endo-β-1,4-glucanase KORRIGAN1 from Arabidopsis thaliana. J Biol Chem 288:22270–22280CrossRefPubMedCentralPubMedGoogle Scholar
  24. Lopez-Casado G, Urbanowicz BR, Damasceno CM, Rose JK (2008) Plant glycosyl hydrolases and biofuels: a natural marriage. Curr Opin Plant Biol 11:329–337CrossRefPubMedGoogle Scholar
  25. Maloney VJ, Mansfield SD (2010) Characterization and varied expression of a membrane-bound endo-β-1,4-glucanase in hybrid poplar. Plant Biotechnol J 8:294–307CrossRefPubMedGoogle Scholar
  26. Maloney VJ, Lacey Samuels A, Mansfield SD (2012) The endo-1,4-β-glucanase Korrigan exhibits functional conservation between gymnosperms and angiosperms and is required for proper cell wall formation in gymnosperms. New Phytol 193:1076–1087CrossRefPubMedGoogle Scholar
  27. Mansfield SD, Kibblewhite RP, Riddell M (2004) Characterization of the reinforcement potential of different softwood Kraft fibres in softwood/hardwood pulp mixtures. Wood Fiber Sci 36:344–358Google Scholar
  28. Mansoori N, Timmers J, Desprez T, Kamei C, Dees D, Vincken J, Visser R, Höfte H, Vernhettes S, Trindade LM (2014) KORRIGAN1 interacts specifically with integral components of the cellulose synthase machinery. PLoS One 9:e112387. doi: 10.1371/journal.pone.0112387 CrossRefPubMedCentralPubMedGoogle Scholar
  29. Mitchell-Olds T, Clauss MJ (2002) Plant evolutionary genomics. Curr Opin Plant Biol 5:74–79CrossRefPubMedGoogle Scholar
  30. Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New YorkGoogle Scholar
  31. Porth I, Klápště J, Skyba O, Lai BS, Geraldes A, Muchero W, Tuskan GA, Douglas CJ, El-Kassaby YA, Mansfield SD (2013) Populus trichocarpa cell wall chemistry and ultrastructure trait variation, genetic control, and genetic correlations. New Phytol 197:777–790CrossRefPubMedGoogle Scholar
  32. Remington DL, Thornsberry JM, Matsuoka Y, Wilson LM, Whitt SR, Doebley J, Kresovich S, Goodman MM, Buckler ES (2001) Structure of linkage disequilibrium and phenotypic associations in the maize genome. Proc Natl Acad Sci USA 98:11479–11484CrossRefPubMedCentralPubMedGoogle Scholar
  33. Ritland K (1996) Estimators for pairwise relatedness and individual inbreeding coefficients. Genet Res Camb 67:175–185CrossRefGoogle Scholar
  34. Rose JK, Bennett AB (1999) Cooperative disassembly of the cellulose–xyloglucan network of plant cell walls: parallels between cell expansion and fruit ripening. Trends Plant Sci 4:176–183CrossRefPubMedGoogle Scholar
  35. Rozas J, Sanchez-Delbarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496–2497CrossRefPubMedGoogle Scholar
  36. Sato S, Kato T, Kakegawa K, Ishii T, Liu YG, Awano T, Takabe K, Nishiyama Y, Kuga S, Sato S, Nakamura Y, Tabata S, Shibata D (2001) Role of the putative membrane-bound endo-1,4-β-glucanase KORRIGAN in cell elongation and cellulose synthesis in Arabidopsis thaliana. Plant Cell Physiol 42:251–263CrossRefPubMedGoogle Scholar
  37. Savard L, Li P, Strauss SH, Chase MW, Michaud M, Bousquet J (1994) Chloroplast and nuclear gene sequences indicate late Pennsylvanian time for the last common ancestor of extant seed plants. Proc Natl Acad Sci USA 91:5163–5167CrossRefPubMedCentralPubMedGoogle Scholar
  38. Shani Z, Dekel M, Roiz L, Horowitz M, Kolosovski N, Lapidot S, Alkan S, Koltai H, Tsabary G, Goren R (2006) Expression of endo-1,4-beta-glucanase (cel1) in Arabidopsis thaliana is associated with plant growth, xylem development and cell wall thickening. Plant Cell Rep 25:1067–1074CrossRefPubMedGoogle Scholar
  39. Stephens M, Scheet P (2005) Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. Am J Hum Genet 76:449–462CrossRefPubMedCentralPubMedGoogle Scholar
  40. Storey JD, Tibshirani R (2003) Statistical significance for genome wide studies. Proc Natl Acad Sci USA 100:9440–9445CrossRefPubMedCentralPubMedGoogle Scholar
  41. Suyama M, Torrents D, Bork P (2006) PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res 34:609–612CrossRefGoogle Scholar
  42. Szyjanowicz PM, McKinnon I, Taylor NG, Gardiner J, Jarvis MC, Turner SR (2004) The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana. Plant J 37:730–740CrossRefPubMedGoogle Scholar
  43. Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585–595PubMedCentralPubMedGoogle Scholar
  44. Takahashi J, Rudsander UJ, Hedenström M, Banasiak A, Harholt J, Amelot N, Immerzeel P, Ryden P, Endo S, Ibatullin FM, Brumer H, del Campillo E, Master ER, Scheller HV, Sundberg B, Teeri TT, Mellerowicz EJ (2009) KORRIGAN1 and its aspen homolog PttCel9A1 decrease cellulose crystallinity in Arabidopsis stems. Plant Cell Physiol 50:1099–1115CrossRefPubMedGoogle Scholar
  45. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefPubMedGoogle Scholar
  46. Taylor NG (2008) Cellulose biosynthesis and deposition in higher plants. New Phytol 178:239–252CrossRefPubMedGoogle Scholar
  47. Thiergart T, Landan G, Schenk M, Dagan T, Martin WF (2012) An evolutionary network of genes present in the eukaryote common ancestor polls genomes on eukaryotic and mitochondrial origin. Genome Biol Evol 4:466–485CrossRefPubMedCentralPubMedGoogle Scholar
  48. Tuskan GA, DiFazio S, Jansson S, Bohlmann J, Grigoriev I et al (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:1596–1604CrossRefPubMedGoogle Scholar
  49. Urbanowicz BR, Bennett AB, del Campillo E, Catala C, Hayashi T, Henrissat B, Hofte H, McQueen-Mason SJ, Patterson SE, Shoseyov O, Teeri TT, Rose JKC (2007a) Structural organization and a standardized nomenclature for plant endo-1,4-β-glucanases (cellulases) of glycosyl hydrolase family 9. Plant Physiol 144:1693–1696CrossRefPubMedCentralPubMedGoogle Scholar
  50. Urbanowicz BR, Catalá C, Irwin D, Wilson DB, Ripoll DR (2007b) A tomato endo-β-1,4-glucanase, SlCel9C1, represents a distinct subclass with a new family of carbohydrate binding modules (CBM49). J Biol Chem 282:12066–12074CrossRefPubMedGoogle Scholar
  51. Van de Peer Y, Maere S, Meyer A (2009) The evolutionary significance of ancient genome duplications. Nat Rev Genet 10:725–732CrossRefPubMedGoogle Scholar
  52. Vandepoele K, Simillion C, Van de Peer Y (2003) Evidence that rice and other cereals are ancient aneuploids. Plant Cell 15:2192–2202CrossRefPubMedCentralPubMedGoogle Scholar
  53. Watterson GA (1975) On the number of segregating sites in genetical models without recombination. Theor Popul Biol 7:188–193CrossRefGoogle Scholar
  54. Wegrzyn JL, Eckert AJ, Choi M, Lee JM, Stanton BJ, Sykes R, Davis MF, Tsai CJ, Neale DB (2010) Association genetics of traits controlling lignin and cellulose biosynthesis in black cottonwood (Populus trichocarpa, Salicaceae) secondary xylem. New Phytol 188:515–532CrossRefPubMedGoogle Scholar
  55. Wilkins O, Nahal H, Foong J, Provart NJ, Campbell MM (2009) Expansion and diversification of the Populus R2R3-MYB family of transcription factors. Plant Physiol 149:981–993CrossRefPubMedCentralPubMedGoogle Scholar
  56. Xie G, Yang B, Xu Z, Li F, Guo K, Zhang M, Wang L, Zou W, Wang Y, Peng L (2013) Global identification of multiple OsGH9 family members and their involvement in cellulose crystallinity modification in rice. PLoS One 8:e50171. doi: 10.1371/journal.pone.0050171 CrossRefPubMedCentralPubMedGoogle Scholar
  57. Yoshida K, Komae K (2006) A rice family 9 glycoside hydrolase isozyme with broad substrate specificity for hemicelluloses in type II cell walls. Plant Cell Physiol 47:1541–1554CrossRefPubMedGoogle Scholar
  58. Yu J, Pressoir G, Briggs WH, Vroh Bi I, Yamasaki M, Doebley JF, McMullen MD, Gaut BS, Nielsen DM, Holland JB, Kresovich S, Buckler ES (2006) A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. Nat Genet 38:203–208CrossRefPubMedGoogle Scholar
  59. Zhang D, Xu B, Yang X, Zhang Z, Li B (2011) The sucrose synthase gene family in Populus: structure, expression, and evolution. Tree Genet Genomes 7:443–451CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Qingzhang Du
    • 1
    • 2
  • Lu Wang
    • 1
    • 2
  • Xiaohui Yang
    • 1
    • 2
  • Chenrui Gong
    • 1
    • 2
  • Deqiang Zhang
    • 1
    • 2
    Email author
  1. 1.National Engineering Laboratory for Tree Breeding, College of Biological Sciences and TechnologyBeijing Forestry UniversityBeijingPeople’s Republic of China
  2. 2.Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, College of Biological Sciences and TechnologyBeijing Forestry UniversityBeijingPeople’s Republic of China

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