Abstract：To provide an important tool for molecular marker-assisted breeding of wood quality traits, a new nuclear genome SSR marker was developed from functional genes related to Cunninghamia lanceolata wood formation. The SSR locus was detected through analyzing the sequences of 10 genes involved in the formation of C. lanceolata. The availability and polymorphism of the designed SSR primers were examined by capillary electrophoresis. Linkage disequilibrium of pairwise loci and the association analysis between developed SSR loci and wood density were performed by using TASSEL (trait analysis by association, evolution and linkage) GLM (general linear model) program. A total of 15 SSR loci were identified in 7 functional genes. There were di-, tri-, tetra- and hexa- nucleotide repeats, with trinucleotide SSRs being the most frequent,accounting for 46.7% of the total loci. Fourteen pairs of PCR amplification primers were designed according to conserved sequences on both sides of SSR loci. Among them, 7 pairs of primers showed polymorphism. A total of 644 SSR loci combinations were found to have a certain degree of linkage disequilibrium (D'≠ 0), with D' values of 183 combinations greater than 0.5. The association analysis revealed that the CLSSR4 locus from the 5' untranslated region (5' UTR) of ClCesA3 (C. lanceolata cellulose synthase 3) was extremely significantly associated with wood density (P<0.01). The wood density variance explained by this locus was 26.07%, and the corresponding alleles of A201 and A205 showed the positive and negative phenotypic effects, respectively. In this study, seven polymorphic SSR primer pairs were developed from genomic sequence of functional genes, and a SSR locus derived from the 5' UTR of ClCesA3 gene was significantly associated with wood density of C. lanceolata. These results will provide an important tool for molecular marker-assisted improvement of C. lanceolata wood quality.
杭芸, 俞金健, 周世水, 程健弘, 黄华宏, 林二培, 童再康. 杉木木材形成功能基因内SSR标记的开发及应用[J]. 农业生物技术学报, 2019, 27(1): 34-42.
HANG Yun, YU Jin-Jian, ZHOU Shi-Shui, CHENG Jian-Hong, HUANG Hua-Hong, LIN Er-Pei, TONG Zai-Kang. Development and Application of SSR Loci from Functional Genes Involved in Wood Formation in Cunninghamia lanceolata. 农业生物技术学报, 2019, 27(1): 34-42.
[1] 胡梦霄. 2016. 基于SSR标记的杉木重要性状关联分析研究[D]. 硕士学位论文, 浙江农林大学, 导师: 童再康, pp. 31-45. (Hu MX.2016. Association analysis of important traits based on SSR markers in Cunninghamia lanceolata[D]. Thesis for M.S., Zhejiang A & F University, Supervisor: Tong Z K, pp. 31-45.) [2] 黄海燕, 杜红岩, 刘攀峰. 2013. 基于杜仲转录组序列的 SSR 分子标记的开发[J]. 林业科学, 49(5): 176-181. (Huang HY, Du HY, Liu PF.2013. Development of SSR molecular markers based on transcriptome sequencing of Eucommia ulmoides[J]. Scientia Silvae Sinicae, 49(5):176-181.) [3] 刘琼瑶. 2015. 杉木NAC基因的克隆、表达与SNP分析[D]. 硕士学位论文, 浙江农林大学, 导师: 黄华宏, pp. 10-28. (Liu QY.2015. Isolation, expression and single nucleotide polymorphisms analysis of NAC genes in Cunninghamia lanceolata[D]. Thesis for M.S., Zhejiang A & F University, Supervisor: Huang H H, pp. 10-28.) [4] 宋志姣, 翁启杰, 周长品, 等. 2016. 细叶桉(Eucalyptus tereticornis)早期生长的SSR 标记关联分析[J]. 分子植物育种, 14(1): 195-203. (Song Z J,Weng Q J,Zhou C P,et al.2016. SSR Markers associated with early growth in Eucalyptus tereticornis[J]. Molecular Plant Breeding,14(10): 173-180) [5] 王保垒, 王博文, 陈清清, 等. 2011. 杨树抗逆转录因子基因内SSR标记的开发[J]. 林业科学, 47(8):67-74. (Wang B L, Wang B W, Chen Q Q, et al.2011. Identification of SSR loci from transcription factor genes expressed under abiotic stresses in Populus[J]. Scientia Silvae Sinicae, 47(8): 67-74.) [6] 文自翔, 赵团结, 郑永战, 等. 2008. 中国栽培和野生大豆农艺及品质性状与SSR标记的关联分析Ⅱ.优异等位变异的发掘[J]. 作物学报, 34(8): 1339-1349. (Wen Z X, Zhao T J, Zheng Y Z, et al.2008. Association analysis of agronomic and quality traits with SSR markers in Glycine max and Glycine soja in China: Ⅱ. Exploration of Elite Alleles[J]. Acta Agronomica Sinica. 34(8):1339-1349.) [7] 文亚峰, 韩文军, 周宏,等. 2015. 杉木转录组SSR挖掘及EST-SSR标记规模化开发[J]. 林业科学, 51(11): 40-49. (Wen Y F, Han W J, Zhou H, et al.2015. SSR mining and development of EST-SSR markers for Cunninghamia lanceolata based on transcriptome sequences[J]. Scientia Silvae Sinicae, 51(11): 40-49.) [8] 张圣, 黄华宏, 林二培, 等. 2013. 杉木与台湾杉EST-SSR标记的开发与应用[J]. 林业科学, 49(10):173-180. (Zhang S, Huang H H, Lin E P, et al.2013. Development and application of EST-SSR markers for Cunninghamia lanceolata and Taiwania cryptomerioides[J]. Scientia Silvae Sinicae, 49(10): 173-180.) [9] Cordeiro G M, Casu R, Mcintyre C L, et al.2001. Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum[J]. Plant Science, 160(6): 1115-1123. [10] Dillon S K, Nolan M, Li W, et al.2010. Allelic variation in cell wall candidate genes affecting solid wood properties in natural populations and land races of Pinus radiate[J]. Genetics, 185: 1477-1487. [11] Du Q, Gong C, Pan W, et al.2013. Development and application of microsatellites in candidate genes related to wood properties in the Chinese White Poplar (Populus tomentosa Carr.)[J]. DNA Research, 20(1): 31-44. [12] Du Q, Pan W, Xu B, et al.2012. Polymorphic simple sequence repeat (SSR) loci within cellulose synthase (PtoCesA) genes are associated with growth and wood properties in Populus tomentosa[J]. New Phytologist, 197(3): 763-776. [13] Elkassaby Y A.2004. Single-copy, species-transferable microsatellite markers developed from loblolly pine ESTs[J]. Theoretical & Applied Genetics, 109(2): 361-369. [14] Ellis J R, Burke J M.2007. EST-SSRs as a resource for population genetic analyses[J]. Heredity, 99(2): 125-132. [15] Ferrão L F V, Caixeta E T, Pena G, et al.2015. New EST-SSR markers of Coffea arabica: Transferability and application to studies of molecular characterization and genetic mapping[J]. Molecular Breeding, 35(1): 31. [16] Huang H H, Xu L L, Tong Z K, et al.2012. De novo characterization of the Chinese fir (Cunninghamia lanceolata) transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis[J]. BMC Genomics. 13: 648. [17] Iglesias A R, Kindlund E, Tammi M, et al.2004. Some microsatellites may act as novel polymorphic cis-regulatory elements through transcription factor binding[J]. Gene, 341: 149-165. [18] Kalia R K, Rai M K, Kalia S, et al.2011. Microsatellite markers: An overview of the recent progress in plants[J]. Euphytica, 177(3): 309-334. [19] Li Y C, Korol A B, Fahima T, et al.2004. Microsatellites within genes: Structure, function, and evolution[J]. Molecular Biology and Evolution, 21(6): 991-1007. [20] Li Y, Wilcox P, Telfer E, et al.2016. Association of single nucleotide polymorphisms with form traits in three New Zealand populations of radiata pine in the presence of genotype by environment interactions[J]. Tree Genetics & Genomes, 12(4): 1-12. [21] Neale D, Kremer A.2011. Forest tree genomics: Growing resources and applications[J]. Nature Reviews Genetics, 12(2): 111-122. [22] Scott K D, Eggler P, Seaton G, et al.2000. Analysis of SSRs derived from grape ESTs[J]. Theoretical & Applied Genetics, 100(5):723-726. [23] Song Q J, Marek L F, Shoemaker R C, et al.2004. A new integrated genetic linkage map of the soybean[J]. Theoretical & Applied Genetics, 109(1): 122-8. [24] Stewart C N, Via L E.1993. A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications[J]. Biotechniques, 14(5): 748-750. [25] Tuskan G A, Gunter L E, Yang Z K.2004. Characterization of microsatellites revealed by genomic sequencing of Populus trichocarpa[J]. Canadian Journal of Forest Research. 34(1): 85-93. [26] Wei Z, Du Q, Zhang J, et al.2013. Genetic diversity and population structure in Chinese indigenous Poplar (Populus simonii) populations using microsatellite markers[J]. Plant Molecular Biology Reporter, 31(3): 620-632.
