Transcriptomic SSR Marker Development and Genetic Diversity Analysis in Celery (Apium graveolens)
CHEN Chang-Long1, DONG Yan2, TIAN Yu1, SHI Miao-Han1,2, GE Xiu-Xiu3, XIE Hua1*
1 Beijing Agro-Biotechnology Research Center/Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China; 2 Chengde Academy of Agriculture and Forestry Sciences, Chengde 067000, China; 3 College of Bioscience and Resources Environment, Beijing University of Agriculture, Beijing 102206, China
Abstract Celery (Apium graveolens) is one of the most economically important leafy vegetables worldwide. Current celery genetic research requires some valuable molecular markers. A total of 4 034 SSR loci were identified in 39 619 unigenes assembled from 3 sets of high quality reads of celery transcriptome data in GenBank. Of these, di-nucleotide repeat motifs were found to be the most abundant, which accounted for 43.9% (1772), followed by mono- (26.7%, 1078) and tri- (21.0%, 846) nucleotide repeat motifs. Tetra-, penta-, and hexa- nucleotide repeats accounted for 1.3% (52) and the other was compound type (7.1%, 286). A set of 39 pairs of primers from the designed 375 SSR primers were successfully validated to yield clear and stable high quality specific PCR products in 5 selected representative celery accessions from different sources. Using fluorenscently labelled PCR and multiplex capillary electrophoresis, all the 39 markers detected 110 alleles in 76 celery accessions, ranging from 2~4 with an average number of 2.821 alleles and 2~9 genotypes with an average of 4.308 per locus. Average polymorphism information content (PIC) and discrimination power (PD) were 0.256 (0.013~0.581) and 0.379 (0.026~0.764) for per locus, respectively. Genetic diversity analysis of the 76 celery accessions indicated that for local celery and celery, the average numbers of alleles were 2.641 and 2.436, the average Nei's indices were 0.347 and 0.194 and the average Shannon's indices were 0.589 and 0.351, suggesting greater genetic diversity in local celery than that in celery. Analysis of molecular variance (AMOVA) of A. graveolens showed that 61.72% of genetic variation was within celery individuals, 22.07% among individuals within local celery or celery population, and 16.22% between local celery and celery populations. The cluster results of PCA (principal component analysis) and UPGMA (unweighted pair-group method with arithmetic means) dendrogram separated the 76 celery cultivars into 2 groups mainly corresponding to celery and local celery, respectively. However, compared to celery, local celery formed a more discrete cluster, which indicated local celery probably had more complex genetic background. The study could enrich SSR markers and provide multiplex capillary electrophoresis detection system based on fluorescence labeling for genetic diversity analysis and identification of a large number of celery germplasm resources.
CHEN Chang-Long,DONG Yan,TIAN Yu, et al. Transcriptomic SSR Marker Development and Genetic Diversity Analysis in Celery (Apium graveolens)[J]. 农业生物技术学报, 2020, 28(4): 616-628.
[1] 陈雅琼, 李凤霞, 李锡坤, 等. 2011. 烟草SSR荧光标记与毛细管电泳检测技术研究[J]. 中国烟草科学, 32(2): 66-70, 80. (Chen Y Q, Li F X, Li X K, et al.2011. Fluorescent detection and capillary electrophoresis in tobacco SSR[J]. Chinese Tobacco Science, 32(2): 66-70, 80.) [2] 郝晨阳, 王兰芬, 贾继增, 等. 2005. SSR荧光标记和银染技术的比较分析[J]. 作物学报, 31(2): 144-149. (Hao C Y, Wang L F, Jia J Z, et al.2005. Comparison of fluorescence and silver-staining detection systems of microsatellite markers[J]. Acta Agronomica Sinica, 31(2): 144-149.) [3] 刘庞源, 张宝海, 何伟明, 等. 2013. 基于RAPD分子标记芹菜品种亲缘关系分析研究[J]. 安徽农业科学, 41(27): 10923-10925. (Liu P Y, Zhang B H, He W M, et al.2013. Analytical method of celery cultivars relationship based on RAPD molecular markers[J]. Journal of Anhui Agricultural Sciences, 41(27): 10923-10925.) [4] 武文艳, 易红梅, 王凤格, 等. 2012. 玉米SSR分子标记的荧光多重PCR体系的构建及优化[J]. 作物杂志, (5): 59-62. (Wu W Y, Yi H M, Wang F G, et al. 2012. Construction and optimization of multiplex PCR of maize with SSR marker[J]. Crops, (5): 59-62.) [5] Acquadro A, Magurno F, Portis E, et al.2006. dbEST-derived microsatellite markers in celery (Apium graveolens L. var. dulce)[J]. Molecular Ecology Notes, 6(4): 1080-1082. [6] Castillo A, Budak H, Varshney R K, et al.2008. Transferability and polymorphism of barley EST-SSR markers used for phylogenetic analysis in Hordeum chilense[J]. BMC Plant Biology, 8(1): 97. [7] Ding Q, Li J, Wang F, et al.2015. Characterization and development of EST-SSRs by deep transcriptome sequencing in Chinese cabbage (Brassica rapa L. ssp. pekinensis)[J]. International Journal of Genomics, 2015: 473028. [8] Excoffier L, Lischer H E L.2010. Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows[J]. Molecular Ecology Resources, 10(3): 564-567. [9] Fu N, Wang P, Liu X, et al.2014. Use of EST-SSR markers for evaluating genetic diversity and fingerprinting celery (Apium graveolens L.) cultivars[J]. Molecules, 19(2): 1939-1955. [10] Fu N, Wang Q, Shen H L.2013. De novo assembly, gene annotation and marker development using Illumina paired-end transcriptome sequences in celery (Apium graveolens L.)[J]. PLOS ONE, 8(2): e57686. [11] Gao L F, Jing R L, Huo N X, et al.2004. One hundred and one new microsatellite loci derived from ESTs (EST-SSRs) in bread wheat[J]. Theoretical and Applied Genetics, 108(7): 1392-1400. [12] Grabherr M G, Haas B J, Yassour M, et al.2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J]. Nature Biotechnology, 29(7): 644-652. [13] Han Z, Ma X, Wei M, et al.2018. SSR marker development and intraspecific genetic divergence exploration of Chrysanthemum indicum based on transcriptome analysis[J]. BMC Genomics, 19(1): 291. [14] Huestis G M, McGrath J M, Quiros C F.1993. Development of genetic markers in celery based on restriction fragment length polymorphisms[J]. Theoretical and Applied Genetics, 85(6-7): 889-896. [15] Jombart T.2008. adegenet: A R package for the multivariate analysis of genetic markers[J]. Bioinformatics, 24(11): 1403-1405. [16] Li G, Quiros C F.2000. Use of amplified fragment length polymorphism markers for celery cultivar identification[J]. HortScience, 35(4): 726-728. [17] Li M, Wang F, Jiang Q, et al.2014. Identification of SSRs and differentially expressed genes in two cultivars of celery (Apium graveolens L.) by deep transcriptome sequencing[J]. Horticulture research, 1(10): 10. [18] Li M Y, Hou X L, Wang F, et al.2018. Advances in the research of celery, an important Apiaceae vegetable crop[J]. Critical Reviews in Biotechnology, 38(2): 172-183. [19] Paradis E, Claude J, Strimmer K.2004. APE: Analyses of phylogenetics and evolution in R language[J]. Bioinformatics, 20(2): 289-290. [20] Pritchard J K, Stephens M, Donnelly P.2000. Inference of population structure using multilocus genotype data[J]. Genetics, 155(2): 945-959. [21] Ryder E J.1979. Leafy Salad Vegetables[M]. Avi publishing company, inc. Westport. pp. 266. [22] Sowbhagya H B.2014. Chemistry, technology, and nutraceutical functions of celery (Apium graveolens L.): An overview[J]. Critical Reviews in Food Science and Nutrition, 54(3): 389-398. [23] Wang S, Yang W, Shen H.2011. Genetic diversity in Apium graveolens and related species revealed by SRAP and SSR markers[J]. Scientia Horticulturae, 129(1): 1-8. [24] Xie H, Sui Y, Chang F Q, et al.2006. SSR allelic variation in almond (Prunus dulcis Mill.)[J]. Theoretical and Applied Genetics, 112(2): 366-372 [25] Xu Y, Ma R, Xie H, et al.2004. Development of SSR markers for the phylogenetic analysis of almond trees from China and the Mediterranean region[J]. Genome, 47(6): 1091-1104 [26] Yang X, Quiros C.1993. Identification and classification of celery cultivars with RAPD markers[J]. Theoretical and Applied Genetics, 86(2-3): 205-212.
