Abstract：Phalaenopsis spp. has extremely high economic and ornamental value. The breeding of its new varieties generally adopts interspecific and intraspecific hybridization, resulting in complex genetic background and ploidy. In this study, expressed sequence tag-simple sequence repeat (EST-SSR) molecular marker technology was used to conduct genetic diversity analysis, hybrid identification, cluster analysis and ploidy prediction on 31 Phalaenopsis germplasm resources as well as the reciprocal F? hybrids of 'Treasure Map' and '075'. The aim was to provide a basis for the utilization and innovation of Phalaenopsis germplasm resources, molecular marker-assisted breeding, ploidy identification and authenticity identification of hybrid progeny. Based on 19 reported SSR primer pairs combined with the M13 universal primer, capillary fluorescence electrophoresis was performed. Ten pairs of EST-SSR primers were screened for genetic diversity, genetic relationship analysis and ploidy prediction of 31 germplasm resources of Phalaenopsis. Seven pairs of EST-SSR primers with paternal characteristic bands were screened for authenticity, cluster analysis and ploidy prediction of 50 F1 hybrid progeny lines, and 50 F1 hybrid progeny lines were identified by flow cytometry combined with chromosome counting. The results showed that a total of 233 alleles were detected in 10 pairs of primers, the average number of alleles detected at each locus was 23.3, the total number of effective alleles was 137.65, the average number of effective alleles at each locus was 13.76, and the polymorphism information content (PIC) value of 10 pairs of primers varied in the range of 0.32~0.91, only primer Pap-3754 had moderate polymorphism, and the other 9 primers had high polymorphism. The cluster analysis of 31 Phalaenopsis germplasm resources showed that the 31 Phalaenopsis germplasm resources were divided into 3 categories at the genetic similarity coefficient of 0.23, among which the classⅠand class Ⅱwere native to Phalaenopsis orchid, and the 25 materials of class Ⅲ were Phalaenopsis horticultural species. The authenticity of the hybrid F1 progeny showed that all 50 hybrid F1 progeny lines were identified as true hybrids. Cluster analysis of 50 crosses showed that the 49 of them tested were closely related to the maternal parent, and only one line, L25, was closely related to the paternal parent. Flow cytometry and chromosome counting results showed that the hybrid offspring of Phalaenopsis were tetraploid, and compared with the molecular marker results, the accuracy of EST-SSR molecular marker was as high as 98%. The results indicated that the EST-SSR molecular marker could be used for the analysis of genetic diversity, ploidy prediction and early identification of hybrid offspring of Phalaenopsis orchid. This study is of positive significance for improving the breeding efficiency of new varieties and exploring the genetic background of Phalaenopsis.
王铫铃, 张文滔, 李嘉铭, 阎冉, 裴子俊, 李艳冬, 崔永一. 基于EST-SSR分子标记的蝴蝶兰种质资源遗传多样性分析和倍性预测[J]. 农业生物技术学报, 2026, 34(4): 743-758.
WANG Yao-Ling, ZHANG Wen-Tao, LI Jia-Ming, YAN Ran, PEI Zi-Jun, LI Yan-Dong, CUI Yong-Yi. Genetic Diversity Analysis and Ploidy Prediction of Phalaenopsis Germplasm Resources Based on EST-SSR Molecular Markers. 农业生物技术学报, 2026, 34(4): 743-758.
[1] 曹玉芬, 刘凤之, 高源, 等. 2007. 梨栽培品种SSR鉴定及遗传多样性[J]. 园艺学报, (02):305-310. (Cao Y F, Liu F Z, Gao Y, et al. 2007. SSR analysis of genetic diversity of pear cultivars[J]. Acta Horticulturae Sinica, (02):305-310.) [2] 高华, 樊红科, 万怡震, 等. 2011. 苹果栽培品种的SSR鉴定及遗传多样性分析[J]. 西北农业学报, 20(02):153-158. (Gao H, Fan H K, Wan Y Z, et al.2011. SSR analysis of genetic diversity of apple cultivars[J]. Acta Agriculturae Boreali-occidentalis Sinica, 20(02):153-158.) [3] 胡文舜, 陈秀萍, 郑少泉. 2019. 龙眼EST-SSR标记开发及无患子科5个属种质遗传多样性分析[J]. 园艺学报, 46(07):1359-1372. (Hu W S, Chen X P, Zhen S Q.2019. EST-SSR markers developed from Dimocarpus longan and their application in genetic diversity analysis of five genera of Sapindaceae[J]. Acta Horticulturae Sinica, 46(07):1359-1372. [4] 林榕燕, 罗远华, 樊荣辉, 等. 2021. 文心兰杂交后代的EST-SSR和SRAP分子标记鉴定[J]. 福建农业学报, 36(12):1439-1446. (Lin R Y, Luo Y H, Fan R H, et al.2021. Identification of hybrid progenies from Oncidium hybridum with EST-SSR and SRAP molecular markers[J]. Fujian Journal of Agricultural Sciences, 36(12):1439-1446.) [5] 刘华, 曾凡佩, 王倩, 等. 2024. 花生栽培种和野生种Arachis sp. 30119六倍体杂种的创制与鉴定[J]. 中国农业科学, 57(10):1870-1881. (Liu H, Zeng F P, Wang Q, et al.2024. Development and identification of an interspecific hexaploid hybrid between an A. hypogaea cultivar and a wild species Arachis sp. 30119 in peanut[J]. Scientia Agricultura Sinica, 57(10):1870-1881.) [6] 刘颖鑫, 李沛曈, 迟天华, 等. 2019. 菊花脑×甘菊种间F1杂种的鉴定和遗传多样性分析[J]. 园艺学报, 46(08):1553-1564. (Liu Y X, Li P T, Chi T H, et al.2019. Identification and genetic diversity analysis of interspecific F1 hybrids between Chrysanthemum nankingense × C. lavandulifolium[J]. Acta Horticulturae Sinica, 46(08):1553-1564.) [7] 刘遵春, 刘大亮, 崔美, 等. 2012. 整合农艺性状和分子标记数据构建新疆野苹果核心种质[J]. 园艺学报, 39(06):1045-1054. (Liu Z C, Liu D L, Cui M, et al.2012. Combining agronomic traits and molecular marker data for constructing Malus sieversii core collection[J]. Acta Horticulturae Sinica, 39(06):1045-1054.) [8] 卢俊. 2022. 蝴蝶兰类原球茎诱导增殖及DhGA3ox遗传转化[D]. 硕士学位论文, 浙江农林大学, 导师:崔永一, pp. 61. (Lu J. 2022. Induction and proliferation of protocorm-like bodies of Phalaenopsis and genetic transformation of DhGA3ox[D]. Thesis for M.S., Zhejiang A&F University, Suppervisor:Cui Y Y, pp. 61.) [9] 鲁振华, 沈志军, 牛良, 等. 2020. 122份栽培桃品种(系)黄白肉性状的分子标记辅助鉴定[J]. 中国农业科学, 53(14):2929-2940. (Lu Z H, Shen Z J, Niu L, et al.2020. Molecular marker-assisted identification of yellow/white flesh trait for 122 peach cultivars (lines)[J]. Scientia Agricultura Sinica, 53(14):2929-2940.) [10] 王瑞, 洪文娟, 罗华, 等. 2023. 石榴品种SSR指纹图谱构建及杂种父本鉴定[J]. 园艺学报, 50(02):265-278. (Wang R, Hong W J, Luo H, et al.2023. Construction of SSR fingerprints of pomegranate cultivars and male parent identification of hybrids[J]. Acta Horticulturae Sinica, 50(02):265-278.) [11] 王衍莉, 杨义明, 范书田, 等. 2021. 基于SSR分子标记的73份山葡萄及杂交后代的遗传多样性分析[J].生物技术通报, 37(01):189-197. (Wang Y L, Yang Y M, Fan S T, et al.2021. Genetic diversity analysis of 73 Vitis amurensis and its hybrids offsprings based on SSR molecular markers[J]. Biotechnology Bulletin, 37(01):189-197.) [12] 张水明, 陈程, 龚凌燕,等. 2012. 蝴蝶兰EST资源SSR标记分析与开发[J]. 园艺学报, 39(06):1191-1198. (Zhang S M, Chen C, Gong L Y, et al.2012. Analysis of SSRs information and development of SSR markers from Phalaenopsis ESTs[J]. Acta Horticulturae Sinica, 39(06):1191-1198.) [13] 张文滔, 李嘉铭, 王铫铃, 等. 2024. 蝴蝶兰种质资源倍性鉴定及育性相关性研究[J]. 核农学报, 38(06):1012-1023. (Zhang W T, Li J M, Wang Y L, et al.2024. Study on ploidy identification and fertility correlation of Phalaenopsis germplasm resources[J]. Journal of Nuclear Agricultural Sciences, 38(06):1012-1023.) [14] 周建金, 曾瑞珍, 刘芳, 等. 2009. 不同倍性蝴蝶兰杂交后代的染色体倍性研究[J]. 园艺学报, 36(10):1491-1497. (Zhou J J, Zeng R Z, Liu F, et al.2009. Investigation on chromosome ploidy of the hybrids of Phalaenopsis polyploids[J]. Acta Horticulturae Sinica, 36(10):1491-1497.) [15] 周艳萍. 2020. 百合遗传多样性和亲缘关系的研究[D]. 博士学位论文, 北京林业大学, 导师:贾桂霞, pp. 81-82; 117-120. (Zhou Y P.2020. Studies on genetic diversity and genetic relationship of Lilium[D]. Thesis for Ph.D., Beijing Forestry University, Suppervisor:Jia G X, pp. 81-82; 117-120.) [16] Chang Y K, Veilleux R E, Iqbal M J.2009. Analysis of genetic variability among Phalaenopsis species and hybrids using amplified fragment length polymorphism[J]. Journal of the American Society for Horticultural Science, 134(1):58-66. [17] Chen M, Nie G, Li X, et al.2023. Development of EST-SSR markers based on transcriptome sequencing for germplasm evaluation of 65 lilies (Lilium)[J]. Molecular Biology Reports, 50(4):3259-3269. [18] Chiou C Y, Chiang Y C, Chen C H, et al.2012. Development and characterization of 38 polymorphic microsatellite markers from an economically important fruit tree, the Indian jujube[J]. American Journal of Botany, 99(5):E199-E202. [19] Deperi S I, Tagliotti M E, Bedogni M C, et al.2018. Discriminant analysis of principal components and pedigree assessment of genetic diversity and population structure in a tetraploid potato panel using SNPs[J]. PLOS ONE, 13(3):e0194398. [20] Fattmah N, Dewi S.2011. Development of sequence-based microsatellite marker for Phalaenopsis orchid[J]. HAYATI Journal of Biosciences, 18(2):71-76. [21] Gao X F, Hao Y, Yang X Y, et al.2025. Transcriptomics and metabolomics analysis reveal the mechanism of hormones regulation of the flower bud formation and development in Phalaenopsis orchid[J]. Journal of Plant Growth Regulation, 44(7):4073-4089. [22] Guo W, Hou J, Yin T M, et al.2016. An analytical toolkit for polyploid willow discrimination[J]. Scientific Reports, 6:37702. [23] Ko Y Z, Shih H C, Tsai C C, et al.2017. Screening transferable microsatellite markers across genus Phalaenopsis (Orchidaceae)[J]. Botanical Studies, 58(1):48. [24] Kong F, Liu J J, Chen Y N, et al.2013. Marker-aided selection of polyploid poplars[J]. BioEnergy Research, 6(3):984-990. [25] Lai J M, Tsai C C, Yen C R, et al.2015. Molecular characterization of twenty polymorphic microsatellite markers in the polyploid fruit tree species Syzygium samarangense (Myrtaceae)[J]. Genetics and Molecular Research, 14(4):13013-13021. [26] Li H, Ma Y S, Pei F Y, et al.2021. Large-scale advances in SSR markers with high-throughput sequencing in Euphorbia fischeriana Steud[J]. Electronic Journal of Biotechnology, 49:50-55. [27] Li Z, Yun L, Gao Z Q, et al.2022. EST-SSR Primer development and genetic structure analysis of Psathyrostachys juncea Nevski[J]. Frontiers in Plant Science, 13:837787. [28] Liang T, Sun W, Ren H, et al.2019. Genetic diversity of Ziziphus mauritiana germplasm based on SSR markers and ploidy level estimation[J]. Planta, 249(6):1875-1887. [29] Lin J Y, Lin B Y, Chang C D, et al.2015. Evaluation of chloroplast DNA markers for distinguishing Phalaenopsis species[J]. Scientia Horticulturae, 192:302-310. [30] Moriya S, Iwanami H, Okada K, et al.2010. A practical method for apple cultivar identification and parent-offspring analysis using simple sequence repeat markers[J]. Euphytica, 177:135-150. [31] Niu Y Y, Zhou W Q, Chen X Y, et al.2020. Genome size and chromosome ploidy identification in pear germplasm represented by Asian pears - local pear varieties[J]. Scientia Horticulturae, 265:109-202. [32] Park P H, Park Y J, Kim M S, et al.2013. Analysis of genetic diversity and identification of domestic bred Phalaenopsis varieties using SRAP and SSR markers[J]. Korean Journal of Horticultural Science and Technology, 31(3):337-343. [33] Preethi P, Rahman S, Naganeeswaran S, et al.2020. Development of EST-SSR markers for genetic diversity analysis in coconut (Cocos nucifera L.)[J]. Molecular Biology Reports, 47(12):9385-9397. [34] Su M, Zhang C, Feng S.2022. Identification and genetic diversity analysis of hybrid offspring of azalea based on EST-SSR markers[J]. Scientific Reports, 12(1):15239. [35] Sun Q, Shuaipeng M A, Liu W, et al.2015. Identification and analysis on true or false hybrids from hybridization populations of litchi (Litchi chinensis Sonn.) using EST-SSR markers[J]. Agricultural Biotechnology, (4):43. [36] Tsai C C, Chen Y K, Chen C H, et al.2013. Cultivar identification and genetic relationship of mango (Mangifera indica) in Taiwan using 37 SSR markers[J]. Scientia Horticulturae, 164:196-201. [37] Tsai C C, Shih H C, Wang H V, et al.2015. RNA-Seq SSRs of moth orchid and screening for molecular markers across genus Phalaenopsis (Orchidaceae)[J]. PLOS ONE, 10(11):e0141761. [38] Tsai C C, Wu P Y, Kuo C C, et al.2014. Analysis of microsatellites in the vulnerable orchid Gastrodia flavilabella:The development of microsatellite markers, and cross-species amplification in Gastrodia[J]. Botanical Studies, 55(1):72. [39] Wu J, Li L T, Li M, et al.2014. High-density genetic linkage map construction and identification of fruit-related QTLs in pear using SNP and SSR markers[J]. Journal of Experimental Botany, 65(20):5771-5581.
