Development and Application of Universal SSR Molecular Marker Primers in Phalaenopsis and Rhynchostylis

doi:10.16420/j.issn.0513-353x.2023-0285

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (5): 1056-1068.doi: 10.16420/j.issn.0513-353x.2023-0285

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Development and Application of Universal SSR Molecular Marker Primers in Phalaenopsis and Rhynchostylis

XIAO Wenfang, LI Zuo^*(), CHEN Heming, LÜ Fubing^*()

Environmental Horticulture Research Institute of Guangdong Academy of Agricultural Sciences，Guangdong Key Lab of Ornamental Plant Germplasm Innovation and Utilization，Guangzhou 510640，China

Received:2023-10-26 Revised:2024-02-28 Online:2024-05-25 Published:2024-05-29

Abstract

Abstract:

Based on simplified genome sequencing data of 38 germplasm resources from 28 native species of Phalaenopsis and nine germplasm resources from three native species of Rhynchostylis，19 pairs of universal SSR primers in Phalaenopsis，12 pairs of universal SSR primers in Rhynchostylis，and 12 pairs of universal SSR primers between the two genera were developed and selected for their high polymorphism and universality. Fluorescent-labeled capillary electrophoresis was used for allele variation detection. Among the 47 germplasm resources，a total of 134 alleles were detected by the 12 pairs of universal primers between genera，with 6-17 alleles amplified per pair of primers. The mean observed number of alleles（N_a）was 11.17，the mean effective number of alleles（N_e）was 6.36，and the expected Heterozygosity（H_e） ranged from 0.66 to 0.88，while the observed heterozygosity（H_o） ranged from 0.07 to 0.38. All primer sites showed high polymorphism，with Shannon's index ranging from 1.30 to 2.43 and a mean of 1.88，indicating that the primers have high polymorphism and were suitable for identifying germplasm resources of both two genera. Cluster analysis and population structure analysis of the 47 germplasm resources could clearly reveal the genetic relationships between Phalaenopsis and Rhynchostylis species，which is consistent with the results of botanical classification. In the detection of F₁ hybrid populations between Phalaenopsis Jiuhbao Red Rose and Rhynchostylis retusa 23 pairs showed polymorphism，and a total of 75 polymorphic bands were amplified. The genetic similarity coefficient between hybrids ranged from 0.58 to 1.00，with an average genetic similarity coefficient between maternal parents and intergeneric hybrids（0.62）higher than that between paternal parents（0.54），indicating that the F₁ generation of intergeneric hybrids showed maternal inheritance，which is consistent with the phenotypic observation results.

Key words: Phalaenopsis, Rhynchostylis, cross-genus hybridization, universal microsatellite marker

XIAO Wenfang, LI Zuo, CHEN Heming, LÜ Fubing. Development and Application of Universal SSR Molecular Marker Primers in Phalaenopsis and Rhynchostylis[J]. Acta Horticulturae Sinica, 2024, 51(5): 1056-1068.

Figures/Tables 9

References 39

[1]	Ai Ye, Chen Lu, Xie Tai-xiang, Chen Juan, Lan Si-ren, Peng Dong-hui. 2019. Construction of core collection of Cymbidium ensifolium cultivars based on SSR fluorescent markers. Acta Horticulturae Sinica, 46 (10):1999-2008. (in Chinese)
	艾叶, 陈璐, 谢泰祥, 陈娟, 兰思仁, 彭东辉. 2019. 基于SSR荧光标记构建建兰品种核心种质. 园艺学报, 46 (10):1999-2008. doi: 10.16420/j.issn.0513-353x.2019-0327
[2]	Bateman R M, Murphy A R M, Tattersall B G. 2017. Dactylodenia lacerta(Orchidaceae):a morphologically cryptic hybrid orchid new to science from the Lizard Peninsula, Cornwall. New Journal of Botany, 7:64-77.
[3]	Botstein D R, White R L, Skolnick M H, Davis R W. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Society of Human Genetics, 32 (3):314-331.
[4]	Cardoso J C. 2017. Ionocidium‘Cerrado 101’:intergeneric orchid hybrid with high quality of blooming. Ornamental Horticulture, 23 (3):351-356.
[5]	Chen He-ming, Lü Fu-bing, Xiao Wen-fang, Li Zuo, Jiang Ming-dian. 2019. Preliminary study on the intergeneric hybridization breeding between Phalaenopsis and Renanthera. Journal of China Agricultural University, 24 (8):60-71. (in Chinese)
	陈和明, 吕复兵, 肖文芳, 李佐, 蒋明殿. 2019. 蝴蝶兰与火焰兰远缘杂交育种初探. 中国农业大学学报, 24 (8):60-71.
[6]	Chen He-ming, Lü Fu-bing, Li Zuo, Xiao Wen-fang. 2021. Aseptic seeding and F₁ phenotypic analysis of intergeneric hybrid between Phalaenopsis spp. and Rhybchostylis retusa. Journal of China Agricultural University, 26 (3):28-37. (in Chinese)
	陈和明, 吕复兵, 李佐, 肖文芳. 2021. 蝴蝶兰(Phalaenopsis spp.)与钻喙兰(Rhynchostylis retusa)远缘杂交种子无菌播种及后代表型分析. 中国农业大学学报, 26 (3):28-37.
[7]	Chen Ming-kun, Chen Lu, Sun Wei-hong, Ma Shan-hu, Lan Si-ren, Peng Dong-hui, Liu Zhong-jian, Ai Ye. 2022. Genetic diversity analysis and core collection of Cymbidium ensifolium germplasm resources. Acta Horticulturae Sinica, 49 (1):175-186. (in Chinese)
	陈明堃, 陈璐, 孙维红, 马山虎, 兰思仁, 彭东辉, 刘仲健, 艾叶. 2022. 建兰种质资源遗传多样性分析及核心种质构建. 园艺学报, 49 (1):175-186. doi: 10.16420/j.issn.0513-353x.2021-0312
[8]	Dong G K, Kang K K, Chul G B. 2015. Development of intergenetic hybrids between wind orchids(Sedirea japonica and Neofinetia falcata)and moth orchids (Phalaenopsis alliances). Horticulture,Environment,and Biotechnology, 56 (1):67-78.
[9]	Fatimah, Dewi S. 2011. Development of sequence-based microsatellite marker for Phalaenopsis orchid. Hayati Journal of Biosciences, 18 (2):71-76.
[10]	Guo Peng-hui, Wu Jing-zhi, Wang Yu-ying, Zheng Ran-ran, Wu Hong-zhi. 2019. Compatibility and overcoming of distant hybridization of Lilium nepalense and hybridization obstacles. Journal of Southern Agriculture, 50 (90):1903-1912. (in Chinese)
	郭朋辉, 吴景芝, 王玉英, 郑冉冉, 吴红芝. 2019. 紫斑百合远缘杂交亲和性分析及杂交障碍克服. 南方农业学报, 50 (90):1903-1912.
[11]	Guo Xiao-can, Han Jia-hui, Tong Wei-ming. 2019. Research progress of incompatibility overcome and DNA methylation of distant hybridization between radish and Brassica species. Jiangsu Agricultural Sciences, 47 (22):12-16. (in Chinese)
	郭晓灿, 韩佳慧, 童未名. 2019. 萝卜与芸薹属远缘杂交不亲和性克服及DNA甲基化的研究进展. 江苏农业科学, 47 (22):12-16.
[12]	Hartati S. 2010. The intergeneric crossing of Phalaenopsis sp. and Vanda tricolor. Journal of Biotechnology and Biodiversity, 1 (1):32-36.
[13]	Hu Guang-ming, Zhang Qiong, Han Fei, Li Da-wei, Li Zuo-zhou, Wang Zhi, Zhao Ting-ting, Tian Hua, Liu Xiao-li, Zhong Cai-hong. 2022. Screening and application of universal SSR molecular marker primers in Actinidia. Scientia Agricultura Sinica, 55 (17):3411-3425. (in Chinese)
	胡光明, 张琼, 韩飞, 李大卫, 李作洲, 汪志, 赵婷婷, 田华, 刘小莉, 钟彩虹. 2022. 猕猴桃属植物通用型SSR分子标记引物的筛选及应用. 中国农业科学, 55 (17):3411-3425. doi: 10.3864/j.issn.0578-1752.2022.17.012
[14]	Huylenbroecka J V, Eeckhaut T, Leus L, Laere K V, Dhooghe E. 2020. Bridging the gap:tools for interspecific and intergeneric hybridization in ornamentals. Acta Horticulturae, 1283:161-168.
[15]	Jin X L, Tsai C C, Ko Y Z, Chiang Y C. 2021. Phylogeny,polymorphism,and SSR markers of Phalaenopsis∥Chen F C,Chin S W. The Orchid Genome,Compendium of Plant Genomes. Switzerland:Springer:145-160.
[16]	Kao Y Y, Chang S B, Lin T Y, Hsieh C H, Chen Y H, Chen W H, Chen C C. 2001. Differential accumulation of heterochromatin as a cause for karyotype variation in Phalaenopsis orchids. Annals of Botany, 87:387-395.
[17]	Kim H T, Kim J S, Moore M J, Neubig K M, Williams N H, Whitten W M, Kim J H. 2015. Seven new complete plastome sequences reveal rampant independent loss of the ndh gene family across orchids and associated instability of the inverted repeat/small single-copy region boundaries. PLoS ONE, 10 (11):e0142215.
[18]	Ko Y Z, Shih H C, Tsai C C, Ho H H, Liao P C, Chiang Y C. 2017. Screening transferable microsatellite markers across genus Phalaenopsis (Orchidaceae). Botanical Studies, 58:48.
[19]	Li Xin-lei. 2004. Studies on self-cross,hybridization and embryo rescue of interspecific hybrid in Dendranthema[M. D. Dissertation]. Nanjing: Nanjing Agricultural University. (in Chinese)
	李辛雷. 2004. 菊属植物自交、杂交及远缘杂种幼胚拯救研究[硕士论文]. 南京: 南京农业大学.
[20]	Liu W L, Shih H C, Weng I S, Ko Y Z, Tsai C C, Chou C H, Chiang Y C. 2016. Characterization of genomic inheritance of intergeneric hybrids between Ascocenda and Phalaenopsis cultivars by GISH,PCR-RFLP and RFLP. PLoS ONE, 11 (4):e0153512.
[21]	Liu Xiong-lun. 2001. Studies on xinjiang wild rape:genetic relationships with Brassica species and its status in Brassica evolution[Ph. D. Dissertation]. Hunan: Hunan Agricultural University. (in Chinese)
	刘雄伦. 2001. 新疆野生油菜与芸薹属物种种间亲缘关系及其进化地位研究[博士论文]. 湖南: 湖南农业大学.
[22]	Liu Zhong-jian, Wang Jie-yu, Olaf Gruss, Lan Si-ren. 2021. The genus Phalaenopsis in world. Beijing: Higher Education Press. (in Chinese)
	刘仲健, 王洁雨, 奥拉夫·格鲁斯, 兰思仁. 2021. 世界蝴蝶兰属植物. 北京: 高等教育出版社.
[23]	Mega W, Minangsari D, Erniawti D, Fitri R, Dewi S, Sudarsono. 2022. Genetic diversity based on SSR markers of 30 Aeridinae subtribe orchid genetic resources of Indonesian Ornamental Crop Research Institute,Cianjur,Indonesia. Biodiversitas, 23 (6):2943-2956.
[24]	Mo Rao, Leng Qing-yun, Huang Ming-zhong, Peng Bin, Tang Yan-qiong, Luo Yuan-hua, Deng Xiao-guo. 2009. A karyological study of fourteen species in eleven genera of the Orchidaceae. Acta Botanica Yunnanica, 31 (6):504-508. (in Chinese)
	莫饶, 冷青云, 黄明忠, 彭彬, 唐燕琼, 罗远华, 邓小果. 2009. 兰科11 属14 种植物核型分析. 云南植物研究, 31 (6):504-508.
[25]	Murni D, Agus H S. 2021. Intergeneric hybridization between Phalaenopsis 2166 and Vanda‘Saint Valentine’:characterization of parents using ndhE cpDNA partial sequence. Journal of Tropical Biodiversity and Biotechnology, 6 (3):jtbb65658.
[26]	Murni D, Agus H S, Lucky P. 2020. Intergeneric hybrids of Phalaenopsis 2166 × Vanda‘Saint Valentine’showing maternal inheritance:Genetic analysis based on ndhE partial gene. Biodiversitas, 21:5138-5145.
[27]	Regy Y, Jose M, Dariusz L S. 2018. Rhynchostylis cymifera(Orchidaceae),a new species from south western Ghats,India. Jordan Journal of Biological Sciences, 11 (3):257-259.
[28]	Shi Li-ting, Zhou Xin-yang, Ye Jian-feng, Zhou Jia-hao, Wang Gang, Xia Guo-hua. 2021. Advances in distant hybridization breeding of woody ornamental plants. Acta Horticulturae Sinica, 48 (9):1827-1838. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0565
	施丽婷, 周鑫洋, 叶建丰, 周家豪, 王刚, 夏国华. 2021. 木本观赏植物远缘杂交育种研究进展. 园艺学报, 48 (9):1827-1838. doi: 10.16420/j.issn.0513-353x.2020-0565
[29]	Shindo S, Kamemoto H. 1963. Karyotype analysis of some species of Phalaenopsis. Cytologia, 28:390-398.
[30]	Tsai C C, Chiang Y C, Huang S C, Chen C H, Chou C H. 2010. Molecular phylogeny of Phalaenopsis Blume(Orchidaceae)on the basis of plastid and nuclear DNA. Plant Systematics and Evolution, 288:77-98.
[31]	Tsai C C, Shih H C, Wang H V, Lin Y S, Chang C H, Chiang Y C, Chou C H. 2015. RNA-seq SSRs of moth orchid and screening for molecular markers across genus Phalaenopsis(Orchidaceae). PLoS ONE, 10 (11):e0141761.
[32]	UPOV. 2021. Guidelines for DNA-profiling:Molecular marker selection and database construction(“BMT Guidelines”). https://www.upov.int/edocs/mdocs/upov/en/twv_55/upov_inf_17_2_draft_5.pdf.
[33]	Wang L X, Chen H L, Bai P, Wu J X, Wang S H, Matthew W B, Cheng X Z. 2015. The transferability and polymorphism of mung bean SSR markers in rice bean germplasm. Molecular Breeding, 35:77.
[34]	Wesley E H. 2012. A new name for a distinctive Rhynchostylis(Orchidaceae) and the misapplication of Rhynchostylis praemorsa. Selbyana, 31 (1):40-43.
[35]	Xiao Wen-fang, Li Zuo, Chen He-ming, Lü Fu-bing. 2016. Detection of Phalaenopsis in somaclonal variation using fluorescent labeled SSR markers. Chinese Journal of Tropical Crops, 37 (10):1938-1944. (in Chinese)
	肖文芳, 李佐, 陈和明, 吕复兵. 2016. 利用SSR 荧光标记毛细管电泳法检测蝴蝶兰组培突变体. 热带作物学报, 37 (10):1938-1944.
[36]	Xiao Zhi-juan, Zhai Mei-zhi, Wang Zhen-yuan, Xu Jing, Li Li, Yang Hui. 2014. Application of microsatellite DNA on analyzing genetic diversity of Juglans regia. Journal of Central South University of Forestry & Technology, 34 (2):55-61. (in Chinese)
	肖志娟, 翟梅枝, 王振元, 许静, 李丽, 杨惠. 2014. 微卫星DNA在分析核桃遗传多样性上的应用. 中南林业科技大学学报, 34 (2):55-61.
[37]	Xu Feng, Song Jie, Zhang Yi-ping, Zhang Bao-qiong, Yang Xiu-mei, Zhang Li-fang, Wang Li-hua, Xie Wei-jia. 2020. 22 distant hybrid seeds of Rhododendron:germination characteristics. Chinese Agricultural Science Bulletin, 36 (14):64-68. (in Chinese) doi: 10.11924/j.issn.1000-6850.casb19010062
	许凤, 宋杰, 张艺萍, 张宝琼, 杨秀梅, 张丽芳, 王丽花, 解玮佳. 2020. 22个常绿杜鹃远缘杂交种子萌发特性. 中国农学通报, 36 (14):64-68. doi: 10.11924/j.issn.1000-6850.casb19010062
[38]	Yin Guo, Lu Zheng-ying, Sun Lu, Zhang Yan-bo, Li Shi-yun. 2021. Chromosome ploidy analysis and interspecific hybridization of Abelmoschus manihot. Chinese Agricultural Science Bulletin, 37 (9):16-21. (in Chinese) doi: 10.11924/j.issn.1000-6850.casb2020-0205
	尹国, 路正营, 孙璐, 张彦波, 李世云. 2021. 金花葵染色体倍性分析及属间远缘杂交研究. 中国农学通报, 37 (9):16-21. doi: 10.11924/j.issn.1000-6850.casb2020-0205
[39]	Zhong Min, Cheng Xu-zhen, Wang Li-xia, Wang Su-hua, Wang Xiao-bao. 2012. Transferability of Mungbean genomic-SSR markers in other Vigna species. Acta Agronomica Sinica, 38 (2):223-230. (in Chinese)
	钟敏, 程须珍, 王丽侠, 王素华, 王小宝. 2012. 绿豆基因组SSR引物在豇豆属作物中的通用性. 作物学报, 38 (2):223-230.

编号 No.	学名 Scientific name	编号 No.	学名 Scientific name
R01	海南钻喙兰Rhynchostylis gigantea‘Shen’s Suns’	Ph15	华西蝴蝶兰（变型）Phal. wilsonii f. alba
R02	海南钻喙兰Rhy. gigantea	Ph16	小尖囊兰Phal. taenialis
R03	细斑海南钻喙兰（变型）Rhy. gigantea f. maculata	Ph17	红河蝴蝶兰Phal. honghenensis
R04	纯白海南钻喙兰（变种）Rhy. gigantea var. petotiana	Ph18	滇西蝴蝶兰Phal. stobartiana
R05	全红海南钻喙兰（变型）Rhy. gigantea f. rubra	Ph19	象耳蝴蝶兰Phal. gigantea
R06	立梗狐尾兰Rhy. coelestis	Ph20	白花象耳蝴蝶兰（变型）Phal. gigantea f. decolorata
R07	纯白立梗狐尾兰（变型）Rhy. coelestis f. alba	Ph21	版纳蝴蝶兰Phal. mannii
R08	粉色立梗狐尾兰（变型）Rhy. coelestis f. rosea	Ph22	深红鹿角蝴蝶兰（变型）Phal. cornu-cervi f. sanguinea
R09	钻喙兰Rhy. retusa	Ph23	黄花鹿角蝴蝶兰（变型）Phal. cornu-cervi f. flava
Ph01	白花蝴蝶兰Phalaenopsis amabilis	Ph24	苏门答腊蝴蝶兰Phal. sumatrana
Ph02	香花白花蝴蝶兰（变型）Phal. amabilis h. v. java	Ph26	美花蝴蝶兰Phal. pulchra
Ph03	蝴蝶兰Phal. aphrodite	Ph27	巴氏蝴蝶兰Phal. bastianii
Ph04	西蕾利蝴蝶兰Phal. schilleriana	Ph28	康宁蝴蝶兰Phal. corningiana
Ph05	桑德蝴蝶兰Phal. sanderiana	Ph29	羞花蝴蝶兰Phal. modesta
Ph06	白花小兰屿蝴蝶兰（变型）Phal. equestris f. aurea	Ph30	棕色盾花蝴蝶兰（变型）Phal. tetraspis f. brunneola
Ph07	蓝唇小兰屿蝴蝶兰（变型）Phal. equestris f. cyanochila	Ph31	盾花蝴蝶兰Phal. tetraspis
Ph08	林登蝴蝶兰Phal. lindenii	Ph32	荧光蝴蝶兰Phal. violacea
Ph09	五唇兰Phal. pulcherrima	Ph33	白花荧光蝴蝶兰（变型）Phal. violacea f. alba
Ph10	大尖囊蝴蝶兰Phal. deliciosa	Ph34	紫花荧光蝴蝶兰（浅紫变型）Phal. violacea f. coerulea
Ph11	罗比蝴蝶兰Phal. lobbii	Ph35	纯红花荧光蝴蝶兰（变型）Phal. violacea h.v. sumatrana
Ph12	麻栗坡蝴蝶兰Phal. malipoensis	Ph36	紫花荧光蝴蝶兰（全紫变型）Phal. violacea f. coerulea
Ph13	洛氏蝴蝶兰Phal. lowii	Ph37	安汶蝴蝶兰Phal. amboinensis
Ph14	华西蝴蝶兰Phal. wilsonii	Ph38	紫花贝雷娜蝴蝶兰（变型）Phal. bellina f. coerulea

编号 No.	学名 Scientific name	编号 No.	学名 Scientific name
R01	海南钻喙兰Rhynchostylis gigantea‘Shen’s Suns’	Ph15	华西蝴蝶兰（变型）Phal. wilsonii f. alba
R02	海南钻喙兰Rhy. gigantea	Ph16	小尖囊兰Phal. taenialis
R03	细斑海南钻喙兰（变型）Rhy. gigantea f. maculata	Ph17	红河蝴蝶兰Phal. honghenensis
R04	纯白海南钻喙兰（变种）Rhy. gigantea var. petotiana	Ph18	滇西蝴蝶兰Phal. stobartiana
R05	全红海南钻喙兰（变型）Rhy. gigantea f. rubra	Ph19	象耳蝴蝶兰Phal. gigantea
R06	立梗狐尾兰Rhy. coelestis	Ph20	白花象耳蝴蝶兰（变型）Phal. gigantea f. decolorata
R07	纯白立梗狐尾兰（变型）Rhy. coelestis f. alba	Ph21	版纳蝴蝶兰Phal. mannii
R08	粉色立梗狐尾兰（变型）Rhy. coelestis f. rosea	Ph22	深红鹿角蝴蝶兰（变型）Phal. cornu-cervi f. sanguinea
R09	钻喙兰Rhy. retusa	Ph23	黄花鹿角蝴蝶兰（变型）Phal. cornu-cervi f. flava
Ph01	白花蝴蝶兰Phalaenopsis amabilis	Ph24	苏门答腊蝴蝶兰Phal. sumatrana
Ph02	香花白花蝴蝶兰（变型）Phal. amabilis h. v. java	Ph26	美花蝴蝶兰Phal. pulchra
Ph03	蝴蝶兰Phal. aphrodite	Ph27	巴氏蝴蝶兰Phal. bastianii
Ph04	西蕾利蝴蝶兰Phal. schilleriana	Ph28	康宁蝴蝶兰Phal. corningiana
Ph05	桑德蝴蝶兰Phal. sanderiana	Ph29	羞花蝴蝶兰Phal. modesta
Ph06	白花小兰屿蝴蝶兰（变型）Phal. equestris f. aurea	Ph30	棕色盾花蝴蝶兰（变型）Phal. tetraspis f. brunneola
Ph07	蓝唇小兰屿蝴蝶兰（变型）Phal. equestris f. cyanochila	Ph31	盾花蝴蝶兰Phal. tetraspis
Ph08	林登蝴蝶兰Phal. lindenii	Ph32	荧光蝴蝶兰Phal. violacea
Ph09	五唇兰Phal. pulcherrima	Ph33	白花荧光蝴蝶兰（变型）Phal. violacea f. alba
Ph10	大尖囊蝴蝶兰Phal. deliciosa	Ph34	紫花荧光蝴蝶兰（浅紫变型）Phal. violacea f. coerulea
Ph11	罗比蝴蝶兰Phal. lobbii	Ph35	纯红花荧光蝴蝶兰（变型）Phal. violacea h.v. sumatrana
Ph12	麻栗坡蝴蝶兰Phal. malipoensis	Ph36	紫花荧光蝴蝶兰（全紫变型）Phal. violacea f. coerulea
Ph13	洛氏蝴蝶兰Phal. lowii	Ph37	安汶蝴蝶兰Phal. amboinensis
Ph14	华西蝴蝶兰Phal. wilsonii	Ph38	紫花贝雷娜蝴蝶兰（变型）Phal. bellina f. coerulea

用途 Usage	引物名称 Primer name	重复基序 Repeat motif	引物序列（5′-3′） Primer sequence	有效扩增/% Effective amplification
钻喙兰属	5-N1001614	(GAA)₇	F：TTTGGTGGAGGATGGAGAAG；R：GCTTCCTTGAGGTGAAGAGC	100.00
Rhynchostylis	14-N1007590	(CT)₅	F：TGACATGGAAGTTATGGCATTC；R：TGTCGCAATTCTATGTCAGGA	100.00
	25-N101022	(ATT)₅	F：TGGACCACCTATCTCAAGGC；R：ACGACATGCTCATGATTCCA	100.00
	28-N1012378	(AAG)₇	F：GGAAGAAGGAGGAGGTGAGG；R：TCCTAGAGAAAGAGCTTGGAGC	100.00
	37-N3601419	(AAG)₈	F：AATGGAAAATCTGGCAGCAC；R：TCTCCATCACTTCAATGGCA	100.00
	45-N1438823	(GAA)₅	F：ATTTGGCTCAAATCCACCAA；R：ATACCTGAAAGCATGGTGCC	100.00
	82-N100023	(AG)₆	F：AGGGTGGGAAGGGAGATAAA；R：CTCTGCCTTCTCCATCTCTCA	100.00
	88-N1001036	(AG)₅	F：AGGGAGGGAGTGAGAGAGGA；R：TCCCGCTTTCTATCGCTATC	100.00
	89-N100105	(TA)₅	F：TTTCCTGCTCTCCATTCCTG；R：TGTCATCTTCTCACACTCCCA	100.00
	91-N1001523	(AG)₈	F：TGATGGAATGAAGTTGAGGGA；R：GGCACATATGCTTTGTGGTG	100.00
	96-N1007386	(TCC)₆	F：AAGTCAGCCACTATGCCAGG；R：CAAGCGACATTTGCGTCTAA	80.00
	97-N1844635	(GGC)₅	F：GTGGATATACCGCTTCGTCG；R：GAAGAAAGTGGCTGCGTCTC	100.00
蝴蝶兰属	6-N10137440	(TTTG)₆	F：TGGCTTCAGTGAGACAATCG；R：CCTCGCAGACCATCAAACTA	92.11
Phalaenopsis	9-N10001991	(TGA)₅	F：TAGCAAAAGGGCGAGGAGTA；R：ATAAGTGGTTGGGCTTCACG	92.11
	31-N10013849	(TTC)₈	F：CCTTAGCTGCCGCTCTTCTA；R：CATCACAGCCCTTCACAGAA	100.00
	37-N10016842	(GGA)₅	F：GAGGAGGTGGTAGGGAGGAG；R：AGTCCTCTGATCGTTAGCCG	100.00
	52-N11200965	(CCT)₇	F：AACAAACACAAAAACAAACTGCAT；R：CGGGATGAAGATGAGGAAGA	92.11
	97-N032454	(AGG)₉	F：ACCTGCAAATGAACTGAAGTGG；R：GCCAACATCAAACAAACAGACC	92.11
	98-N034003	(AG)₉	F：GTGTCTACTTCTAACGCATCTTGT；R：GGAGCTGAGTATATCAGACTTCG	100.00
	99-N034673	(CT)₉	F：GATATTATGTGATCAATGCGACG；R：CCAAAATAAACCGCTTAATAAGC	97.37
	101-N032870	(CT)₈	F：TCCGCGCTATCTTTCTCTTAACT；R：TTCCTGGTAATTAGAAAGGACGG	97.37
	102-N033385	(CT)₈	F：CTCCGCGCTATCTTTCTCTTAAC；R：TCCTGGTAATTAGAAAGGACGGT	100.00
	106-N035168	(GA)₈	F：GCCTTCCAACCTTTTCTTCTTT；R：CGTATGAGTCCCGATGAATCC	76.32
	018164443-13	(CT)₉	F：ATTGCACGCTCTCATTCCTT；R：TCTTCCCATTTCTCAATGCC	92.11
	018164443-20	(ATT)₅	F：CAGAAGCCGGAGAAGACAAG；R：ATAAGCGAATCAGCAGCGTT	100.00
	018164443-30	(AAT)₇	F：CGTTCAGTCCATCCCAAGAT；R：ACTGGCGATGTTTGGTGATT	92.11
	018164443-31	(TTG)₁₃	F：CATGTAGCCGGTCTCACGTA；R：TGCCTCAGGAATCCTATTGA	81.58
	018164443-34	(CCG)₅	F：ACACAGACGTCTCCCAATCC；R：GCTACGGTGAGGGAGTTCAG	97.37
	018164443-43	(AAT)₅	F：AAGCGGGAAACTTAAGCTGG；R：TGCAAAAGGCCATAGAGCTT	84.21
	018164443-52	(CATG)₅	F：AAGAGGGAACGAGGAAGCA；R：AATGGCTGTAGACCACCTGC	92.11
	U6190	(TTAG)₅	F：CCATTAAAGCTCAGGAGGGA；R：CGAAGAAATAGCCATCGAGC	92.11
两属间	2-N10002234	(AAC)₅	F：CCTGCAGGTTAAACAACAACAA；R：ATTTAGACCGTGGGAAGCCT	93.75
Between Phal.	7-N1001895	(TCT)₈	F：ATCGCCATTTTTGTGCTCTT；R：ATTGAAATGATGGCTTTCGC	93.75
and Rhy.	22-N10009521	(AT)₉	F：TGAAGAAAAGGTCTAACAAGCAGA；R：TGAGTTAAAGGAAGAAATGGCCT	100.00
	28-N1001301	(ATT)₁₉	F：TCCTTCCAACATTTATTGCTCC；R：TCACATCCTTCAATATTGCCC	95.83
	42-N8802928	(TA)₈	F：CCCAAAACTTTACTTTTCCAGC；R：CTGTAAAAGCGTACCTGGCA	100.00
	43-N5725511	(TA)₆	F：CTGTAAAAGCGTACCTGGCG；R：AGGACCCCCAACATACAACA	97.92
	68-N2965390	(AAT)₆	F：TCAATGATATCACATTCCTTGTACG；R：GCTAGGATTTTGTTAATTATTGTGC	77.08
	74-N8009753	(TTC)₁₀	F：TGTTTGCAGGGAAATTTGTTTCA；R：TCTTCAACTTATGTGATTGCAGG	100.00
	88-CB033847	(AT)₃₁	F：GCTCCGCTAGCTCTGACAGA；R：CATCCGAGCTGATGAAAGGT	91.67
	90-CB034920	(TGA)₁₂	F：TGTTGGTCTTCGGAAAGAAGTAT；R：AGCGCAGATAATAAAATCCTACC	100.00
	92-CK858704	(GA)₆	F：GAGGCTGAGCAAAAGATTATGAG；R：CGAATCATCGGATTCTTACTCTT	95.83
	94-CB033577	(TC)₁₀	F：CTCCTTCCCTGATTTCTTACTTG；R：TCCACTCAAAAGACCTCCTTTAG	91.67

用途 Usage	引物名称 Primer name	重复基序 Repeat motif	引物序列（5′-3′） Primer sequence	有效扩增/% Effective amplification
钻喙兰属	5-N1001614	(GAA)₇	F：TTTGGTGGAGGATGGAGAAG；R：GCTTCCTTGAGGTGAAGAGC	100.00
Rhynchostylis	14-N1007590	(CT)₅	F：TGACATGGAAGTTATGGCATTC；R：TGTCGCAATTCTATGTCAGGA	100.00
	25-N101022	(ATT)₅	F：TGGACCACCTATCTCAAGGC；R：ACGACATGCTCATGATTCCA	100.00
	28-N1012378	(AAG)₇	F：GGAAGAAGGAGGAGGTGAGG；R：TCCTAGAGAAAGAGCTTGGAGC	100.00
	37-N3601419	(AAG)₈	F：AATGGAAAATCTGGCAGCAC；R：TCTCCATCACTTCAATGGCA	100.00
	45-N1438823	(GAA)₅	F：ATTTGGCTCAAATCCACCAA；R：ATACCTGAAAGCATGGTGCC	100.00
	82-N100023	(AG)₆	F：AGGGTGGGAAGGGAGATAAA；R：CTCTGCCTTCTCCATCTCTCA	100.00
	88-N1001036	(AG)₅	F：AGGGAGGGAGTGAGAGAGGA；R：TCCCGCTTTCTATCGCTATC	100.00
	89-N100105	(TA)₅	F：TTTCCTGCTCTCCATTCCTG；R：TGTCATCTTCTCACACTCCCA	100.00
	91-N1001523	(AG)₈	F：TGATGGAATGAAGTTGAGGGA；R：GGCACATATGCTTTGTGGTG	100.00
	96-N1007386	(TCC)₆	F：AAGTCAGCCACTATGCCAGG；R：CAAGCGACATTTGCGTCTAA	80.00
	97-N1844635	(GGC)₅	F：GTGGATATACCGCTTCGTCG；R：GAAGAAAGTGGCTGCGTCTC	100.00
蝴蝶兰属	6-N10137440	(TTTG)₆	F：TGGCTTCAGTGAGACAATCG；R：CCTCGCAGACCATCAAACTA	92.11
Phalaenopsis	9-N10001991	(TGA)₅	F：TAGCAAAAGGGCGAGGAGTA；R：ATAAGTGGTTGGGCTTCACG	92.11
	31-N10013849	(TTC)₈	F：CCTTAGCTGCCGCTCTTCTA；R：CATCACAGCCCTTCACAGAA	100.00
	37-N10016842	(GGA)₅	F：GAGGAGGTGGTAGGGAGGAG；R：AGTCCTCTGATCGTTAGCCG	100.00
	52-N11200965	(CCT)₇	F：AACAAACACAAAAACAAACTGCAT；R：CGGGATGAAGATGAGGAAGA	92.11
	97-N032454	(AGG)₉	F：ACCTGCAAATGAACTGAAGTGG；R：GCCAACATCAAACAAACAGACC	92.11
	98-N034003	(AG)₉	F：GTGTCTACTTCTAACGCATCTTGT；R：GGAGCTGAGTATATCAGACTTCG	100.00
	99-N034673	(CT)₉	F：GATATTATGTGATCAATGCGACG；R：CCAAAATAAACCGCTTAATAAGC	97.37
	101-N032870	(CT)₈	F：TCCGCGCTATCTTTCTCTTAACT；R：TTCCTGGTAATTAGAAAGGACGG	97.37
	102-N033385	(CT)₈	F：CTCCGCGCTATCTTTCTCTTAAC；R：TCCTGGTAATTAGAAAGGACGGT	100.00
	106-N035168	(GA)₈	F：GCCTTCCAACCTTTTCTTCTTT；R：CGTATGAGTCCCGATGAATCC	76.32
	018164443-13	(CT)₉	F：ATTGCACGCTCTCATTCCTT；R：TCTTCCCATTTCTCAATGCC	92.11
	018164443-20	(ATT)₅	F：CAGAAGCCGGAGAAGACAAG；R：ATAAGCGAATCAGCAGCGTT	100.00
	018164443-30	(AAT)₇	F：CGTTCAGTCCATCCCAAGAT；R：ACTGGCGATGTTTGGTGATT	92.11
	018164443-31	(TTG)₁₃	F：CATGTAGCCGGTCTCACGTA；R：TGCCTCAGGAATCCTATTGA	81.58
	018164443-34	(CCG)₅	F：ACACAGACGTCTCCCAATCC；R：GCTACGGTGAGGGAGTTCAG	97.37
	018164443-43	(AAT)₅	F：AAGCGGGAAACTTAAGCTGG；R：TGCAAAAGGCCATAGAGCTT	84.21
	018164443-52	(CATG)₅	F：AAGAGGGAACGAGGAAGCA；R：AATGGCTGTAGACCACCTGC	92.11
	U6190	(TTAG)₅	F：CCATTAAAGCTCAGGAGGGA；R：CGAAGAAATAGCCATCGAGC	92.11
两属间	2-N10002234	(AAC)₅	F：CCTGCAGGTTAAACAACAACAA；R：ATTTAGACCGTGGGAAGCCT	93.75
Between Phal.	7-N1001895	(TCT)₈	F：ATCGCCATTTTTGTGCTCTT；R：ATTGAAATGATGGCTTTCGC	93.75
and Rhy.	22-N10009521	(AT)₉	F：TGAAGAAAAGGTCTAACAAGCAGA；R：TGAGTTAAAGGAAGAAATGGCCT	100.00
	28-N1001301	(ATT)₁₉	F：TCCTTCCAACATTTATTGCTCC；R：TCACATCCTTCAATATTGCCC	95.83
	42-N8802928	(TA)₈	F：CCCAAAACTTTACTTTTCCAGC；R：CTGTAAAAGCGTACCTGGCA	100.00
	43-N5725511	(TA)₆	F：CTGTAAAAGCGTACCTGGCG；R：AGGACCCCCAACATACAACA	97.92
	68-N2965390	(AAT)₆	F：TCAATGATATCACATTCCTTGTACG；R：GCTAGGATTTTGTTAATTATTGTGC	77.08
	74-N8009753	(TTC)₁₀	F：TGTTTGCAGGGAAATTTGTTTCA；R：TCTTCAACTTATGTGATTGCAGG	100.00
	88-CB033847	(AT)₃₁	F：GCTCCGCTAGCTCTGACAGA；R：CATCCGAGCTGATGAAAGGT	91.67
	90-CB034920	(TGA)₁₂	F：TGTTGGTCTTCGGAAAGAAGTAT；R：AGCGCAGATAATAAAATCCTACC	100.00
	92-CK858704	(GA)₆	F：GAGGCTGAGCAAAAGATTATGAG；R：CGAATCATCGGATTCTTACTCTT	95.83
	94-CB033577	(TC)₁₀	F：CTCCTTCCCTGATTTCTTACTTG；R：TCCACTCAAAAGACCTCCTTTAG	91.67

引物 Primer	产物大小/bp Fragment size	观测等位基因数 N_a	有效等位基因数 N_e	观测杂合度 H_o	期望杂合度 H_e	多态性信息含量 PIC	Shannon指数 I
2-N10002234	212 ~ 267	12.50	8.29	0.53	0.88	0.89	2.27
7-N1001895	137 ~ 217	17.00	10.06	0.46	0.72	0.94	2.07
22-N10009521	119 ~ 155	9.50	4.43	0.33	0.77	0.78	1.76
28-N1001301	135 ~ 183	13.00	7.88	0.29	0.80	0.91	2.06
42-N8802928	96 ~ 108	7.00	4.75	0.27	0.79	0.77	1.71
43-N5725511	120 ~ 146	6.50	3.66	0.31	0.72	0.73	1.52
68-N2965390	96 ~ 117	8.50	4.65	0.48	0.73	0.88	1.61
74-N8009753	101 ~ 148	14.50	7.33	0.64	0.86	0.85	2.27
88-CB033847	123 ~ 163	8.00	3.17	0.33	0.68	0.70	1.46
90-CB034920	130 ~ 166	14.50	9.20	0.44	0.79	0.93	2.12
92-CK858704	157 ~ 174	6.00	3.11	0.07	0.66	0.78	1.30
94-CB033577	157 ~ 225	17.00	9.76	0.40	0.88	0.93	2.43
均值 Mean		11.17	6.36	0.38	0.77	0.84	1.88

Development and Application of Universal SSR Molecular Marker Primers in Phalaenopsis and Rhynchostylis

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 9

References 39

Related Articles 9

Recommended Articles

Metrics

Comments

引物 Primer	观测等位基因数 N_a	有效等位基因数 N_e	观测杂合度 H_o	期望杂合度 H_e	多态性信息含量 PIC	Shannon指数 I
2-N10002234	3.00	2.58	0.04	0.61	0.53	1.00
7-N1001895	4.00	3.00	0.02	0.67	0.62	1.24
9-N10001991	5.00	2.07	0.06	0.52	0.48	1.04
14-N1007590	2.00	1.99	0.45	0.50	0.37	0.69
22-N10009521	2.00	2.00	0.01	0.50	0.38	0.69
25-N101022	3.00	2.67	0	0.63	0.55	1.04
28-N1012378	5.00	2.35	0.23	0.57	0.49	1.02
31-N10013849	2.00	1.05	0	0.05	0.05	0.11
37-N3601419	2.00	1.06	0.01	0.05	0.05	0.12
43-N5725511	2.00	1.05	0.01	0.05	0.05	0.12
45-N1438823	4.00	3.48	0.51	0.71	0.66	1.30
52-11200965	5.00	2.69	0.29	0.63	0.56	1.15
68-N2965390	3.00	1.27	0.01	0.21	0.20	0.42
74-N8009753	3.00	2.80	0.48	0.64	0.57	1.06
82-N100023	12.00	5.99	0.51	0.83	0.81	1.94
88-CB033847.1	2.00	1.90	0.23	0.47	0.36	0.67
88-N1001036	2.00	1.98	0.38	0.49	0.37	0.69
90-CB034920.1	3.00	1.86	0.01	0.46	0.40	0.79
92-CK858704.1	2.00	1.25	0	0.20	0.18	0.35
94-CB033577.1	2.00	1.47	0.05	0.32	0.27	0.50
102-CB033385.1	2.00	1.83	0	0.45	0.35	0.65
01816443-13	3.00	1.68	0.17	0.41	0.34	0.65
018164444-43	2.00	1.80	0	0.44	0.35	0.64
均值 Mean	3.26	2.17	0.15	0.45	0.39	0.43

[1]	LÜ Fubing, CHEN Heming, XIAO Wenfang, LI Zuo. A New Phalaenopsis Cultivar‘Jiaohong’ [J]. Acta Horticulturae Sinica, 2024, 51(3): 701-702.
[2]	LÜ Fubing, CHEN Heming, LI Zuo, XIAO Wenfang. A New Phalaenopsis Cultivar‘Yufen’ [J]. Acta Horticulturae Sinica, 2023, 50(7): 1605-1606.
[3]	XU Shenping, YUAN Xiuyun, LIANG Fang, ZHANG Yan, JIANG Suhua, NIU Suyan, CUI Bo. A New Phalaenopsis Cultivar‘Zhengshi Zhaoxia’ [J]. Acta Horticulturae Sinica, 2023, 50(3): 691-692.
[4]	CHEN Heming, LÜ Fubing, XIAO Wenfang, LI Zuo. A New Cultivar‘Xiaobaima’of Intergeneric Hybridization Between Holcoglossum and Rhynchostylis [J]. Acta Horticulturae Sinica, 2023, 50(10): 2301-2302.
[5]	WU Ting, JIA Ruidong, YANG Shuhua, ZHAO Xin, YU Xiaonan, GUO Yuan, GE Hong. Research Advances and Prospects on Phalaenopsis Polyploid Breeding [J]. Acta Horticulturae Sinica, 2022, 49(2): 448-462.
[6]	HOU Tianze, YI Shuangshuang, ZHANG Zhiqun, WANG Jian, LI Chonghui. Selection and Validation of Reference Genes for RT-qPCR in Phalaenopsis- type Dendrobium Hybrid [J]. Acta Horticulturae Sinica, 2022, 49(11): 2489-2501.
[7]	CHEN Heming, WANG Zhen, LÜ Fubing, XIAO Wenfang, LI Zuo, YOU Yi. A New Phalaenopsis Cultivar "Minixiang" [J]. Acta Horticulturae Sinica, 2021, 48(12): 2527-2528.
[8]	LÜ Fubing, CHEN Heming, LI Zuo, XIAO Wenfang, You Yi. A New Phalaenopsis Cultivar‘Ruyu’ [J]. Acta Horticulturae Sinica, 2021, 48(11): 2323-2324.
[9]	CHEN Chun, ZHUANG Xiqing, CHEN Xiaochou, ZHANG Yizhi, LIN Chenli, and WANG Yong. A New Phalaenopsis Cultivar‘Red Swan’ [J]. Acta Horticulturae Sinica, 2020, 47(S2): 3049-3050.