基于表型性状与SSR标记的香橼种质资源遗传多样性分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0846

摘要/Abstract

摘要：

目的明确我国香橼资源果实表型变异程度及其种质遗传多样性、群体结构、遗传分化特点，为香橼种质资源保护、优良品种选育提供重要依据。方法实地调查我国16个省（市）、37个市（县）的香橼种质资源分布，对采集40份香橼果实进行15个表型性状描述分析，并利用筛选的10对SSR引物对90份香橼叶片进行遗传标记试验。结果不同产区香橼果实表型的7个质量性状Shannon-Wiener指数H范围为0.526‒1.365，Simpson指数D范围为0.349‒0.711；8个数量性状变异系数为21%（囊瓣数）-99%（单果重）；表型性状聚类将香橼分为3类：Ⅰ（香圆与枳雀）、Ⅱ（香泡）和Ⅲ（枸橼）。10对SSR引物对90份香橼样本进行PCR扩增，结果显示，位点水平香橼等位基因数（Na）、有效等位基因数（Ne）、Shannon’s多样性指数（I）、观察杂合度（Ho）、期望杂合度（He）分别为1.832、1.354、0.331、0.824、0.210；SSR聚类、群体结构和PCoA分析均将香橼分为4类，其中，香圆与枳雀被进一步分开；群体遗传多样性水平由高到低依次为枳雀（Ho=0.682，He=0.199）、枸橼（Ho=0.659，He=0.175）、香泡（Ho=0.436，He=0.133）、香圆（Ho=0.391，He=0.127）；Mantel检验显示，香橼遗传距离与地理距离存在显著正相关性（P<0.05）；遗传分化分析显示，种质遗传变异主要存在于群体内（73%）。结论我国香橼种质资源存在广泛的遗传变异和丰富的多样性。

关键词: 香橼, 种质资源, 表型鉴定, 聚类分析, 遗传多样性

Abstract:

Objective Citri fructus germplasm resources in China were investigated to analyze phenotypic variation of fruit traits, genetic diversity, population structure and genetic differentiation characteristics, providing a scientific basis for germplasm conservation and superior cultivar breeding. Method The total of 40 batches of fruits and 90 leaves samples of plants from 16 provinces (37 cities) of China were used as the test materials, and then the genetic diversity analysis was conducted using 15 phenotypic traits and screened 10 pairs of SSR markers. Result The Shannon-Wiener index H and Simpson’s diversity index D of 7 quality traits of citri fructus from different regions varied from 0.526 to 1.365 and 0.349 to 0.711, respectively. Among the 8 quantitative traits coefficient of variation ranged from 21% (number of segments) to 99% (single-fruit weight). According to phenotypic traits, the tested materials could be divided into three groups: Ⅰ (Xiangyuan and Zhique), Ⅱ (Xiangpao) and Ⅲ (Juyuan). The 10 pairs of primers with good polymorphism and stable bands were screened for PCR of SSR primers. The results showed that tthe number of alleles (Na), the number of effective alleles (Ne), Shannon’s diversity index (I), observed heterozygosity (Ho), and expected heterozygosity (He) at the locus level were 1.832, 1.354, 0.331, 0.824, and 0.210, respectively. By clustering, population structure and PCoA analysis, the 90 germplasm were divided into four groups. Xiangyuan and Zhique were further separated. The genetic diversity of the four groups was different with the descending order of Zhique (Ho=0.682, He=0.199), Juyuan (Ho=0.659, He=0.175), Xiangpao (Ho=0.436, He=0.133) and Xiangyuan (Ho=0.391, He=0.127). The Mantel test showed that the genetic distance of the citri fructus and geographical distance of the provenances were significantly correlated (r=0.558, P=0.010). The molecular variance analysis of xiang yuan population exhibited 73% of the variation within the population. Conclusion Citri fructus germplasm resources in China have rich phenotypic genetic variation and high diversity.

Key words: citri fructus, germplasm resource, phenotypic identification, cluster analysis, genetic diversity

魏丽娜, 蒋景龙, 邓茜茜, 李岩, 李丽, 邓家锐, 丁德宽. 基于表型性状与SSR标记的香橼种质资源遗传多样性分析[J]. 生物技术通报, 2026, 42(4): 202-215.

WEI Li-na, JIANG Jing-long, DENG Xi-xi, LI Yan, LI Li, DENG Jia-rui, DING De-kuan. Genetic Diversity Analysis of Citri Fructus Germplasm Resources Based on Phenotypic Traits and SSR Markers[J]. Biotechnology Bulletin, 2026, 42(4): 202-215.

图/表 20

参考文献 35

[1]	国家药典委员会. 中华人民共和国药典-一部: 2025年版 [M]. 北京: 中国医药科技出版社, 2025.
	People’s republic of China (PRC) pharmacopoeia-part I: 2025 edition [M]. Beijing: China Medical Science Press, 2025.
[2]	严辉, 高明亮, 查玉玲, 等. 香橼化学成分和药理作用研究进展及其质量标志物预测分析 [J]. 中国现代应用药学, 2022, 39(7): 976-988.
	Yan H, Gao ML, Zha YL, et al. Research progress on chemical components and pharmacological effects of citri fructus and predictive analysis on its quality marker [J]. Chin J Mod Appl Pharm, 2022, 39(7): 976-988.
[3]	Zhao P, Duan L, Guo L, et al. Chemical and biological comparison of the fruit extracts of Citrus wilsonii Tanaka and Citrus medica L. [J]. Food Chem, 2015, 173: 54-60.
[4]	Conforti F, Statti GA, Tundis R, et al. In vitro activities of Citrus medica L. cv. Diamante (Diamante citron) relevant to treatment of diabetes and Alzheimer’s disease [J]. Phytother Res, 2007, 21(5): 427-433.
[5]	杨辉, 杨培君, 李会宁. 中药材香圆挥发油成分GC-MS分析与比较 [J]. 食品与生物技术学报, 2010, 29(2): 219-229.
	Yang H, Yang PJ, Li HN. Comparison of volatile oils from both fructus aurantii and fructus aurantii immaturus of Citrus wilsonii in Shaanxi by GC-MS [J]. J Food Sci Biotechnol, 2010, 29(2): 219-229.
[6]	Chhikara N, Kour R, Jaglan S, et al. Citrus medica: nutritional, phytochemical composition and health benefits - a review [J]. Food Funct, 2018, 9(4): 1978-1992.
[7]	任丽, 张余, 邓姗, 等. 基于形态标记的新育成辣椒品种特性分析 [J]. 植物遗传资源学报, 2023, 24(6): 1676-1689.
	Ren L, Zhang Y, Deng S, et al. Characterization of new Capsicum spp. varieties based on morphological markers [J]. J Plant Genet Resour, 2023, 24(6): 1676-1689.
[8]	郭方其, 吕萍, 吴超, 等. 基于表型性状的切花多头菊种质资源遗传多样性分析 [J]. 分子植物育种, 2020, 18(18): 6205-6215.
	Guo FQ, Lü P, Wu C, et al. Phenotypic diversity analysis of cutting flower Chrysanthemum germplasm resources based on phenotypic traits [J]. Mol Plant Breed, 2020, 18(18): 6205-6215.
[9]	Laosatit K, Amkul K, Chankaew S, et al. Molecular genetic diversity of winged bean gene pool in Thailand assessed by SSR markers [J]. Hortic Plant J, 2022, 8(1): 81-88.
[10]	Ramadugu C, Keremane ML, Hu XL, et al. Genetic analysis of citron (Citrus medica L.) using simple sequence repeats and single nucleotide polymorphisms [J]. Sci Hortic, 2015, 195: 124-137.
[11]	Yang XM, Li H, Liang M, et al. Genetic diversity and phylogenetic relationships of citron (Citrus medica L.) and its relatives in southwest China [J]. Tree Genet Genomes, 2015, 11(6): 129.
[12]	刘航秀, 冯迪, 龙春瑞, 等. 枸橼药用植物果实变异及地理分布研究 [J]. 中国中药杂志, 2021, 46(23): 6289-6293.
	Liu HX, Feng D, Long CR, et al. Fruit variation and geographical distribution of citron [J]. China J Chin Mater Med, 2021, 46(23): 6289-6293.
[13]	徐静, 谭李梅, 符红艳, 等. 利用分子标记对14份枸橼种质进行多样性分析 [J]. 分子植物育种, 2021, 19(14): 4726-4737.
	Xu J, Tan LM, Fu HY, et al. Genetic diversity analysis of 14 citron genotypes based on molecular markers [J]. Mol Plant Breed, 2021, 19(14): 4726-4737.
[14]	Maurya A, Mishra BP, Mohan R, et al. Analysis of genetic diversity and environmental associations of wild citron (Citrus medica L.) in northeast India [J]. Sci Hortic, 2024, 338: 113690.
[15]	魏丽娜, 胡佳, 敖义俊, 等. 陕南香圆资源调查与产业开发现状研究 [J]. 中国南方果树, 2024, 53(6): 15-23.
	Wei LN, Hu J, Ao YJ, et al. Investigation of Citrus wilsonii resources and its industrial development in Southern Shaanxi province [J]. South China Fruits, 2024, 53(6): 15-23.
[16]	孙旺, 蒋景龙, 胡选萍, 等. 濒危植物秦岭石蝴蝶的SCoT遗传多样性分析 [J]. 西北植物学报, 2020, 40(3): 425-431.
	Sun W, Jiang JL, Hu XP, et al. Genetic diversity analysis of endangered plant Petrocosmea qinlingensis based on SCoT [J]. Acta Bot Boreali Occidentalia Sin, 2020, 40(3): 425-431.
[17]	Peakall R, Smouse PE. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research‒an update [J]. Bioinformatics, 2012, 28(19): 2537-2539.
[18]	Earl DA, VonHoldt BM. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method [J]. Conserv Genet Resour, 2012, 4(2): 359-361.
[19]	何庆海, 杨少宗, 李因刚, 等. 枫香树种群种子与果实表型性状变异分析 [J]. 植物生态学报, 2018, 42(7): 752-763.
	He QH, Yang SZ, Li YG, et al. Phenotypic variations in seed and fruit traits of Liquidambar formosana populations [J]. Chin J Plant Ecol, 2018, 42(7): 752-763.
[20]	Sivakumar J, Sridhar Reddy M, Sergeant K, et al. Principal component analysis-assisted screening and selection of salt-tolerant tomato genotypes [J]. Plant Physiol Rep, 2023, 28(2): 272-288.
[21]	Thakur N, Dogra BS, Kaur J, et al. Study on genetic variability and heritability in cherry tomato (Solanum lycopersicum var. cerasiforme) for yield and quality traits [J]. Agric Res J, 2022, 59(6): 1026-1033.
[22]	Deviona, Syukur M, Zuhry E, et al. Principal component and cluster analyses of 20 genotypes of chilli (Capsicum sp.) planted on peatlands [J]. IOP Conf Ser: Earth Environ Sci, 2023, 1228(1): 12003.
[23]	Bihon W, Ognakossan KE, Tignegre JB, et al. Evaluation of different tomato (Solanum lycopersicum L.) entries and varieties for performance and adaptation in Mali, west Africa [J]. Horticulturae, 2022, 8(7): 579.
[24]	翟彩娇, 葛礼姣, 程玉静, 等. 基于表型性状与SSR标记的冬瓜、节瓜种质资源遗传多样性分析 [J]. 中国农业科学, 2024, 57(17): 3440-3467.
	Zhai CJ, Ge LJ, Cheng YJ, et al. Genetic diversity analysis of wax gourd and chieh-qua germplasm resources based on phenotypic traits and SSR markers [J]. Sci Agric Sin, 2024, 57(17): 3440-3467.
[25]	林存学, 杨晓华, 刘海荣. 东北寒地96份李种质资源表型性状遗传多样性分析 [J]. 园艺学报, 2020, 47(10): 1917-1929.
	Lin CX, Yang XH, Liu HR. Genetic diversity analysis of 96 plum germplasm resources by phenotypic traits in northeast cold area [J]. Acta Hortic Sin, 2020, 47(10): 1917-1929.
[26]	Li JW, Li H, Liu ZW, et al. Molecular markers in tea plant (Camellia sinensis): Applications to evolution, genetic identification, and molecular breeding [J]. Plant Physiol Biochem, 2023, 198: 107704.
[27]	Fu YR, Liu FL, Li S, et al. Genetic diversity of the wild Asian Lotus (Nelumbo nucifera) from Northern China [J]. Hortic Plant J, 2021, 7(5): 488-500.
[28]	刘冬峰, 陈巍, 林绍生, 等. 基于SSR标记的浙江地方柚类种质资源遗传关系分析 [J]. 果树学报, 2017, 34(2): 166-174.
	Liu DF, Chen W, Lin SS, et al. Analysis of genetic relationship of pummelo germplasms by SSR markers in Zhejiang province [J]. J Fruit Sci, 2017, 34(2): 166-174.
[29]	Wang Y, Qi YH, He W, et al. Genetic diversity of pummelo (Citrus grandis osbeck) germplasms in Sichuan Basin inferred from SSR markers [C]//2018 International Conference on Biotechnology and Bioengineering: 8TH Icbb, Budapest, Hungary, 2019: 020004.
[30]	Hu Y, Tian L, Shi JW, et al. Genetic structure of cultivated Zanthoxylum species investigated with SSR markers [J]. Tree Genet Genomes, 2018, 14(6): 89.
[31]	Lu M, Zhang HS, An HM. Chloroplast DNA-based genetic variation of Rosa roxburghii in southwest China: phylogeography and conservation implications [J]. Hortic Plant J, 2021, 7(4): 286-294.
[32]	Liu QW, Song Y, Liu L, et al. Genetic diversity and population structure of pear (Pyrus spp.) collections revealed by a set of core genome-wide SSR markers [J]. Tree Genet Genomes, 2015, 11(6): 128.
[33]	Boris KV, Trifonova AA, Dubrovsky ML, et al. Genetic diversity of apple clonal rootstocks from the collection of the michurinsk state agrarian university based on SSR markers [J]. Plants, 2023, 12(16): 2991.
[34]	Özkan G, Haliloğlu K, Türkoğlu A, et al. Determining genetic diversity and population structure of common bean (Phaseolus vulgaris L.) landraces from türkiye using SSR markers [J]. Genes, 2022, 13(8): 1410.
[35]	徐皓, 周天华, 张智强, 等. 基于SSR分子标记的延胡索遗传多样性研究 [J]. 西北植物学报, 2018, 38(6): 1032-1038.
	Xu H, Zhou TH, Zhang ZQ, et al. Genetic diversity of Corydalis yanhusuo W.T.Wang based on SSR molecular markers [J]. Acta Bot Boreali Occidentalia Sin, 2018, 38(6): 1032-1038.

质量性状 Quality trait	性状描述及分级 Character description and classification
质量性状 Quality trait	1	2	3	4	5	6
平滑度 Peel smoothness	光滑 Smooth	较光滑 Slightly smooth	粗糙 Rough
果实形状 Fruit shape	类球形 Spheroid	高扁圆形 High oblate	倒卵圆形 Obovoid	纺锤形 Fusiform
果顶形状 Apex shape	凸 Protruding	浅凹 Slightly depressed	平 Flatten	乳突 Mammillated	圆 Rounded
果顶印圈 Areole ring	明显 Obvious	不明显 Indistinct	无 None
果基形状 Base shape	深凹 Depressed	凸 Protruding	浅凹 Slightly depressed	平 Flatten	圆 Rounded	颈领 Necked
外果皮颜色 Peel color	黄色 Yellow	淡黄 Pale yellow	黄绿 Green-yellow	橙红 Orange-red	橙黄 Orange-yellow
白皮层颜色 Albedo color	白色 White	淡黄 Pale yellow

质量性状 Quality trait	性状描述及分级 Character description and classification
质量性状 Quality trait	1	2	3	4	5	6
平滑度 Peel smoothness	光滑 Smooth	较光滑 Slightly smooth	粗糙 Rough
果实形状 Fruit shape	类球形 Spheroid	高扁圆形 High oblate	倒卵圆形 Obovoid	纺锤形 Fusiform
果顶形状 Apex shape	凸 Protruding	浅凹 Slightly depressed	平 Flatten	乳突 Mammillated	圆 Rounded
果顶印圈 Areole ring	明显 Obvious	不明显 Indistinct	无 None
果基形状 Base shape	深凹 Depressed	凸 Protruding	浅凹 Slightly depressed	平 Flatten	圆 Rounded	颈领 Necked
外果皮颜色 Peel color	黄色 Yellow	淡黄 Pale yellow	黄绿 Green-yellow	橙红 Orange-red	橙黄 Orange-yellow
白皮层颜色 Albedo color	白色 White	淡黄 Pale yellow

引物名称 Primer name	正向引物序列 Forward primer （5′‒3′）	反向引物序列 Reverse primer （5′‒3′）	退火温度 Tm （℃）
Cs2	TCCACAAGGAGAAGAAACGG	GCGTCTTTACTGTTACCGGG	56
Cs3	GTTGCGCAGTTATTCTCAAA	CCGACCACTTTTACCCACTG	54
Cs6	AGGTCTACATTGGCATTGTC	ACATGCAGTGCTATAATGAATG	52
Cs9	TAAAAACCAACGTCCCCTCA	CGGGCGAGGTAGAAGTAATG	54
Cs11	CGCAAATGACTTCCCAGAAT	GCTCCCTCCGATTCTCTCTC	52
Cs13	CACGCAGCTTGAGTTTGAAG	GTGCCGTTTAGGGTTTTCCT	55
Cs14	CAGGCAGTAACCTCCCAGAC	AGCGAAAGCTAATGATGGTG	54
Cs15	CGGGCTAGGCTGAGAGATA	TTCTTTGGAGCCGAACAACT	52
Cs20	AGTGAACTGTCCATTGGATTTTCG	GTGTTGAATCCCGACCTTCTACC	54
Cs21	GCAGCGCAACAACATAACTA	GGCCAATAGCTTCCATTCA	52

引物名称 Primer name	正向引物序列 Forward primer （5′‒3′）	反向引物序列 Reverse primer （5′‒3′）	退火温度 Tm （℃）
Cs2	TCCACAAGGAGAAGAAACGG	GCGTCTTTACTGTTACCGGG	56
Cs3	GTTGCGCAGTTATTCTCAAA	CCGACCACTTTTACCCACTG	54
Cs6	AGGTCTACATTGGCATTGTC	ACATGCAGTGCTATAATGAATG	52
Cs9	TAAAAACCAACGTCCCCTCA	CGGGCGAGGTAGAAGTAATG	54
Cs11	CGCAAATGACTTCCCAGAAT	GCTCCCTCCGATTCTCTCTC	52
Cs13	CACGCAGCTTGAGTTTGAAG	GTGCCGTTTAGGGTTTTCCT	55
Cs14	CAGGCAGTAACCTCCCAGAC	AGCGAAAGCTAATGATGGTG	54
Cs15	CGGGCTAGGCTGAGAGATA	TTCTTTGGAGCCGAACAACT	52
Cs20	AGTGAACTGTCCATTGGATTTTCG	GTGTTGAATCCCGACCTTCTACC	54
Cs21	GCAGCGCAACAACATAACTA	GGCCAATAGCTTCCATTCA	52

质量性状 Quality trait	分级及频率 Classification and frequency （%）						Shannon-Wiener指数 Shannon-Wiener index H	Simpson指数 Simpson index D
质量性状 Quality trait	1	2	3	4	5	6	Shannon-Wiener指数 Shannon-Wiener index H	Simpson指数 Simpson index D
平滑度 Peel smoothness	30.0	50.0	20.0	-	-	-	1.030	0.620
果实形状 Fruit shape	35.0	37.5	17.5	10.0	-	-	1.277	0.696
果顶形状 Apex shape	30.0	12.5	40.0	15.0	2.5	-	1.365	0.711
果顶印圈 Areole ring	30.0	12.5	57.5	-	-	-	0.939	0.564
果基形状 Base shape	10.0	5.0	65.0	5.0	12.5	2.5	1.161	0.546
外果皮颜色 Peel color	65.0	12.5	12.5	2.5	7.5	-	1.084	0.540
白皮层颜色 Albedo color	77.5	22.5	-	-	-	-	0.526	0.349