伊犁郁金香cpDNA-PCR体系构建与遗传多样性分析

doi:10.3969/j.issn.1004-1524.20240378

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (1): 78-89.DOI: 10.3969/j.issn.1004-1524.20240378

伊犁郁金香cpDNA-PCR体系构建与遗传多样性分析

秦斗文¹^,²(), 刘伟强¹^,², 田吉婷¹^,², 巨秀婷¹^,²^,^*()

1.青海大学农牧学院,青海西宁 810016
2.青海省园林植物与观赏园艺重点实验室,青海西宁 810016

收稿日期:2024-04-26 出版日期:2025-01-25 发布日期:2025-02-14
作者简介:秦斗文(1996—),女,河南信阳人,硕士,研究方向为观赏植物遗传育种。E-mail:qindouwen@163.com
通讯作者: *巨秀婷,E-mail:juxiuting@163.com
基金资助:
2021年度“西部之光”人才培养计划(中科人字〔2022〕4号)

Establishment of cpDNA-PCR reaction system and genetic diversity analysis of Tulipa iliensis

QIN Douwen¹^,²(), LIU Weiqiang¹^,², TIAN Jiting¹^,², JU Xiuting¹^,²^,^*()

1. College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, China
2. Key Laboratory of Landscape Plants and Ornamental Horticulture of Qinghai Province, Xining 810016, China

Received:2024-04-26 Online:2025-01-25 Published:2025-02-14

摘要/Abstract

摘要： 为明确伊犁郁金香(Tulipa iliensis)的遗传背景,在叶绿体基因组水平开展遗传多样性研究。本研究以伊犁郁金香为研究对象,对cpDNA-PCR反应体系中的Mg²⁺、dNTPs、引物浓度、Taq DNA酶和模板DNA浓度在L₁₆(4⁵)正交试验设计的基础上结合单因素优化筛选试验构建伊犁郁金香cpDNA-PCR反应体系,在最优体系的基础上进行引物筛选并通过引物筛选完成cpDNA-PCR扩增,以验证cpDNA-PCR反应体系的稳定性,并对伊犁郁金香野生群体进行遗传多样性分析。结果表明,构建的伊犁郁金香最佳cpDNA-PCR反应体系(25 μL)为Mg²⁺ 2.00 mmol·L^-1,dNTPs 0.20 mmol·L^-1,引物浓度0.40 μmol·L^-1,Taq DNA酶0.50 U,模板DNA浓度110 ng。利用最佳扩增体系,从16对候选引物组合中筛选出可用于后续伊犁郁金香cpDNA-PCR扩增的14对引物(trnK-rps16, M3-M4, F71-R1516, trnD-trnE, psbB-psbH, trnL-trnF, atpB-rbcL, a1-b1, 1F-1R, rps8-rp116, atpI-atpH, accD-psaI, petG-trnP, 2F-2R)。建立的伊犁郁金香cpDNA-PCR反应体系扩增稳定,条带清晰,对54个伊犁郁金香个体进行遗传多样性分析,引物psbB-psbH扩增片段平均长度为582 bp,其中多态性位点数284个,核苷酸多态性P_i为0.055、平均核苷酸差异数K为30.261,基因流(Nm=1.21>1),表明伊犁郁金香居群遗传多样性高,不同居群间基因交流频繁。该研究建立的cpDNA-PCR反应体系可用于伊犁郁金香遗传多样性和种群遗传结构研究,研究结果可以为野生郁金香种质资源的合理保护、可持续利用提供理论依据和技术支撑,为科学保护伊犁郁金香种质资源提供依据。

关键词: 伊犁郁金香, cpDNA, 体系构建, 遗传多样性

Abstract:

To clarify the genetic background of Tulipa iliensis, the genetic diversity was studied at the level of chloroplast genome. The cpDNA-PCR reaction system of T. iliensis was constructed by using the L₁₆(4⁵) orthogonal experiment design and single factor optimization screening experiment. The system was optimized by adjusting the Mg²⁺, dNTPs, primer concentration, Taq DNA polymerase, and template DNA concentration in the cpDNA-PCR reaction system. With the optimal system, primers were screened and performed cpDNA-PCR amplification to verify the stability of the reaction system, and the genetic diversity of the wild population of T. iliensis was also analyzed. The results showed that the optimal cpDNA-PCR reaction system for T. iliensis consisted of 2.00 mmol·L^-1 Mg²⁺, 0.20 mmol·L^-1 dNTPs, 0.40 μmol·L^-1 primer concentration, 0.50 U Taq DNA polymerase, and 110 ng template DNA concentration. Using the optimal amplification system, 14 pairs of candidate primers (trnK-rps16, M3-M4, F71-R1516, trnD-trnE, psbB-psbH, trnL-trnF, atpB-rbcL, a1-b1, 1F-1R, rps8-rp116, atpI-atpH, accD-psaI, petG-trnP, 2F-2R) were selected from 16 pairs of candidate primer combinations for subsequent cpDNA-PCR amplification of T. iliensis. The established cpDNA-PCR reaction system for T. iliensis was stable in amplification and produced clear bands, genetic diversity analysis was performed on 54 T. iliensis individuals, The average length of amplified fragments using primer psbB-psbH was 582 bp, with 284 identified polymorphic sites, the nucleotide polymorphism P_i was 0.055, the average nucleotide difference number K was 30.261, and the gene flow (Nm=1.21>1) indicated that the genetic diversity of the T. iliensis population was high, and gene exchange among different populations was frequent. The cpDNA-PCR reaction system established in this study can be used to study the genetic diversity and population genetic structure of T. iliensis, providing a theoretical basis and technical support for the rational protection and sustainable utilization of wild tulip germplasm resources, and to provide a basis for scientific protection of T. iliensis germplasm resources.

Key words: Tulipa iliensis, cpDNA, establishment of PCR system, genetic diversity

中图分类号:

S682.2.63

秦斗文, 刘伟强, 田吉婷, 巨秀婷. 伊犁郁金香cpDNA-PCR体系构建与遗传多样性分析[J]. 浙江农业学报, 2025, 37(1): 78-89.

QIN Douwen, LIU Weiqiang, TIAN Jiting, JU Xiuting. Establishment of cpDNA-PCR reaction system and genetic diversity analysis of Tulipa iliensis[J]. Acta Agriculturae Zhejiangensis, 2025, 37(1): 78-89.

图/表 12

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序号 Number	居群 Population	居群个体数 Individual number of population	采样地 Sample location
1	1 300	4	巩留县吉尔格郎乡Jilgelang Township, Gongliu County, China
2	1 400	4	巩留县吉尔格郎乡Jilgelang Township, Gongliu County, China
3	1 600	29	昭苏县喀夏加尔镇Kazhagar Township, Zhaosu County, China
4	1 700	5	昭苏县喀夏加尔镇Kazhagar Township, Zhaosu County, China
5	1 800	2	昭苏县喀夏加尔镇Kazhagar Township, Zhaosu County, China
6	1 900	5	昭苏县乌尊布拉克镇Uzunbulak Township, Zhaosu County, China
7	2 000	5	昭苏县乌尊布拉克镇Uzunbulak Township, Zhaosu County, China
合计Total		54

序号 Number	居群 Population	居群个体数 Individual number of population	采样地 Sample location
1	1 300	4	巩留县吉尔格郎乡Jilgelang Township, Gongliu County, China
2	1 400	4	巩留县吉尔格郎乡Jilgelang Township, Gongliu County, China
3	1 600	29	昭苏县喀夏加尔镇Kazhagar Township, Zhaosu County, China
4	1 700	5	昭苏县喀夏加尔镇Kazhagar Township, Zhaosu County, China
5	1 800	2	昭苏县喀夏加尔镇Kazhagar Township, Zhaosu County, China
6	1 900	5	昭苏县乌尊布拉克镇Uzunbulak Township, Zhaosu County, China
7	2 000	5	昭苏县乌尊布拉克镇Uzunbulak Township, Zhaosu County, China
合计Total		54

水平 Level	Taq DNA酶 Taq DNA polymerase/U	镁离子 Mg²⁺/(mmol·L^-1)	脱氧核糖核苷酸 dNTPs/(mmol·L^-1)	引物 Primer/(μmol·L^-1)	DNA/ng
1	0.25	1.0	0.05	0.2	30
2	0.50	1.5	0.10	0.3	50
3	0.75	2.0	0.15	0.4	80
4	1.00	2.5	0.20	0.5	110

水平 Level	Taq DNA酶 Taq DNA polymerase/U	镁离子 Mg²⁺/(mmol·L^-1)	脱氧核糖核苷酸 dNTPs/(mmol·L^-1)	引物 Primer/(μmol·L^-1)	DNA/ng
1	0.25	1.0	0.05	0.2	30
2	0.50	1.5	0.10	0.3	50
3	0.75	2.0	0.15	0.4	80
4	1.00	2.5	0.20	0.5	110

编号No.	引物Primer	序列Sequence	参考文献Reference	退火温度Annealing temperature/℃
1	trnK	5'-TACTCTACCATTGAGTTAGCAAC-3'	[31]	51.3
	rps16	5'-AAAGGTGCTCAACCTACAAGAAC-3'	[31]	55.0
2	AtpI	5'-TATTTACAAGYGGTATTCAAGCT-3'	[31]	49.8
	AtpH	5'-CCAAYCCAGCAGCAATAAC-3'	[31]	52.9
3	rps8	5'-TGAACAATATTTTCGGTAAT-3'	[32]	42.7
	rpl16	5'-AACCAGATTTCGTAAACAAC-3'	[32]	47.3
4	PetB	5'-CTGCCGTATTTATGTTATG-3'	[31]	44.7
	PetD	5'-GTCTAGCCCCTGTTCTTCCT-3'	[31]	56.4
5	M3	5'-GCAACAATACTTCCTATATCCGCTTCT-3'	[33]	56.2
	M4	5'-GAACTCTTCTAATAATCCCGAACCTAA-3'	[33]	53.4
6	F71	5'-GCTATGCTTAGTGTGTGACTCGTTG-3'	[34]	57.9
	R1516	5'-CCCTTCATTCTTCCTCTATGTTG-3'	[34]	52.7
7	accD	5'-AATYGTACCACGTAATCYTTTAAA-3'	[31]	49.0
	psaI	5'-AGAAGCCATTGCAATTGCCGGAAA-3'	[31]	60.2
8	trnV	5'-GTCTACGGTTCGARTCCGTA-3'	[31]	55.0
	ndhC	5'-TATTATTAGAAATGYCCARAAAATATCATAT-3'	[31]	47.7
9	petG	5'-GGTCTAATTCCTATAACTTTGGC-3'	[35]	50.4
	trnP	5'-GGGATGTGGCGCAGCTTGG-3'	[35]	63.5
10	trnD	5'-ACCAATTGAACTACAATCCC-3'	[36]	49.3
	trnE	5'-AGGACATCTCTCTTTCAAGGAG-3'	[36]	53.4
11	psbH	5'-TCAAYRGTYTGTGTAGCCAT-3'	[36]	52.1
	psbB	5'-TCCAAAAANKKGGAGATCCAAC-3'	[36]	53.9
12	trnL	5'-CGAAATCGGTAGACGCTACG-3'	[37]	55.8
	trnF	5'-ATTTGAACTGGTGACACGAG-3'	[37]	52.4
13	atpB	5'-ACATCKARTACKGGACCAATAA-3'	[38]	51.2
	rbcL	5'-AACACCAGCTTTRAATCCAA-3'	[38]	50.3
14	a1	5'-CATTACAAATGCGATGCTCT-3'	[38]	50.2
	b1	5'-TCTACCGATTTCGCCATATC-3'	[38]	51.3
15	1F	5'-TGCCTCTTGTCCTATGTCTC-3'	[38]	53.6
	1R	5'-TTTCCAAATGGATAGGATGGGGT-3'	[38]	55.8
16	2F	5'-TGGACTCCGTGGGCACATCAA-3'	[38]	61.9
	2R	5'-ATGCATCCTGTACGGTTGAGACC-3'	[38]	59.4

伊犁郁金香cpDNA-PCR体系构建与遗传多样性分析

Establishment of cpDNA-PCR reaction system and genetic diversity analysis of Tulipa iliensis

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参考文献 50

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参数 Parameter	Taq DNA酶 Taq DNA polymerase/U	镁离子 Mg²⁺/(mmol·L^-1)	脱氧核糖核苷酸 dNTPs/(mmol·L^-1)	引物 Primer/(μmol·L^-1)	模板DNA Template DNA/ng
K₁	35.00	26.00	35.00	35.00	37.00
K₂	36.00	57.00	31.00	36.00	39.00
K₃	50.00	40.00	43.00	43.00	38.00
K₄	43.00	13.00	55.00	50.00	50.00
k₁	8.75	6.50	8.75	8.75	9.25
k₂	9.00	14.25	7.75	9.00	9.75
k₃	12.50	10.00	10.75	10.75	9.50
k₄	10.75	10.25	13.75	12.50	12.50
R	3.75	7.75	6.00	3.75	3.25

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