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

doi:10.3969/j.issn.1004-1524.20240378

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (1): 78-89.DOI: 10.3969/j.issn.1004-1524.20240378

• Horticultural Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S682.2.63

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.

Figures/Tables 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

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

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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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