杨柳科单拷贝核基因引物的开发与利用

doi:10.3969/j.issn.1000-2006.201811015

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南京林业大学学报（自然科学版） ›› 2019, Vol. 43 ›› Issue (5): 44-50.doi: 10.3969/j.issn.1000-2006.201811015

杨柳科单拷贝核基因引物的开发与利用

刘舒宇¹(), 刘霞², 李金花¹^,^*(), 王兆山¹^,^*()

1.中国林业科学研究院林业研究所,北京 100091
2.重庆文理学院特色植物研究院,重庆 402160

收稿日期:2018-11-06 修回日期:2019-05-05 出版日期:2019-10-08 发布日期:2019-10-08
通讯作者: 李金花,王兆山
基金资助:
国家自然科学基金面上项目(31470665)

Development of single copy nuclear gene primers for the family Salicaceae

LIU Shuyu¹(), LIU Xia², LI Jinhua¹^,^*(), WANG Zhaoshan¹^,^*()

1. Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
2. Institute of Special Plants, Chongqing University of Arts and Sciences, Chongqing 402160, China

Received:2018-11-06 Revised:2019-05-05 Online:2019-10-08 Published:2019-10-08
Contact: LI Jinhua,WANG Zhaoshan

摘要/Abstract

摘要：

【目的】由于现有的遗传标记比较单一且变异太少,杨柳科(Salicaceae)属间系统发育关系还存在较大的争议,需要开发在种间变异大且信息量多的标记来准确揭示杨柳科属间系统位置。相比胞质基因组,单拷贝核基因具有双亲遗传、携带大量信息位点和直系同源的特点,可以满足不同系统发育问题及不同分类阶元研究的需要。【方法】根据先前研究中发表的15个杨柳科(杨属、柳属)单拷贝核基因标记以及核糖体DNA的转录间隔区序列(ITS),选取杨柳科4个属的代表物种:箣柊(Scolopia chinensis)、大果刺篱木(Flacourtia ramontchi)、爪哇脚骨脆(Casearia velutina)、天料木(Homalium cochinchinense)进行PCR扩增测序,以测序结果为参考序列,设计合适的引物。利用杨柳科6个不同属的代表物种:山桂花(Bennettiodendron leprosipes)、柞木(Xylosma congesta)、锡兰莓(Dovyalis hebecarpa )、栀子皮(Itoa orientalis)、山桐子(Idesia polycarpa)、山拐枣(Poliothyrsis sinensis)来验证引物的通用性。利用箣柊一个自然居群中的20个个体对新引物进行多态性计算以及中性检验。将单拷贝核基因序列和ITS序列组合成联合片段,分别用最大简约法和贝叶斯法构建系统发育树。【结果】在10个属中成功筛选设计出6对合适的引物,分别为5对单拷贝核基因标记以及ITS遗传标记,各引物扩增出的序列长度为297~716 bp。多态性检测结果表明:单倍型数目(H)为3~9个,平均核苷酸差异数(κ)为0.644~2.278,核苷酸多样性(π)和Watterson核苷酸多样性参数(θ_w)分别为0.001 04~0.004 51和0.001 19~0.004 75,所有位点在箣柊中核苷酸多样性较高。中性检验结果表明所有位点均符合中性进化假设。利用位点联合方法构建的最大简约树和贝叶斯树的拓扑结构基本一致,各分支均有较高的支持率。【结论】筛选出的6对通用性高的引物,丰富了杨柳科的分子标记,可为杨柳科属间系统发育学以及杨柳科植物遗传多样性的研究提供参考。

关键词: 杨柳科, 单拷贝核基因, 多态性, 系统发育关系

Abstract:

【Objective】 The phylogenetic relationships among genera in the Salicaceae family have remained controversial, largely because the available genetic markers lack polymorphisms. More efficient and highly variable genetic markers are needed to address lingering questions about taxonomic relationships within the Salicaceae family. Compared with the cytoplasmic genome, single-copy nuclear genes (SCNGs) are inherited bi-parentally, carry a large number of information sites, and are orthologous, and therefore can better reflect phylogenetic relationships among species and satisfy the requirements of different taxa. 【Method】 Based on the 15 SCNG markers of old Salicaceae members (Populus and Salix) and the internal transcribed spacer (ITS) sequences published in previous studies, PCR amplification and sequencing were carried out in 4 species in the Salicaceae family: Scolopia chinensis, Flacourtia ramontchi, Casearia velutina and Homalium cochinchinense. The primers were designed based on the sequencing results. Then, we used the representative species of 6 different genera of Salicaceae (Bennettiodendron leprosipes, Xylosma congesta, Dovyalis hebecarpa, Itoa orientalis, Idesia polycarpa and Poliothyrsis sinensis) to verify the application of the primers. Polymorphism and neutral tests were performed on the new primers using 20 individuals in a population of S. chinensis. Maximum parsimony and Bayesian inference methods were used to construct the phylogenetic trees by combining data (SCNGs+ITS). 【Result】 Six pairs of primers (5 SCNG markers and ITS) with an aligned length ranging from 297 to 716 bp were successfully identified from 10 genera. The results of polymorphism analysis of the 6 primers showed that the number of haplotypes (H) ranged from 3 to 9, the average nucleotide difference (κ) was 0.644-2.278, the nucleotide diversity (π) and the Watterson’s parameter (θ_w) were 0.001 04-0.004 51 and 0.001 19-0.004 75, respectively. Neutral test results indicated that all loci were consistent with neutral evolutionary assumptions. Two phylogenetic trees showed similar topology and both could be classified well with high support values based on combined data. 【Conclusion】 The six markers developed in this study are anticipated to enrich molecular markers for species in the Salicaceae family, and provide useful information for elucidating the phylogenetic relationships and genetic diversity within Salicaceae.

Key words: Salicaceae, single-copy nuclear gene, polymorphism, phylogenetic relationship

中图分类号:

刘舒宇,刘霞,李金花,等. 杨柳科单拷贝核基因引物的开发与利用[J]. 南京林业大学学报（自然科学版）, 2019, 43(5): 44-50.

LIU Shuyu, LIU Xia, LI Jinhua, WANG Zhaoshan. Development of single copy nuclear gene primers for the family Salicaceae[J].Journal of Nanjing Forestry University (Natural Science Edition), 2019, 43(5): 44-50.DOI: 10.3969/j.issn.1000-2006.201811015.

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sample No. 样本编号	species 物种	collection locality 采集地	geographic coordinates 地理坐标	sample number 个体数
CZ11	Scolopia chinensis箣柊	Baise, Guangxi	106°36'E, 24°51″N	21
DGCLIM2	Flacourtia ramontchi大果刺篱木	Jinhong, Yunnan	101°25'E, 21°41'N	1
ZWJGC1	Casearia velutina瓜哇脚骨脆	Huizhou, Guangdong	114°11″E, 23°48″N	1
TLML1	Homalium cochinchinense天料木	Guangzhou, Guangdong	113°20'E, 23°04'N	1
SGH	Bennettiodendron leprosipes山桂花	Guangzhou, Guangdong	114°35'E, 24°21'N	1
ZM	Xylosma congesta柞木	Anshun, Guizhou	105°92'E, 26°25'N	1
XLM	Dovyalis hebecarpa锡兰莓	Jinhong, Yunnan	101°18'E, 22°01'N	1
ZZP	Itoa orientalis栀子皮	Kunming, Yunnan	114°40'E, 27°36'N	1
STZ	Idesia polycarpa山桐子	Kunming, Yunnan	102°76'E, 25°05'N	1
SGZ	Poliothyrsis sinensis山拐枣	Taian, Shandong	117°15'E, 36°16'N	1
DF	Hydnocarpus hainanensis海南大风子	Jinhong, Yunnan	101°33'E, 22°43'N	1

locus 位点	forward primer sequence (5'-3') 正向引物序列	reverse primer sequence (5'-3') 反向引物序列	PCR product size/bp 产物长度	T_a/℃ 退火温度
DSH1	ATTGAGGCTTTTGTTCAGCGGTTA	CCTGTACTTGTTTGTCTGGCTTTG	550-589	58
DSH2	CATCTTTTGCCTTATTGTCTGCT	TGCGTTAAATGATCTTTCTGGTA	410-451	56
DSH3	TCTGCTTTCCACTTCTTGC	CATACTCTCCCATTGTCCC	654-680	55
DSH4	CCACCGCTACTCCTCCG	TCCACCCCTCCATCCAC	401-465	58
DSH5	TGGCAGAATCACCAGACCCTC	CCAATTTAGCATCTTCAGCCTCAT	583-622	59
DSH6	GCCTCCTGATTATTATGC	TATTACAAGCCCTTCCAG	456-526	54
DSH7	TGTCCACAAACGCATCC	CAAACTTTACCACCCCA	512-574	58
DSH8	GTTTGTTGTTCTGTTGATTGT	GGCTTCTCTTCTCTGATATTT	531-586	56
DSH10	TACAAAAGCATTAAAGATCACCA	GGAGCACACTTATCAATAAAACT	780-842	54
DSH11	GTGGCAAGACCAGCTGCTAGT	ATGGAGGAAGGGTGGACAATG	912-1005	55
DSH12	CACCACATCCCGCTTTCTCTCTTC	TAAACCCCAGGAGGCAAAACAG	496-558	57
DSH14	TGTTTGATGGACCTGGCTGCT	CGGTTTATTGCCTTGTGGAGA	845-921	55
DSH15	CTGAAAGGGAAAATAGTGGACA	GGATAACAGTAGCATGGAGATAT	798-861	56
DSH19	AAGTCTGGTCAAGGCAGTGGTC	TCTGTGCTGTGATGTTTGGGGG	796-845	54
DSH21	CATGCTTATGAAGGTGTGGGCTT	TGCAAACATCTCACTGGTGACTG	701-775	53

species 物种	primer 引物
species 物种	DF-1 (DSH1)	DF-4 (DSH4)	DF-6 (DSH6)	DF-8 (DSH8)	DF-12 (DSH15)	ITS
S. chinensis箣柊	+	+	*	+	*	+
F. ramontchi大果刺篱木	+	+	+	*	+	+
H. cochinchinense天料木	*	*	*	+	+	+
C. velutina瓜哇脚骨脆	+	*	+	+	+	+
B. leprosipes山桂花	+	+	+	+	+	+
X. congesta柞木	+	+	+	+	+	+
D. hebecarpa锡兰莓	+	+	+	+	+	+
Itoa orientalis栀子皮	+	+	+	+	+	+
Idesia polycarpa山桐子	+	+	+	+	+	+
P. sinensis山拐枣	+	+	+	+	+	+

primer name 引物名称	forward primer sequences (5'-3') 正向引物序列	reverse primer sequences (5'-3') 反向引物序列	T_a/℃ 退火温度	PCR product in size/bp 产物长度
DF1	GCCACGCACTGGAAGAGGAAGTC	GAGTGCTTGATCATCGCCTCCAC	55	297
DF4	CCACCACGTCATCACAACACCCT	CCCTCCCCCTCCTCCTCCCAATC	56	457
DF6	GAGGAGGCTGAGGAGAGAAGGAA	TTATCACCTCTTCCTTTTGTTAT	54	394
DF8	TGGAATTGATTTACATGCGTTCT	GCTCCATTTTAGTAGCTAAAAGA	55	525
DF12	TGAGGAAGATATGATGCAGGATT	ATCATCACGCTGAGGCATGTGAA	58	716
ITS	TCGTAACAAGGTTTCCGTAGG	TTCCTTCCGCTTATTGATATGC	57	595

primer name 引物名称	N	S	H	π	θ_w	κ	R_m	D^*	F^*	Tajima D	GenBank accession No. GenBank序列号
DF1	20	6	6	0.004 51	0.004 75	1.338	1	1.191 92	0.914 16	-0.136 45	MH619740-MH619759
DF4	20	8	6	0.004 25	0.004 12	1.942	0	-0.048 25	-0.004 31	0.093 30	MH619760-MH619779
DF6	19	2	3	0.001 64	0.001 21	0.644	0	0.777 10	0.866 32	0.677 15	MH619780-MH619798
DF8	18	9	9	0.004 34	0.004 13	2.278	1	-0.294 08	-0.298 73	-0.168 93	MH619799-MH619816
DF12	18	7	6	0.001 04	0.002 36	0.746	0	-1.191 72	-0.724 97	-1.580 99	MH619817-MH619834
ITS	20	3	6	0.002 06	0.001 19	1.223	1	0.915 72	1.299 31	1.600 27	MH619835-MH619854

杨柳科单拷贝核基因引物的开发与利用

Development of single copy nuclear gene primers for the family Salicaceae

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