野生玫瑰多态性cpDNA和ITS引物的筛选与验证

doi:10.3969/j.issn.1004-1524.2022.05.18

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (5): 1032-1038.DOI: 10.3969/j.issn.1004-1524.2022.05.18

野生玫瑰多态性cpDNA和ITS引物的筛选与验证

许建军(), 马燕, 吴其超, 王宝盛, 臧德奎()

黄河下游国家林业局林业重点实验室,山东农业大学林学院,山东泰安 271018

收稿日期:2021-03-23 出版日期:2022-05-25 发布日期:2022-06-06
通讯作者: 臧德奎
作者简介:* 臧德奎,E-mail: zangdk@sdau.edu.cn
许建军(1995—),男,山东临沂人,硕士研究生,研究方向为特色园林植物种质资源。E-mail: 1049057931@qq.com
基金资助:
国家自然科学基金(31870688);山东省农业良种工程(2019LZGC01802)

Screening and analysis of genome sequence difference in wild rose

XU Jianjun(), MA Yan, WU Qichao, WANG Baosheng, ZANG Dekui()

Key Laboratory of State Forestry Administration for Silviculture of the Lower Yellow River,Forestry College, Shandong Agricultural University, Tai’an 271018, Shandong, China

Received:2021-03-23 Online:2022-05-25 Published:2022-06-06
Contact: ZANG Dekui

摘要/Abstract

摘要：

为了筛选出具有高多态性的cpDNA 和ITS序列引物,用于研究野生玫瑰自然种群的分子谱系地理,以野生玫瑰8个种群为试材,通过12对叶绿体基因组引物和4对核基因组ITS引物的PCR扩增和PCR产物测序,筛选出trnL-trnF、rpl20-rps12、rbcl、ITS1四对具有高多态性的引物。序列变异计算与系统发育树结果显示,8个野生玫瑰种群变异较低,可划分为2支。筛选出高多态性的cpDNA引物3对和ITS引物1对,适用于研究野生玫瑰系统发育和谱系地理结构,能够进一步探究濒危物种野生玫瑰的谱系地理。

关键词: 野生玫瑰, ITS序列, 引物筛选, 系统发育树

Abstract:

In order to screen out highly polymorphic cpDNA and ITS sequence primers for studying the molecular pedigree of natural populations of wild rose, 8 wild rose populations were used as test materials, 12 pairs of chloroplast genome primers and 4 pairs of nuclear genome ITS primers were adopted. PCR amplification and PCR product sequencing, four pairs of high polymorphic primers trnL-trnF, rpl20-rps12, rbcl, and ITS1 were screened out. Sequence variation calculation and phylogenetic tree results showed that 8 wild rose populations had low variation and could be divided into 2 branches. Three pairs of highly polymorphic cpDNA primers and 1 pair of ITS primers were screened, which were suitable for studying the phylogeny and pedigree of wild rose, and could further explore the pedigree of endangered species of wild rose.

Key words: wild rose, ITS sequence, sequence screening, phylogenetic tree

中图分类号:

S685.12

许建军, 马燕, 吴其超, 王宝盛, 臧德奎. 野生玫瑰多态性cpDNA和ITS引物的筛选与验证[J]. 浙江农业学报, 2022, 34(5): 1032-1038.

XU Jianjun, MA Yan, WU Qichao, WANG Baosheng, ZANG Dekui. Screening and analysis of genome sequence difference in wild rose[J]. Acta Agriculturae Zhejiangensis, 2022, 34(5): 1032-1038.

图/表 6

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代码 Code	采样地点 Locality	经度 Longitude(E)	纬度 Latitude(N)	数量 Quantity
GEF	山东省烟台市大窑镇Dayao Town, Yantai City, Shandong Province	121.406 0	37.273 2	25
JGC	山东省烟台市酒馆村Tavern Village, Yantai City, Shandong Province	121.522 0	37.277 0	10
CSZ	山东省威海市成山镇Chengshan Town, Weihai City, Shandong Province	122.303 9	37.241 0	25
DWGC	山东省威海市大伟广场Dawei Square, Weihai City, Shandong Province	120.005 8	37.501 2	12
HYK	辽宁省庄河市明阳镇Mingyang Town, Zhuanghe City, Liaoning Province	122.408 4	39.311 4	20
SCD	辽宁省庄河市石城岛Shicheng Island, Zhuanghe City, Liaoning Province	121.563 9	40.060 2	20
JSP	吉林省珲春市九沙坪村Jiushaping Village, Hunchun City, Jilin Province	130.320 8	42.365 7	25
SQGY	吉林省珲春市沙丘公园Shaqiu Park, Hunchun City, Jilin Province	130.345 4	42.275 1	20

代码 Code	采样地点 Locality	经度 Longitude(E)	纬度 Latitude(N)	数量 Quantity
GEF	山东省烟台市大窑镇Dayao Town, Yantai City, Shandong Province	121.406 0	37.273 2	25
JGC	山东省烟台市酒馆村Tavern Village, Yantai City, Shandong Province	121.522 0	37.277 0	10
CSZ	山东省威海市成山镇Chengshan Town, Weihai City, Shandong Province	122.303 9	37.241 0	25
DWGC	山东省威海市大伟广场Dawei Square, Weihai City, Shandong Province	120.005 8	37.501 2	12
HYK	辽宁省庄河市明阳镇Mingyang Town, Zhuanghe City, Liaoning Province	122.408 4	39.311 4	20
SCD	辽宁省庄河市石城岛Shicheng Island, Zhuanghe City, Liaoning Province	121.563 9	40.060 2	20
JSP	吉林省珲春市九沙坪村Jiushaping Village, Hunchun City, Jilin Province	130.320 8	42.365 7	25
SQGY	吉林省珲春市沙丘公园Shaqiu Park, Hunchun City, Jilin Province	130.345 4	42.275 1	20

引物名称 Primers	引物序列 Primer sequences(5'-3')	退火温度 Temperature/ ℃	参考文献 References
psbA-trnH	F:GTTATGCATGAACGTAATGCTC	52	[17]
	R:CGCGCATGGTGGATTCACAATCC
trnL-trnF	F:GGTTCAAGTCCCTCTATCCC	55	[18]
	R:ATTTGAACTGGTGACACGAG
trnS-trnG	F:AGATAGGGATTCGAACCCTCGGT	52	[17]
	R:GTAGCGGGAATCGAACCCGCATC
accD-psal	F:TTATTCGATCCAATCGTACCAC	58	[17]
	R:AGAAGCCATTGCAATTGCCGGAAA
ndhF-rpl32	F:CCAATATCCCTTYYTTTTCCAA	52	[17]
	R:GAAAGGTATKATCCAYGMATATT
rpl20-rps12	F:TTT GTTCTA CGTCTCCGA GC	53	[16]
	R:GTCGAGGAACATGTACTA GG
rpl16	F:GCTATGCTTAGTGTGTGACTCGTTG	56	[17]
	R:CCCTTCATTCTTCCTCTATGTTG
atpF-atpH	F:ACTCGCACACACTCCCTTTCC	59	[16]
	R:GCTTTTATGGAAGCTTTAACAAT
rbcl	F:ATGTCACCACAAACAGAAAC	52	[17]
	R:TCGCATGTACCTGCAGTAGC
psbJ-petA	F:ATAGGTACTGTARCYGGTATT	52	[17]
	R:AACARTTYGARAAGGTTCAATT
psbD-trnT	F:CTCCGTARCCAGTCATCCATA	52	[17]
	R:CCCTTTTAACTCAGTGGTAG
trnV-trnW	F:ACT GCC TTG ATC CAC TTG GC	51	[9]
	R:CGA AGCTCCATCTACAAA TGG
ITS5	F:GGAAGTAAAAGCGTAACAAGC	56	[19]
ITS4	R:TCCTCCGCTATATGATATGC
ITSA	F:T CCTCCGCTTATTGATATGC	53	[19]
	R:GGAAGGAGAAGTCGTAACAAGG
ITS2	F:CGAGTCTTTGAACGCAAGTTGC	56	[19]
	R:TTAAATTCCAGCGGGTAACCCCGC
ITS1	F:GTAGGTGAACCTGCGGAAGGAT	55	[19]
	R:GCCGAGATATCCGTTGCCG

引物名称 Primers	引物序列 Primer sequences(5'-3')	退火温度 Temperature/ ℃	参考文献 References
psbA-trnH	F:GTTATGCATGAACGTAATGCTC	52	[17]
	R:CGCGCATGGTGGATTCACAATCC
trnL-trnF	F:GGTTCAAGTCCCTCTATCCC	55	[18]
	R:ATTTGAACTGGTGACACGAG
trnS-trnG	F:AGATAGGGATTCGAACCCTCGGT	52	[17]
	R:GTAGCGGGAATCGAACCCGCATC
accD-psal	F:TTATTCGATCCAATCGTACCAC	58	[17]
	R:AGAAGCCATTGCAATTGCCGGAAA
ndhF-rpl32	F:CCAATATCCCTTYYTTTTCCAA	52	[17]
	R:GAAAGGTATKATCCAYGMATATT
rpl20-rps12	F:TTT GTTCTA CGTCTCCGA GC	53	[16]
	R:GTCGAGGAACATGTACTA GG
rpl16	F:GCTATGCTTAGTGTGTGACTCGTTG	56	[17]
	R:CCCTTCATTCTTCCTCTATGTTG
atpF-atpH	F:ACTCGCACACACTCCCTTTCC	59	[16]
	R:GCTTTTATGGAAGCTTTAACAAT
rbcl	F:ATGTCACCACAAACAGAAAC	52	[17]
	R:TCGCATGTACCTGCAGTAGC
psbJ-petA	F:ATAGGTACTGTARCYGGTATT	52	[17]
	R:AACARTTYGARAAGGTTCAATT
psbD-trnT	F:CTCCGTARCCAGTCATCCATA	52	[17]
	R:CCCTTTTAACTCAGTGGTAG
trnV-trnW	F:ACT GCC TTG ATC CAC TTG GC	51	[9]
	R:CGA AGCTCCATCTACAAA TGG
ITS5	F:GGAAGTAAAAGCGTAACAAGC	56	[19]
ITS4	R:TCCTCCGCTATATGATATGC
ITSA	F:T CCTCCGCTTATTGATATGC	53	[19]
	R:GGAAGGAGAAGTCGTAACAAGG
ITS2	F:CGAGTCTTTGAACGCAAGTTGC	56	[19]
	R:TTAAATTCCAGCGGGTAACCCCGC
ITS1	F:GTAGGTGAACCTGCGGAAGGAT	55	[19]
	R:GCCGAGATATCCGTTGCCG

种群名称 Name of population	引物及变异位点碱基 Primers and bases at variant sites
种群名称 Name of population	trnL-trnF				rpl20-rps12				rbcl				ITS1
GEF	T	C	A	-	-	C	T	-	T	A	C	T	C
JGC	A	-	A	-	-	C	C	-	T	A	C	T	-
CSZ	A	-	A	-	T	T	T	C	T	A	C	T	-
DWGC	T	C	A	C	-	T	T	-	T	A	C	T	C
HYK	A	-	A	-	-	T	T	-	T	A	C	T	-
SCD	A	-	C	-	-	T	T	-	G	G	T	A	-
JSP	A	-	C	-	-	T	T	-	T	G	T	T	-
SQGY	A	-	A	-	-	T	T	-	T	A	C	T	-
比对长度	1 795												285
Comparative length/bp

野生玫瑰多态性cpDNA和ITS引物的筛选与验证

Screening and analysis of genome sequence difference in wild rose

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