基于SSR分子标记的福建百香果品种（系）鉴定及指纹图谱构建

魏秀清; 李亮; 熊亚庆; 许玲; 章希娟; 陈志峰; 宋奇洁; 许家辉

doi:10.19303/j.issn.1008-0384.2022.010.007

基于SSR分子标记的福建百香果品种（系）鉴定及指纹图谱构建

doi: 10.19303/j.issn.1008-0384.2022.010.007

福建省农业科学院果树研究所，福建　福州　350013

基金项目: 福建省科技计划公益类专项（2019R1028-9）；福建省种业创新与产业化工程项目（zycxny2021010-1）；福建省农业科学院科技创新团队建设项目（CXTD2021008-2）

详细信息

作者简介:
魏秀清（1982−），女，硕士，副研究员，主要从事果树育种研究（E-mail：18720941@qq.com）

通讯作者:
许家辉(1971−)，男，硕士，研究员，主要从事果树遗传育种及生理生化研究 (E-mail：xjhui577@163.com)

中图分类号: S 667.9
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出版历程
- 收稿日期: 2022-09-11
- 修回日期: 2022-10-08
- 网络出版日期: 2022-11-29
- 刊出日期: 2022-10-30

Research on the identification of passion fruit varieties in Fujian province and construction of fingerprint based on SSR markers

Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China

摘要

摘要: 目的基于SSR分子标记鉴别福建百香果品种的遗传多样性，构建福建百香果品种的分子指纹图谱。方法基于转录组测序，开发西番莲SSR分析标记，并筛选出15个重复性好、多态性高的分子标记用于17个福建百香果品种（系）鉴定及遗传多态性分析，构建百香果品种（系）的SSR分子指纹图谱。结果利用 MISA 软件对1 kb 以上的24319条西番莲（Passiflora caerulea L.） Unigene进行搜索，于 8742条 Unigene 上检测出 11385个 SSR 位点，出现频率为 46.82%，平均分布距离为 7.15 kb。单核苷酸、二核苷酸和三核苷酸分别占总 SSR 的 63.72%、20.40%和14.28%，为数量较大的优势重复基序，其优势重复基元分别为A/T、 AG/CT和AAG/CTT 。通过 Primer 3.0 共获得出 28257 对 SSR 引物。从 26 对有效扩增引物中选择重复性好的15对引物，对 17份百香果品种（系）进行多态性验证分析。15对SSR引物共产生235条多态性条带，PIC平均值为0.95。利用 UPGMA 进行聚类分析并作图，在遗传距离0.765处，可将 17个品种（系）分为 7个类群。根据15对多态性引物扩增电泳图，选出PFSSR20和PFSSR29引物组合用于构建DNA指纹图谱，可鉴别出17个百香果品种（系）。结论利用西番莲转录组数据构建的 SSR分子标记具有较高的多态性潜能，类型丰富，本研究基于此构建的DNA指纹图谱可分辨出17个百香果品种（系）。
- 百香果 /
- SSR分子标记 /
- 品种鉴定 /
- 指纹图谱
Abstract: Objective Based on SSR markers, the genetic diversity of passion fruit varieties in Fujian province were analyzed, and SSR fingerprints of passion fruit varieties were constructed. Method The SSR markers of passion fruit were developed using transcriptome sequencing. 15 SSR markers with good repeatability and high polymorphism were screened for the identification and genetic polymorphism analysis of 17 Fujian passion fruit varieties, and the SSR molecular fingerprint was constructed. Result Using the MISA software, 24319 unigenes above 1 kilo base pairs of passion fruit were analyzed, and a total of 11385 SSRs were identified from 8742 unigenes, with an appearing frequency of 46.82 % and an average distribution distance of 7.15 kb. Mononucleotide, dinucleotide and trinucleotide accounted for 63.72 %, 20.40 % and 14.28 % of the total SSR, respectively. Furthermore, the A/T、 AG/CT and AAG/CTT were the predominant dinucleotide repeat types for mononucleotide, dinucleotide and trinucleotide repeat types, respectively. There were 28257 SSR primers designed using Primer 3.0. 15 pairs of primers with good repeatability were selected from 26 pairs of effective primers to verify the polymorphism of 17 passion fruit cultivars. A total of 235 polymorphic bands were generated by 15 pairs of SSR primers, and the average PIC was 0.95. The 17 cultivars were divided into 7 groups at the genetic distance of 0.765 by UPGMA cluster analysis. According to the 15 pairs of polymorphic primer amplification electrophoresis map, PFSSR20 and PFSSR29 primer combinations were selected to construct DNA fingerprinting, and 17 passion fruit varieties could be identified. Conclusion The SSR molecular markers constructed using passion fruit transcriptome data have high polymorphism potential and rich types. 17 Passiflora varieties (lines) were successfully identified based on the DNA fingerprints constructed in this study.
- passion fruit /
- SSR molecular marker /
- verification of variety /
- finger print

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图 1 17个百香果品种（系）的UPGMA聚类图

品种编号名称见表1，下同。

Figure 1. UPGMA dendrogram of 17 passion fruit varieties (lines)

Code name are shown in table 1, the same as below.

下载: 全尺寸图片幻灯片

图 2 引物PFSSR20和PFSSR29在17个百香果（系）材料中的多态性

Figure 2. Polymorphisms showed in 17 passion fruits by primer PFSSR20 and PFSSR29

下载: 全尺寸图片幻灯片

图 3 17个百香果品种（系）的DNA指纹图谱

Figure 3. DNA fingerprints map of 17 passion fruit varieties (lines)

下载: 全尺寸图片幻灯片

表 1 供试品种（系）材料的基本信息

Table 1. Basic information of the tested varieties (lines)

序号 No.	名称或编号 Name or code	来源 Source	果实颜色 Fruit color	序号 No.	名称或编号 Name or code	来源 Source	果实颜色 Fruit color
1	福建1号 Fujian No. 1	福建邵武 Shaowu,Fujian	紫色 Purple	10	三联黄 San Lian Yellow	福建三联 Sanlian,Fujian	黄色 Yellow
2	福建2号 Fujian No. 2	福建赤溪 Chixi,Fujian	紫色 Purple	11	芭乐味 Guava Flavor	福建东兴 Dongxing,Fujian	黄色 Yellow
3	福建3号 Fujian No. 3	福建惠安 Huian,Fujian	黄色 Yellow	12	金蜜味 Golden Honey Flavor	福建象洞 Xiangdong,Fujian	黄色 Yellow
4	武紫 Wu Zi	福建武平 Wuping,Fujian	紫色 Purple	13	三联芭乐味 San Lian Guava Flavor	福建三联 Sanlian,Fujian	黄色 Yellow
5	武蜜 Wu Mi	福建武东 Wudong,Fujian	黄色 Yellow	14	黄金 Gold	福建福州 Fuzhou,Fujian	黄色 Yellow
6	武砧012 Wu Anvil 012	福建武平 Wuping,Fujian	黄色 Yellow	15	巨无霸 Big Mac	福建漳州 Zhangzhou,Fujian	黄色 Yellow
7	民主紫 Democratic Purple	福建民主 Minzhu,Fujian	紫色 Purple	16	荔枝味 Lychee Flavor	福建福州 Fuzhou,Fujian	绿色 Green
8	赤港紫 Chi Gang Purple	福建赤港 Chigang,Fujian	紫色 Purple	17	满天星 Gypsophila	福建福州 Fuzhou,Fujian	紫色 Purple
9	台砧 Tai Anvil	福建南平 Nanping,Fujian	紫色 Purple

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表 2 西番莲SSR的类型、数量及分布频率

Table 2. Type，number and frequency of SSRs in passion fruit

项目 Item	重复次数 Repeat number								总计 Total	比例 Ratio/％
项目 Item	5	6	7	8	9	10	11～20	＞20	总计 Total	比例 Ratio/％
单核苷酸 Mono	0	0	0	0	0	2927	4196	132	7255	63.72
二核苷酸 Di	0	813	517	386	298	193	115	0	2322	20.40
三核苷酸 Tri	981	413	196	30	3	1	2	0	1626	14.28
四核苷酸 Tetra	95	17	4	1	1	0	0	0	118	1.04
五核苷酸 Penta	29	3	6	1	0	0	0	0	39	0.34
六核苷酸 Hexa	18	5	1	0	0	0	1	0	25	0.22
总计 Total	1123	1251	724	418	302	3121	4314	132	11385	100.00
比例 Ratio/%	9.86	11.00	6.36	3.67	2.65	27.41	37.89	1.16	100.00

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表 3 西番莲转录中不同微卫星重复基序（motif）出现的频率

Table 3. Occurrence frequency of different microsatellites motifs of passion fruit

重复基序类型　　　 Repeat type	重复基序 Repeat motif	数量 Number	比例 Ratio/％
单核苷酸 Mono	A/T	7181	63.07
	C/G	74	0.65
二核苷酸 Di	AC/GT	231	2.03
	AG/CT	1385	12.17
	AT/AT	698	6.13
	CG/CG	8	0.07
三核苷酸 Tri	AAC/GTT	74	0.65
	AAG/CTT	286	2.51
	AAT/ATT	91	0.80
	ACA/TGT	29	0.25
	ACC/GGT	124	1..09
	ACG/CGT	74	0.65
	ACT/AGT	59	0.52
	AGA/TCT	116	1.02
	AGC/GCT	105	0.92
	AGG/CCT	171	1.50
	ATA/TAT	21	0.18
	ATC/GAT	76	0.67
	ATG/CAT	69	0.61
	CAC/GTG	38	0.33
	CCG/CGG	58	0.51
	CGC/GCG	13	0.11
	CTC/GAG	110	0.97
	GAC/GTC	112	0.98
四核苷酸 Tetra	AAAG/CTTT, AAAT/ATTT, AACC, AAGA/TCTT, AATA/TATT, AATG, ACAA/TTGT, ACAG, AGCG, AGGA/TCCT, AGTC/GACT, AGTG, ATAG/CTAT, ATCC, CAAA/TTTG, CAGG, CATC/GATG, CCAT, CCGT, CCTC, CTAC/GTAG, CTGA, CTGG, CTGC, CTGG, GAAG, GAGT, GCAG, GCAT, GCCT, GTAT, GTTA, TAGA, TCAA, TCAT, TCCC, TCGT, TGAG, TGCT, TGGA, TGGC, TTCC	118	1.04
五核苷酸 Penta	AACAG, AAGAA, AAGAG, AAGCT, AATCT, ACAGA, ACAGT, ACCCA, AGAAC, AGACC, AGCCC, ATGCC, CAAAG, CACCA, CCTTC, CTCCT, CTTTC, GAAAA, GAAGT, GAGGA, GGAGG, GGGAT, GGTAT, GTTGA, GTTTC, GTTTG, TCCTT, TGGTG, TGTTC, TTCAC, TTCTC, TTGTA, TTTGT	39	0.34
六核苷酸 Hexa	AAAACA, AACACC, AATAGT, ACCACG, AGAGAC, AGGCTC, CAATCT, CCAGCT, CGCACA, CTGACT, CTGGAA, CTGTTT, GACAGG, GAGACC, GCAGAT, GGTTGC, GTAGTG, GTCCGA, TAGCAG, TCAGTA, TGTTCA, TTCTTG, TTTTCT, TTTTTA	25	0.22

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表 4 15对西番莲SSR引物信息

Table 4. Information of 15 pairs of primers developed from passion fruit

引物编号 Primer No.	引物序列（5′→3′） Primer sequence（5′→3′）	SSR基元 SSR motif	产物长度 Length of product/bp	多态性条带数 Number of polymorphic bands	多态信息量 PIC
PFSSR5	F: AACCCCTTCCTCGTGCTAAT; R: GTCCAGGCCTACAACCCATA	(CAATCT)5	253	9	0.92
PFSSR6	F: GGAAGAGAAGAGCGAGCAGA; R: ACCCAGACGAAACTCCACAG	(CTGACT)5	203	13	0.94
PFSSR9	F: CGGTCGTCGTCTTCTTCTTC; R: CGCACAAGAGTACCAGCAAA	(GTTGA)5	141	18	0.96
PFSSR10	F: GTTTCAGCTCCAGTTCCAGC; R: ATCTTGGTCCCAAACCACAC	(CCAGCT)6	132	16	0.95
PFSSR11	F: CGGCTTTTTGACACTGAACA; R: TGATCCTCCCAATTCTTTGC	(ATGCC)6	146	13	0.94
PFSSR12	F: GTGAGGACAGAGAGCGATCC R: GCACTGATGCAAGGTTCTGA	(GGTAT)5	280	12	0.94
PFSSR14	F: GTCTCGGGTTCGTTCAACAT; R: AAAAAGCAGATGCCAACCAC	(CT)10	126	19	0.96
PFSSR17	F: CAATTGAAGATTCCAGCGGT; R: CGGTCTTCTTTCTCTCGACG	(GAGACC)5	254	8	0.91
PFSSR20	F: TCCAAACAATTCACCATTGC; R: AGTTTGACTGGGGAGCTTCA	(TCCT)7	147	20	0.96
PFSSR21	F: GGTTGCAACGATGGAGTTTT; R: GCTAGGATTTCGATGGGTCA	(CT)10	128	17	0.96
PFSSR26	F: TAAAGGACTCGATGCAGGCT R: AGGAGGCATGGTTTGAATTG	(ATCC)8	146	23	0.96
PFSSR29	F: CTGCAGTTTCTTGAGGAGGG; R: GCTGAGAAGAAGGGTCAACG	(CAAAG)5	220	16	0.95
PFSSR30	F: GCTTAGAACAAGGGCGAGC; R: CCTAGCTTGACAACGCTTCC	(CT)10	258	26	0.97
PFSSR32	F: CTACAGACTCTCCGTTCCCG; R: GCAGACAGGACAATCAAGCA	(ACCACG)5	254	11	0.93
PFSSR43	F: GAGGGACCAATGAAGCAAGA R: CAATCAGGTTCCAGGCTTGT	(TGGC)6	156	14	0.95

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