Research on the identification of passion fruit varieties in Fujian province and construction of fingerprint based on SSR markers
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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）。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.
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Keywords:
- passion fruit /
- SSR molecular marker /
- verification of variety /
- finger print
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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
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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)
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 color1 福建1号
Fujian No. 1福建邵武
Shaowu,Fujian紫色
Purple10 三联黄
San Lian Yellow福建三联
Sanlian,Fujian黄色
Yellow2 福建2号
Fujian No. 2福建赤溪
Chixi,Fujian紫色
Purple11 芭乐味
Guava Flavor福建东兴
Dongxing,Fujian黄色
Yellow3 福建3号
Fujian No. 3福建惠安
Huian,Fujian黄色
Yellow12 金蜜味
Golden Honey Flavor福建象洞
Xiangdong,Fujian黄色
Yellow4 武紫
Wu Zi福建武平
Wuping,Fujian紫色
Purple13 三联芭乐味
San Lian Guava Flavor福建三联
Sanlian,Fujian黄色
Yellow5 武蜜
Wu Mi福建武东
Wudong,Fujian黄色
Yellow14 黄金
Gold福建福州
Fuzhou,Fujian黄色
Yellow6 武砧012
Wu Anvil 012福建武平
Wuping,Fujian黄色
Yellow15 巨无霸
Big Mac福建漳州
Zhangzhou,Fujian黄色
Yellow7 民主紫
Democratic Purple福建民主
Minzhu,Fujian紫色
Purple16 荔枝味
Lychee Flavor福建福州
Fuzhou,Fujian绿色
Green8 赤港紫
Chi Gang Purple福建赤港
Chigang,Fujian紫色
Purple17 满天星
Gypsophila福建福州
Fuzhou,Fujian紫色
Purple9 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/％5 6 7 8 9 10 11～20 ＞20 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
PICPFSSR5 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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