Universality of Simple Sequence Repeat (SSR) Markers from Malvaceae plants to Roselle (Hibiscus sabdariffa L.)
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摘要:
目的 丰富玫瑰茄（Hibiscus sabdariffa L.）SSR分子标记的种类和数量，验证锦葵科作物SSR标记在玫瑰茄中的通用性，进而分析玫瑰茄的遗传多样性。 方法 选取锦葵科红麻、棉花、黄秋葵和木槿等4种作物共62对SSR引物（木槿17对，其他各15对），对8份不同来源的玫瑰茄种质进行多态性分析。 结果 62对SSR引物中有58对具有通用性，占比为93.5%；通用性引物中有48对具有多态性，占比为82.8%；这些引物共扩增出246条条带，其中多态性条带239条，占比97.2%。同时，棉花有14对SSR引物具有多态性，多态性引物比率最高，达86.7%；其次是木槿，有82.4%的引物具有多态性，且其引物多态信息含量值（PIC）最高，为0.685；而黄秋葵和红麻多态性引物比率较低，分别为73.3%和66.7%。另外，聚类分析结果表明，在相似系数 0.49处可将 8 份玫瑰茄种质分为3个各具特点的亚类，其中19FZ-76被单独聚为一类，表明这一种质与其他7份种质亲缘关系最远，遗传差异最大，聚类结果与其表型性状表现一致；而来自缅甸的19FZ-74和漳州本地种遗传相似系数最高，说明这两份种质的亲缘关系较近，遗传差异较小，表明我国福建种植的玫瑰茄品种与东南亚的品种有较近的血缘关系，有可能从东南亚引种而来。 结论 锦葵科作物的SSR引物在玫瑰茄中具有较好的通用性，筛选出的SSR标记可为玫瑰茄品种鉴定、亲缘关系分析、遗传图谱构建等遗传学研究提供可靠方法。 Abstract:Objective Universality of simple sequence repeat (SSR) markers of members of Malvaceae family and roselle herbal plants was examined to extend the genetic information bank. Method A total of 62 pairs of SSR primers were selected from Hibiscus cannabinus, Gossypium spp, Hibiscus esculentus L, and Hibiscus syriacus L. of Malvaceae family. They included 17 pairs from rosemallow and 15 pairs from each of the other crops to compare for polymorphism with those of 8 accessions of Hibiscus sabdariffa L. germplasms. Result Of the 62 SSR primers, 58, or 93.5%, were found common and 48, or 82.8%, polymorphic between the two groups. And out of 246 bands amplified, 239, or 97.2%, were polymorphic. Among the Malvaceae family members, cotton had 14 primer pairs polymorphic at the highest rate of 86.7%, while the rate with rosemallow primers 82.4%, with okra primers 73.3%, and with kenaf primers 66.7%. The PIC of rosemallow primers was the highest at 0.685 among all. A cluster analysis classified the 8 roselle accessions into 3 groups at a similarity coefficient of 0.49. The strain, 19FZ-76, was clustered by itself in the genetic relationship significantly far from other accessions that was reflected by the phenotypic traits as well. The greatest genetic similarity coefficient was observed in the 19FZ-74 cultivars from Myanmar and Zhangzhou, indicating a close genetic relationship of the two accessions and the likely origin of the roselle in Fujian. Conclusion The SSR primers of Malvaceae plants including roselle showed a certain universality. The selected markers could provide a reliable venue for genetic studies, such as variety identification, relationship analysis, and map construction on the herbal plant. -
Key words:
- Roselle /
- SSR markers /
- universality /
- genetic diversity
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图 1 3对不同引物在8份玫瑰茄种质中的扩增结果
M：100 bp ladder marker；1：FAFU1；2：H150；3：19FZ-74；4：白桃K；5：漳州本地种；6：MG5；7：MG6；8：19FZ-76。引物1：HA-1；引物2：HA-2；引物3：HA-3。
Figure 1. Amplified 3 SSR primers for 8 roselle accessions
M: 100 bp ladder marker; 1: FAFU1; 2: H150; 3: 19FZ-74; 4: Baitao K; 5: Zhangzhou native cultivar; 6: MG5; 7: MG6; 8:19FZ-76; Primer 1: HA-1; Primer 2: HA-2; Primer 3: HA-3.
表 1 供试玫瑰茄的名称、来源地及主要性状
Table 1. Names, origins and main phenotypes of roselle germplasms
编号 No. 名称
Name来源地
Origin主要性状
Main phenotypes1 FAFU1 中国福建 紫色肉质花萼，掌状浅裂叶，茎紫红色 2 H150 中国湖南 无肉质花萼，掌状全裂叶，茎紫红色 3 19FZ-74 缅甸仰光 无肉质花萼，掌状深裂叶，茎紫红色 4 白桃K Baitao K 中国广东 绿色肉质花萼，掌状浅裂叶，茎浅绿色 5 漳州本地种
Zhangzhou native cultivar中国福建 紫色肉质花萼，掌状深裂叶，茎紫红色 6 MG5 中国福建 红色肉质花萼，掌状深裂叶，茎紫红色 7 MG6 中国福建 紫色肉质花萼，掌状浅裂叶，茎紫红色 8 19FZ-76 中国福建 紫色肉质花萼，掌状全裂叶，茎紫红色 
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表 2 供试引物名称和序列信息
Table 2. Names and sequence information of SSR primers
来源
Origin名称
Name引物上游序列(5′-3′)
Foward primer sequence (5′-3′)引物下游序列(5′-3′)
Reverse primer sequence(5′-3′)木槿 Rosemallow HA-1 TTGAACATAAACAAGCGG AAAACAAGTTGGGGAGG HA-2 CTGAATGCCAGAATGACT CAGGCGAAAGAGGAAGAT HA-3 ATCATTATCATCTTCGTTTC AAGGGACCAAAGTCTCAA HA-4 CACCAAATCCTGGAGAAG GCAAACGAGAATAATCAAAA HA-5 GCGTGGATGTTCTTTCTT TCGAACCCTATGGATGTA HA-6 GAACAAGCCTGTCACTAA CACAAACCGATTTACGAT HA-7 CAGCAGTTAGAGCAGGAGGT TTCGGACATGAGTATGGGAT HA-8 CACTTCCACGAAGCTCTTAC GGAGATAAACAGAAAAGGGTA HA-9 TATGGGTTTAGTGCCTGTAT TAGGTTGCTTGAATCTTTTC HA-10 CCCAAACCTCTATCATCT ATATCCCTTAGTTCTGCT HA-11 CACCAAATCCTGGAGAAGTA GGCAAACGAGAATAATCAAA HA-12 AAGGAGAAGCCAAGGTGAAA GACAAACCCACATACAGGAA HA-13 ACTTTTATCGTATAGACCAG GAACACCTTTATTTCAGTGT HA-14 GAAATGGCAAGGTTTTAGAT CTCAACTTTTGTGATGTGGC HA-15 CAGCCACAATCCTCCTAACT GAAGGGTAACTTGTTTCACG HDAT-1 CCCTTCAAGTGCTCCTCT TCAATTCACCTTCCGTACCC HDAT-2 TGTCAAGCTGTCAAGGGTGA CCGATCCGTGTTTTTCAAGT 红麻 Kenaf HcES0010 AGCACATTCCCGGTTGCTAA TGCAGCAAAGCAATGTAGGC HcES0011 GCTAAGCTGGGTTTCTGGGA CCCAACAGCTTCTATTCACTGC HcES0012 CCCCCTCCATTTGGGTTTCT AACCCAGCAATCCCTTGGTT 红麻 Kenaf HcES0014 CGTGCATGTCGTGAAATCCG ATTACAAAGTGGGCAGGCGA HcES0015 TGGGAAAGGGATTGGAACCA ACAATTCCGTCACCACCTCC HcES0020 CACGTGTGACCACAGAGCTA AACAAAACATGCAGCTCCCG HcES0041 ATCCAATCCAATCCAAACTCCT TCAGGTATAAGTCGGGGCGT HcES0042 CCCCATTGACTCGAGGCTTT AGCCTTTACAATACTTAGTCCGGT HcES0060 AGGTGTCTGGTGACTGGTCA GTGCAAGAACTGGAGTTGCG HcES0063 AGTTGGGATCTGCTGACACG CTGAACCGAAATCAAGGCACA HcES0064 CCACTAGAAGTTTATCGTTGGCC AGAGTATGTGCACGGGAAGC HcES0070 AGGCTGTTGCTGAGCTGAAT CGCCGTTAGCAATAGCAACC HcES0075 CAGGAAGGAAATGGGCAGGA ATCGACCGAAACTCGAGAGC HcES0085 AGCCATCCCATTGCAAGACA TCACTGCATCACCCTTCCAA HcES0087 CCTCTGATGCATCCGCTTCT ATCTCCAGGGTGTTGCGTTT C2-0004C TTTGTTTTGCGTTCCTTTA TCCGACAATGCCTTACAAG C2-0005B CCCCATTCCTACTCATCC CACAGAAAGGTGCTCATGC CGR5501 TCTCTCTTGCTGGTCACGAA TGCCAAATACCCAAATCCAT CGR5503 GCTGCTTCCATGCCATTATT GGGTCGCTTTGTAAGTGAATG CGR5506 CAGCAACCACAATTCGATCA GAAGTTGCTGTTGGGAAGGA 棉花 Cotton CGR5508 CAACTTTCCGAGCTGGATTC TGATCGAGGAATGAAAGCAA CGR5645 GAGCGGAGAGTCCGGTTT CCCAAACGAATCAAAGATGG DC30003 AGGAGGGAAAGAGTGGTG CCTCCTCACATCCAATCA DC30005 ATGAGAAACGGTGTCGAA TTGACCGAATACTCCCCT DPL0840 GAGTCGTTGCCGCTGTTTA GCTACGACTCGATGTTACGG SHIN0733 GCTTTGCCTTCGGTTCATT GGACTTCGCTTTATGAATGCTT SHIN0745 GCACCGAGTCTCCTATGCTC GGACCCTCAAACTTGTATTACACT CIR102 TAACAACTGGATGAGATGA CATAACTGCAAAGGAGAA DC20067 ATGCAAACCATAAACATCT TGGGTTTGTGTGCTATCT DPL0063 GTTCATACATATGGAGAGGCAGC ATGCTTCTCACATGGCTACTCTTT 黄秋葵 Okra HeSSR002 CGCTGCTCCAGACATAACAA TCTTTCTCTCCGAACCTCCA HeSSR003 GGCGATCAAATTAGAAACCAG GTCACGCTGTAGCTCTCGAA HeSSR009 GAAGAACAAGGCAAGGCAAG AACATCTCCAAGCTCCCTCA HeSSR011 TGCAGGCAAATCAACTGAAAG GTTTCCATTCGACTCGGGTA HeSSR017 ACTGCCACTCAACAGCAATG TGCGACAATATTTCCAGCAG HeSSR020 TTCTCACTGCAGCAACAAGG TGCCACTTCTCTGCCTCTTT HeSSR023 CAAGGAGACAATGCAGGGTT ATTCGATACCCAAAGCTCCC HeSSR030 GAGATCAAGAACGTGTGGCA AATCTTCGTTGGTTTCGGTG HeSSR033 TAAGAGGCGTGAAAGGAAGC AAGCTCAACCTTTACGGCAA HeSSR036 GTGGGTTCAAACTTTGGGTGAAG CAGCAAGGTTATTACACTTTACA HeSSR037 ACGTCATCCTCATTAACGGC TTCCTTTCCGACAATTCGAC HeSSR040 CGTTTGAACGCTACCCCATA CCCGAAACCACCTAACTCAA HeSSR041 GCGAATCTTTTGGTATCGGA ATGGATGATTTGGTGGGATG HeSSR043 GCTGCAGCTAAAACGCTTCT CGTAGTCCATGAGGGCAAAT HeSSR046 GAATGGAAGCCAAAGGATCAT GGAATGAATTTTATGATGATGG
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表 3 不同来源的SSR引物在玫瑰茄中的扩增情况
Table 3. Amplified SSR primers in roselle
标记来源
Origins标记总数
Number of
primers引物通用率
Transferability/
%多态性引物比率
Ratio of polymorphic
primers/%多态性条带比率
Ratio of polymorphic
bands/%有效条带数量范围
Number range of
effective bands平均扩增条带
Average amplified
bands木槿 Rosemallow 17 94.1 82.4 100 3～8 5.5 红麻 Kenaf 15 86.7 66.7 98.0 1～8 4.5 黄秋葵 kra 15 100 73.3 96.0 2～8 5 棉花 Cotton 15 93.3 86.7 93.8 1～9 5 总数 Total 62 93.5 82.8 97.2
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表 4 8份玫瑰茄种质的遗传多样性信息
Table 4. Genetic diversity of 8 roselle accessions
来源
Origin引物编号
Primer code等位基因数
Na有效等位基因数
NeShannon多样性指数
I观测杂合度
Ho期望杂合度
He平均Nei's多样性指数
H多态信息含量
PIC木槿 Rosemallow HA-1 6 4.923 1.684 0.875 0.800 0.797 0.768 HA-2 8 6.737 1.981 0.750 0.850 0.852 0.834 HA-3 3 2.085 0.892 0.750 0.908 0.520 0.464 HA-4 6 4.414 1.635 0.429 0.560 0.773 0.744 HA-5 6 3.657 1.509 0.625 0.825 0.727 0.691 HA-7 6 4.167 1.609 0.571 0.659 0.760 0.730 HA-8 4 1.969 0.951 0.625 0.725 0.492 0.458 HA-9 7 5.333 1.787 0.625 0.775 0.813 0.787 HA-10 6 4.741 1.646 0.625 0.867 0.789 0.757 HA-11 7 4.571 1.721 0.625 0.842 0.781 0.755 HA-12 8 5.818 1.923 0.125 0.525 0.828 0.809 HA-13 3 2.133 0.900 0.500 0.833 0.531 0.468 HA-14 5 3.200 1.332 0.500 0.567 0.688 0.636 HA-15 5 3.657 1.455 0.625 0.883 0.727 0.690 红麻 Kenaf HcES011 4 2.612 1.157 0.375 0.675 0.617 0.572 HcES012 2 1.133 0.234 0.400 0.844 0.117 0.110 HcES014 8 5.565 1.890 0.125 0.125 0.820 0.799 HcES015 6 4.741 1.663 0.625 0.875 0.789 0.759 HcES020 5 2.000 1.037 0.875 0.842 0.500 0.474 HcES041 6 2.370 1.247 0.375 0.533 0.578 0.553 HcES060 7 6.533 1.909 0.375 0.658 0.847 0.828 HcES070 5 2.000 1.037 0.625 0.617 0.500 0.474 HcES075 5 4.000 1.494 0.286 0.912 0.750 0.712 HcES087 3 2.032 0.831 0.500 0.533 0.508 0.428 黄秋葵 Okra HeSSR002 3 2.246 0.882 1.000 0.592 0.555 0.456 HeSSR003 5 3.122 1.354 0.000 0.800 0.680 0.641 HeSSR009 6 2.032 1.124 0.625 0.542 0.508 0.489 HeSSR017 3 1.684 0.736 0.250 0.233 0.406 0.371 HeSSR020 8 5.765 1.909 0.000 0.000 0.827 0.806 HeSSR023 3 1.684 0.736 0.714 0.890 0.406 0.371 HeSSR033 4 2.941 1.221 0.000 0.433 0.660 0.610 HeSSR040 4 3.600 1.330 0.500 0.433 0.722 0.671 HeSSR041 8 7.111 2.014 0.500 0.450 0.859 0.843 HeSSR043 3 1.343 0.509 0.000 0.788 0.255 0.240 HeSSR046 3 2.723 1.043 0.375 0.917 0.633 0.556 棉花 Cotton C20005B 5 4.000 1.494 0.286 0.275 0.750 0.712 CGR5501 6 2.370 1.247 0.375 0.775 0.578 0.553 CGR5503 9 7.000 2.069 0.625 0.617 0.857 0.842 CGR5506 3 1.471 0.602 0.571 0.923 0.320 0.294 CGR5508 2 1.882 0.662 0.125 0.342 0.469 0.359 CGR564 4 2.579 1.091 0.250 0.800 0.612 0.541 SHIN734 5 1.730 0.909 0.750 0.900 0.422 0.404 SHIN745 2 1.280 0.377 0.750 0.800 0.219 0.195 DC20067 7 3.368 1.560 0.200 0.733 0.703 0.678 DC30005 7 6.400 1.895 0.750 0.500 0.844 0.824 DPL0840 3 3.000 1.099 0.625 0.542 0.667 0.593 CIR102 7 4.000 1.661 0.625 0.750 0.750 0.725 C2-0004C 4 2.246 1.041 1.000 0.733 0.555 0.511 总计 Total 245 165.969 62.088 23.707 32.002 30.360 28.583 平均值 Average value 5 3.458 1.293 0.494 0.667 0.633 0.595
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