Parentage assignment of Monopterus albus using multiplex PCR of microsatallites
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摘要: 为助力黄鳝 (Monopterus albus) 良种选育中的亲子鉴定和系谱管理问题，利用黄鳝全基因组预测并筛选获得的16个多态性较高的微卫星标记，建立了2组微卫星多重PCR体系，并成功用于11个家系的亲子鉴定。通过Cervus 3.0软件对132尾黄鳝进行遗传多样性分析，结果显示，16个微卫星标记的平均等位基因数 (Na) 为5.562，平均观测杂合度 (Ho) 和平均期望杂合度 (He) 分别为0.627和0.619，平均多态信息含量 (PIC) 为0.564。亲子鉴定分析结果表明，双亲基因型未知时单亲本的累积排除概率 (CE-1P) 为0.999 999 99，单亲基因型已知时另一亲本的累积排除概率 (CE-2P) 为0.999 999 91，双亲基因型未知时双亲的累积排除概率 (CE-PP) 为0.999 964 76。黄鳝11个家系的模拟鉴定率为99.96%，实际鉴定率为95%。模拟分析不同亲本数的结果显示，实验的16个微卫星标记在双亲性别已知的200对亲本和双亲性别未知的150对亲本的情况下，均可达到95%以上的鉴定率。利用NTSYS软件对11个家系的110尾子代个体进行聚类分析，结果显示除2尾子代外其余108尾子代均可正确聚类，准确率为98.18%。实验构建的2组微卫星多重PCR亲子鉴定技术为黄鳝良种选育及种质资源管理提供了重要技术支持。Abstract: In order to solve the problems in paternity test and genealogical management for improved variety breeding of Monopterus albus, we obtained 16 pairs of microsatellite markers with high polymorphism through whole genome searching of and following confirmation, and established two multiplex PCR sets by using 16 high polymorphic microsatellite markers and successfully applied to the parentage assignment for 11 full-sib M. albus families. The average allele number(Na) of 16 microsatellite was 5.562; the average of abserved heterozygosite(Ho) was 0.627; the average of expected heterozygosite(He) was 0.619; the average of polymorphic information content (PIC) was 0.564. Parentage analysis reveales that the combined exclusion probability of a first parent given only the genotype of the offspring (CE-1P), a second candidate parent given the genotype of a the offspring and one known parent (CE-2P), and combined exclusion probability probability of a parent pair given only the genotype(CE-PP) were 0.999 999 99, 0.999 999 91 and 0.999 964 76, respectively. The simulated identification rate of the 11 M. albus families was 99.96%, and the actual identification rate was 95%. Furthermore, the simulation analysis shows that the identification rate could reach more than 95% in the case of 200 candidate parental individuals with known sexes and 150 candidate parental individuals with unknown sexes. The cluster analysis shows that 108 offspring could be clustered correctly except two, and the accuracy rate was 98.18%. The paternity method of M. albus established in this study provides important technical supports for the future breeding program and management of germplasm resources of M. albus.
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Key words:
- Monopterus albus /
- Parentage assignment /
- Microsatellite /
- Multiplex PCR
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图 3 黄鳝16个微卫星标记基于不同亲本数的模拟分析
Figure 3. Simulation analysis of 16 pairs microsatellite markers of M. albus based on different candidate parent
图 4 黄鳝家系聚类分析图
注：右侧数字代表家系，其中1—07和101—07为聚类错误的子代编号。
Figure 4. Cluster analysis diagram in 110 M. albus
Note: The numbers on the right represent families, in which 1-07 and 101-07 are clustered incorrectly.
图 5 亲子鉴定应用聚类分析
注：“-”前的数字代表家系、后面的数字代表子代标号，M代表家系对应的母本，F代表家系对应的父本，其中10-04为聚类错误的子代编号。
Figure 5. Cluster analysis of parentage test application
Note: The number before "-" represents the family; the number after "-" represents the offspring label; m represents the female parent corresponding to the family; and f represents the male parent corresponding to the family, which 10-04 was clustered incorrectly.
表 1 黄鳝 2 组微卫星多重PCR的引物信息
Table 1. Primer information of two multiplex PCR sets of microsatellites in M. albus
分组
Group位点
Locus重复单元
Reapet motif退火温度
Tm/℃引物序列
Primer sequence荧光标记
Fluorescence label片段长度
Size/bp浓度比例
RatioA Mta027 (GCA)7 52 F: CTGCGGTAACAAGCGTATCA
R: CTGGGGATCCCAGTCAAACTFAM 147~154 3 Mta141 (GT)17 52 F: CAGAGATGGTCGACTGGTGA
R: AGAGCCATGGGAAGCACTTAFAM 185~312 2 Mta031 (ATG)8 52 F: AGAGGCAGCTCATGGACACT
R: TTCATGGCTGAGCTGACTTGFAM 242~265 3 Ma28 (ATC)7 52 F: GGGATTGTCTGGAATGCTGT
R: GTGAACCCTGAACAGACGGTHEX 144~168 3 Ma39 (GAT)8 52 F: AGGTGAAGGGGAACACACTG
R: TTGCCCTGTCCATTTTTCTCHEX 193~224 3 Ma71 (TCA)13 52 F: TCACATTGCCCAGAGAACAG
R: GGCAGCATCAAGAGACCTTCHEX 258~269 2 Ma24 (CTG)9 52 F: TCGTCTGGCTCAGAGGAGAT
4R: AGCTGCAGCAGGAGGAATACTAMRA 125~143 7 Mta026 (CAG)8 52 F5: TGAACATCCCTTCTTCCACC
R: GTCATGGGTTTGTGTTGCTGTAMRA 171~182 4 Ma85 (TGC)9 52 F: GTGGAAGAAGCTGGATGAGC
R: CACATGGGGTGTTCTCACTGTAMRA 208~236 5 Mta013 (AGG)9 52 F: GAGCGTCTTTTCCATCCTTG
R: CCTACTGCTGCTTCTGGTCCROX 131~150 4 Mta025 (GCT)7 52 F: GCCAAGCAAACGTGTGAGTA
R: AACGAGCTGCGTGTTAAGGTROX 172~184 3 Ma78 (TAAGA)5 52 F: CTGACGGTTGTGTGTAACGG
R: CACGTACGCCGGTTAAACTTROX 214~235 3 B Mta033 (TGT)7 52 F: TGGAGGCAGGAGAAGGAGTA
R: TGAAAGACTCCTCGCAACCTFAM 158~170 1 Mta021 (TGT)7 52 F: AGAGTCTTGCTCGGTTTCCA
R: AGAGTCTTGCTCGGTTTCCAFAM 201~210 1 Ma5 (TCTAC)7 52 F: TTCAGGTTCGGGACTTTGAC
R: AGACGGTTGTGTCAGGAAGGFAM 231~256 1 Mta148 (GT)15 52 F: TTTTCCCAGCAGCTGATTTC
R: CATGACAACAGGACGCAAACHEX 158~174 1 
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表 2 16 个微卫星位点的遗传参数
Table 2. Genetic parameters of 16 SSR loci
位点
locus等位基因数
Na观测杂合度
Ho期望杂合度
He多态信息含量
PICNE-1P NE-2P NE-PP 无效等位基因频率
FNullMa5 5 0.758 0.704 0.647 0.723 0.555 0.380 −0.037 6 Ma24 6 0.550 0.546 0.513 0.833 0.662 0.472 −0.018 2 Ma28 9 0.679 0.660 0.613 0.747 0.574 0.384 −0.017 3 Ma39 9 0.771 0.797 0.766 0.570 0.393 0.206 0.004 0 Ma71 5 0.485 0.494 0.444 0.873 0.732 0.579 0.005 4 Ma78 4 0.489 0.490 0.401 0.880 0.780 0.662 −0.001 5 Ma85 8 0.583 0.717 0.676 0.682 0.504 0.308 0.106 6 Mta013 4 0.583 0.647 0.586 0.775 0.615 0.444 0.046 6 Mta021 4 0.667 0.578 0.485 0.830 0.714 0.574 −0.076 9 Mta025 4 0.652 0.588 0.522 0.822 0.676 0.517 −0.055 4 Mta026 4 0.636 0.621 0.545 0.799 0.657 0.498 −0.020 5 Mta027 3 0.432 0.382 0.347 0.928 0.803 0.675 −0.086 5 Mta031 8 0.756 0.746 0.714 0.640 0.457 0.259 −0.019 3 Mta033 4 0.606 0.509 0.430 0.870 0.756 0.625 −0.100 9 Mta141 8 0.735 0.764 0.732 0.620 0.438 0.244 0.018 8 Mta148 5 0.644 0.666 0.603 0.760 0.600 0.430 0.008 3 双亲未知时单个亲本的累积排除率 Combined non-exclusion probability (First parent): 0.013 807 65 已知单亲时另一个亲本的累积排除率 Combined non-exclusion probability (Second parent): 0.000 339 45 双亲未知时父母本组合的累积排除率 Combined non-exclusion probability (Parent pair): 0.000 001 26 注：NE-1P代表双亲基因型未知时，单个位点的单亲排除率；NE-2P表示已知单亲基因型时，另一亲本单个位点的排除率；NE-PP表示双亲基因型未知时，单个位点的双亲排除率。 Note: NE-1P stands for exclusion probability and of the first parent given only the genotype; NE-2P stands for exclusion probability for one candidate parent given the genotype of a known parent of the opposite sex; NE-PP stands for exclusion probability probability of a parent pair given only the genotype.
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表 3 黄鳝子代和亲本群体的遗传多样性
Table 3. Genetic diversities of offsprings and parents of M.albus
群体
Population平均等位基因数
Mean Na平均观测杂合度
Mean Ho平均期望杂合度
Mean He平均多态信息含量
Mean PICOffspring-1 2.750 0.619 0.495 0.408 Offspring-41 2.813 0.688 0.554 0.453 Offspring-68 2.375 0.669 0.475 0.375 Offspring-70 2.063 0.488 0.386 0.301 Offspring-77 2.563 0.719 0.536 0.436 Offspring-79 2.125 0.575 0.430 0.334 Offspring-81 2.563 0.806 0.574 0.457 Offspring-85 2.125 0.494 0.384 0.311 Offspring-88 2.375 0.556 0.466 0.373 Offspring-96 2.438 0.639 0.506 0.411 Offspring-101 3.188 0.700 0.574 0.474 Parents 5.625 0.556 0.636 0.570
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