A METHOD FOR PATERNITY TESTING OF GRASS CARP (CTENOPHARYNGODON IDELLUS) USING MICROHAPLOTYPES
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摘要: 研究选择一种新型分子标记——微单体型用于亲子鉴定, 构建了高效的标记筛选和亲子鉴定流程, 并以草鱼(Ctenopharyngodon idellus) 为例评估了该亲子鉴定方法的效果。结果表明, 利用基因组重测序数据能够准确完成微单体型标记的分型, 效果和适应性明显优于传统的基于群体遗传学推断的分型; 通过信息熵的大小能够高效筛选微单体型标记组合, 3个和5个微单体型标记的亲子鉴定结果与微卫星序列(SSR)鉴定结果的一致性分别达到97.08%和99.42%。研究表明使用微单体型分子标记可以快速而准确地完成鱼类的亲子鉴定工作。Abstract: Paternity testing is a technique of great importance in the genetic breeding of aquatic animals. Currently, the most frequently used type of biomarker in paternity tests is microsatellites (SSRs). However, weaknesses of SSRs lie in the complicated and labor-intensive genotyping process, which leads to low efficiency when such analyses are performed on a large scale. In this study, a new type of molecular biomarker, microhaplotypes (MH), was introduced for paternity testing. For the purpose of marker screening and paternity testing, a more efficient pipeline was constructed and evaluated with data from a grass carp population. The results showed that the genotypes of the MHs can be accurately obtained from genome resequencing data with clearly improved efficiency and compatibility over conventional genotyping methods based on population genetics. It is feasible to screen highly efficient MH combinations using the informative index. The consistency with the paternity test results obtained using SSRs reached 97.08% or 99.42% when 3 or 5 MHs were used, respectively. This research suggests that MHs can be used for the rapid and accurate paternity testing of fishes.
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Keywords:
- Microhaplotype /
- Paternity test /
- Grass carp /
- High-throughput sequencing
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图 1 微单体型获取和亲子鉴定流程
Figure 1. Pipeline for microhaplotype genotyping and paternity testing
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图 3 基于个体序列组装的微单体型分析结果
Figure 3. Analysis of the microhaplotypes assembled using the sequence data of individuals
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图 4 亲子鉴定一致性与MH标记数量(A)和各基因有效标记信息熵之和(B)的关系
Figure 4. Consistency between the SSRs used in the paternity test and MH marker numbers (A) or the sum of the informative indexes of the markers within each gene (B)
表 1 用于亲子鉴定的SSR标记信息
Table 1 Information of the SSRs used for the paternity test
标记ID Marker ID PCR引物PCR primer (5′—3′) 重复片段Repeat motif 信息熵Informative index G5010 CATTTTACTGCTTGCCTCAC AGAAG 2.4464 CCCTTCCTTTCGCATAGA G5011 AAGCCACCAACCTCTACGA TTCTC 2.6464 TAACAGGGATGGGATGAAAT G5012 GATGACATGGGGGTGAGTAA AGAGA 2.7219 CAGAAAGGTAGTAAACAACGAAA G5020 CAACCCTGTTTCTGTCCTGT AAAGG 2.4464 GCAAGCAACTGTCAACCTG G5024 ATTCCTTCCGAAATCAGTG GAGAA 2.1710 AGAGGGAGAAAGATAAGACCA 注: 信息熵计算方法与1.3中一致Note: The informative index is calculated in the same way as 1.3 
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表 2 各基因上的SNP位点与微单体型区域的数量以及亲子鉴定准确率
Table 2 SNP loci and MR in each gene and the accuracy of paternity testing
基因Gene 长度Length (bp) SNP数Number of SNPs MR总数Total number of MRs 有效MR数Effective number of MRs 与用SSR鉴定结果的一致率Consistency with SSR’s results (%) 可鉴定子代占比Ratio of detectable offspring (%) adamts20 51075 188 32 18 99.40 97.08 brca2 16330 167 23 12 98.14 94.15 dlc1 21420 46 9 4 58.08 97.66 gbp 16058 35 6 3 29.46 75.44 lgals9 5112 14 1 0 — — lrp5 18312 89 13 4 38.57 81.87 meis2b 16360 43 9 2 63.75 46.78 mrps23 4348 28 4 1 12.77 54.97 msi2 2859 13 2 0 — — nos2b 11347 48 9 6 91.61 90.64 prtga 25714 158 25 16 99.41 99.42 rpz4 1138 18 1 0 — — snx14 45146 192 38 26 97.14 61.40 thsd4 7511 30 5 0 — — zmym4 12827 59 7 2 65.09 98.83 注: 其中“—”表示由于标记信息不足以完成亲子鉴定Note: “—” indicates the failure to perform the paternity testing due to insufficient information
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表 3 所选用的MR标记与171尾子代个体的亲子鉴定
Table 3 MR markers adopted and paternity test results of 171 offspring
标记数量Number of markers 标记名字
Marker ID预测一致的子代数Number of offspring consistent with prediction 一致率Ratio of consistency (%) 3 prtga_14, gbp_2, brca2_1 166 97.08 4 prtga_14, gbp_2, brca2_1, adamts20_2 169 98.83 5 prtga_14, gbp_2, brca2_1, adamts20_2, snx14_5 170 99.42
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