MICROSATELLITE MARKERS ANALYSIS OF TWO HYBIRDS OF MEGALOBRAMA TERMINALIS×ERYTHROCULTER ILISHAE- FORMIS, MEGALOBRAMA AMBLYCEPHALA×ERYTHROCULTER ILISHAEFORMIS
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摘要: 为了指导三角鲂(Megalobrama terminalis)、团头鲂(Megalobrama amblycephala) 与翘嘴鲌(Erythroculter ilishaeformis)的杂交育种工作, 利用筛选出的16对微卫星引物, 比较分析了团头鲂、三角鲂、翘嘴鲌、团头鲂♀×翘嘴鲌♂、三角鲂♀×翘嘴鲌♂后代群体的遗传结构; 结果显示, 平均等位基因数(Na)分别为3.56、3.63、3.44、4.00和4.31, 平均观测杂合度(Ho)分别为0.3510、0.3757、0.3175、0.3818和0.4079, 平均期望杂合度(He)分别为0.6182、0.6290、0.5921、0.6490和0.6825, 平均多态信息含量(PIC)分别为0.5354、0.5367、0.5258、0.5785和0.6067。杂交群体的平均多态信息含量均大于他们的亲本团头鲂、三角鲂和翘嘴鲌, 表明杂交亲群体的遗传多样性较高。聚类分析显示团头鲂与三角鲂首先聚类, 团头鲂♀×翘嘴鲌♂与三角鲂♀×翘嘴鲌♂首先聚类, 然后这2大类聚为一支, 最后与翘嘴鲌聚类。其中团头鲂与翘嘴鲌遗传距离最远, 为0.5204, 团头鲂和三角鲂遗传距离最近, 为0.0853, 结合遗传相似度分析表明2种杂交子代均具有母本效应。基因型分析表明, 2种杂交后代的等位基因均来自于父母本。引物TTF3、TTF4、TTF10以及Mam25在5个群体中均可产生特异性条带, 可区分5个群体。研究结果对三角鲂×翘嘴鲌和团头鲂×翘嘴鲌的良种选育、种质资源保存以及种群鉴定具有重要意义。Abstract: To guide breeding of Megalobrama terminalis, Megalobrama amblycephala and Erythroculter ilishaeformis, two hybrids Megalobrama amblycephala×Erythroculter ilishaeformis, Megalobrama terminalis×Erythroculter ilishaeformisand their parents were analyzed using sixteen microsatellite markers. The mean value of number of alleles (Na) were 3.56, 3.63, 3.44, 4.00 and 4.31, respectively. The mean value of observed heterozygosity (Ho) were 0.3510, 0.3757, 0.3175, 0.3818, and 0.4079, respectively. The mean value of expected heterozygosity (He) were 0.6182, 0.6290, 0.5921, 0.6490 and 0.6825, respectively. And the average value of polymorphism information content (PIC) were 0.5354, 0.5367, 0.5258, 0.5785, and 0.6067, respectively. The average value of polymorphism information content (PIC) of two hybrid population were higher than their parent Megalobrama amblycephala, Megalobrama terminalis and Erythroculter ilishaeformis, indicating a high genetic diversity. The unweighted pair-group method with arithmetic means (UPGMA) showed that populations of the Megalobrama amblycephalaand Megalobrama terminalisfirst grouped together, and that Megalobrama amblycephala♀×Erythroculter ilishaeformis♂ andMegalobrama terminalis♀×Erythroculter ilishaeformis♂ first grouped together, and then they clustered with the Erythroculter ilishaeformis. In addition, the farthest genetic distance (0.5204) was between Megalobrama amblycephala and Erythroculter ilishaeformis, and the nearest genetic distance (0.0853) was between Megalobrama amblycephalaand Megalobrama terminalis. According to the genetic identity analysis, two hybirds groups showed the maternal effect. The alleles of two hybirds were from their parents based on genetic analysis. Particularly, microsatellite primers TTF3, TTF4, TTF10, and Mam25 identified 5 groups. Our results provide important value in breeding, germplasm resources preservation, and germplasm identification of Megalobrama terminalis×Erythroculter ilishaeformis andMegalobrama amblycephala×Erythroculter ilishaeformis.
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图 1 TTF3在5个群体中的PAGE图谱
M. PBR322 DNA/MspΙ; 1－4. 团头鲂; 5－8. 三角鲂; 9－12. 翘嘴鲌; 13－16. 团头鲂♀×翘嘴鲌♂; 17－20. 三角鲂♀×翘嘴鲌♂; 下同
Figure 1. PAGE analysis by primers TTF3 in 5groups
M. PBR322 DNA/MspΙ; 1－4. Megalobrama amblycephala; 5—8. Megalobrama terminalis; 9—12. Erythroculter ilishaeformis; 13—16. Megalobrama amblycephala♀×Erythroculter ilishaeformis♂; 17—20. Megalobrama terminalis ♀×Erythroculter ilishaeformis♂; the same applies below
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图 3 不同群体的UPGMA聚类图
Figure 3. Dendrogram of among various populations using UPGMA clustering method
表 1 微卫星引物特征
Table 1 Characteristics of microsatellite primers
位点
Locus重复序列
Repeat motif引物序列
Primer sequence (5′－3′)退火温度
Tm (℃)等位基因大小范围
Size range (bp)TTF01 (CA)21 TGGAGATGAAAGCTGAAGGAA
ATGCACGAACTGCCACATAA55.9 252—328 TTF02 (CA)5(CT)21 AAACAGCTGCTACCCTTGGA
TTTGCCAGAAGAGCAAATCA55.9 196—228 TTF03 (TC)27 AAGACGCCACGGAAACTTTA
CTGACCGGATAGCAAAGTGA56.4 214—266 TTF04 (CA)14 GACTGGAGTCGTCAGGCTTC
TGCCCCACATTGTTAGACTG60.5 182—222 TTF05 (CA)15 CTAGTGGGTAGGTGGCAGGT
TGACTGGGAGAGACAGAGGAG60.5 164—188 TTF06 (GA)13 GGCAGGTCAGGCACATTTAT
TCTCTACCTCACATTCTCTCATTCT60.5 186—218 TTF07 (GT)13 ATGGGTAAGCCGATGGATTC
GTGTCAGCATTCCAGCTCCT60.5 285—331 TTF08 (GT)18 GGGGAAATAAAGGGAGAAAGTG
TTTCTCCTGATCCGTTGACC60.5 178—224 TTF09 (TC)19 AAGACGCCACGGAAACTTTA
GAGGTGGGACTGTGTGGAAT56.9 269—319 TTF10 (TC)6(TG)5 FAAACAGGCTCGCCAATTTC
TCACCCACACACTCTTATTCTCTC55.9 255—291 EST13 (AC)14 TCTTTCACAAACAAACCCTT
GGATTATCAAACGCGGACT57 209—266 EST23 (AC)12 GCGATCATCAAGGCAACG
AGATTCATCAGCTCCTGTAGTGT55 265—352 EST37 (TG)8 CACAAACCATAAACACAG
AATGCCCATAAAACACAC62 147—216 EST66 (GT)20 TCAATCAGGCATAAACAT
AACTAACTAGCACGCAAA60 290—349 Mam03 (CA)18 TTGCAGGTACTGTGGGAAAA
AGCAACATGCAAACATCAAA60 231—324 Mam25 (AC)5(AC)14 TCACACCAACAACACCGAAT
CCTTGTTTTCTCCAGGCATC62 166—252 注: TTF1—TTF10、Mam03—Mam25和EST13—66分别来源于文献[11]、[12]、[13]Note: TTF1—TTF10, Mam03—Mam25 and EST13—EST66 are microsatellite primers and are respectively from reference [11], [12] and [13] 
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表 2 五个群体的微卫星位点的等位基因数、多态信息含量
Table 2 Number of alleles (Na) and the polymorphism information content (PIC) of microsatellite loci
位点
Locus等位基因 Na 多态信息含量 PIC A B C D E A B C D E TTF01 4.00 3.00 3.00 5.00 5.00 0.5717 0.5269 0.5269 0.7056 0.7101 TTF02 3.00 4.00 4.00 4.00 5.00 0.4822 0.6075 0.5973 0.6594 0.5714 TTF03 4.00 5.00 3.00 3.00 4.00 0.6387 0.5544 0.5424 0.5713 0.5786 TTF04 4.00 5.00 5.00 5.00 5.00 0.4848 0.7185 0.7220 0.7154 0.6357 TTF05 3.00 4.00 4.00 5.00 4.00 0.4918 0.5637 0.5739 0.6345 0.6799 TTF06 3.00 4.00 3.00 4.00 4.00 0.5599 0.4213 0.4414 0.4848 0.6528 TTF07 3.00 5.00 4.00 4.00 5.00 0.4781 0.6216 0.5039 0.5048 0.5954 TTF08 3.00 1.00 1.00 4.00 5.00 0.4414 0.3750 0.4211 0.4021 0.6765 TTF09 4.00 3.00 3.00 4.00 4.00 0.6038 0.4918 0.4600 0.5882 0.6569 TTF10 3.00 3.00 5.00 4.00 5.00 0.4918 0.4778 0.6254 0.5958 0.6816 EST13 4.00 4.00 4.00 4.00 4.00 0.5747 0.6819 0.6707 0.5945 0.5809 EST23 3.00 5.00 4.00 4.00 3.00 0.5150 0.7185 0.5418 0.5424 0.6207 EST37 5.00 3.00 3.00 4.00 4.00 0.5580 0.5478 0.4414 0.5973 0.5718 EST66 5.00 3.00 3.00 5.00 5.00 0.7427 0.3976 0.4202 0.6454 0.6989 Mam03 3.00 3.00 3.00 3.00 4.00 0.4211 0.4415 0.4414 0.5052 0.4211 Mam25 3.00 3.00 4.00 2.00 3.00 0.5111 0.4414 0.4842 0.5098 0.3750 平均 3.56 3.63 3.44 4.00 4.31 0.5354 0.5367 0.5258 0.5785 0.6067
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表 3 五个群体的微卫星位点的观测杂合度、期望杂合度
Table 3 Observed heterozygosity (Ho) and expected heterozygosity (He) of microsatellite loci
位点
Locus观测杂合度Ho 期望杂合度He A B C D E A B C D E TTF01 0.2384 0.3503 0.2328 0.3847 0.3847 0.6153 0.6497 0.6153 0.7616 0.7672 TTF02 0.2740 0.4164 0.3463 0.3294 0.3153 0.6706 0.5836 0.6847 0.7260 0.6537 TTF03 0.3463 0.2989 0.3362 0.3734 0.3638 0.6266 0.7011 0.6362 0.6537 0.6638 TTF04 0.2294 0.4141 0.2972 0.2249 0.2266 0.7751 0.5859 0.7734 0.7706 0.7028 TTF05 0.2972 0.4102 0.2655 0.3486 0.3734 0.6514 0.5898 0.6266 0.7028 0.7345 TTF06 0.4141 0.3576 0.2842 0.4458 0.4610 0.5542 0.6424 0.5390 0.5859 0.7158 TTF07 0.4435 0.4186 0.3294 0.4000 0.3090 0.6000 0.5814 0.6910 0.5565 0.6706 TTF08 0.5175 0.4458 0.2616 0.4599 1.0000 0.5401 0.5542 0.0000 0.4825 0.7384 TTF09 0.3441 0.3181 0.2780 0.4328 0.4102 0.5672 0.6819 0.5898 0.6559 0.7220 TTF10 0.3429 0.4102 0.2565 0.4328 0.4328 0.6944 0.5898 0.5672 0.6571 0.7435 EST13 0.3435 0.3469 0.3407 0.2661 0.2559 0.7339 0.6531 0.7441 0.6565 0.6593 EST23 0.3734 0.3949 0.3023 0.3695 0.2277 0.6305 0.6051 0.7723 0.6266 0.6977 EST33 0.3379 0.3633 0.3503 0.4458 0.3695 0.5542 0.6367 0.6305 0.6621 0.6497 EST66 0.2949 0.2090 0.2480 0.4616 0.5045 0.5384 0.7910 0.4955 0.7051 0.7520 Mam03 0.4006 0.4599 0.4599 0.4458 0.4458 0.5542 0.5401 0.5542 0.5994 0.5401 Mam25 0.4175 0.3972 0.4915 0.4153 0.4458 0.5847 0.6028 0.5542 0.5825 0.5085 平均 0.3510 0.3757 0.3175 0.3818 0.4079 0.6182 0.6290 0.5921 0.6490 0.6825 注: A. 团头鲂; B.三角鲂; C. 翘嘴鲌; D. 团头鲂♀×翘嘴鲌♂; E. 三角鲂♀×翘嘴鲌♂Note: A. Megalobrama amblycephala; B. Megalobrama terminalis; C. Erythroculter ilishaeformis; D. Megalobrama amblycephala♀×Erythroculter ilishaeformis♂; E. Megalobrama terminalis♀×Erythroculter ilishaeformis♂
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表 4 不同群体的Nei’s遗传相似性(对角线上方)和遗传距离(对角线下方)
Table 4 Nei’s genetic identity (above diagonal) and genetic distance (below diagonal)
群体Population A B C D E A **** 0.9183 0.4207 0.7653 0.6661 B 0.0853 **** 0.5984 0.7117 0.7224 C 0.5204 0.5135 **** 0.6566 0.6213 D 0.3252 0.3401 0.4063 **** 0.8784 E 0.3266 0.2676 0.4207 0.1296 **** 注: A. 团头鲂; B. 三角鲂; C. 翘嘴鲌; D. 团头鲂♀×翘嘴鲌♂; E. 三角鲂♀×翘嘴鲌♂Note: A. Megalobrama amblycephala; B. Megalobrama terminalis; C. Erythroculter ilishaeformis; D. Megalobrama amblycephala♀×Erythroculter ilishaeformis♂; E. Megalobrama terminalis♀×Erythroculter ilishaeformis♂
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