基于IRAP技术的‘磨盘柿’变异单株鉴定

1、基于IRAP技术的磨盘柿变异单株鉴定论文导读:：君迁子作为与柿的亲缘关系较近。许多主栽柿品种其中51.2263条具多态性。19个引物中，11个无多态性，其它引物多态性介于50.0090.00。利用此11个多态引物可将20份试材全部区分。19个逆转座子引物的详细扩增情况见表3。供试12份磨盘柿中，19个引物共扩增109条带，多态性带21条，多态比例19.27。其中，4个引物rtdk4-p3亲缘关系，rtdk2-p6，rtdk13-f5和rtdk16-r10具多态性。利用多态引物能很好地对12份磨盘柿基因型鉴别区分表4站大全。图1箭头所指为可用于鉴别的特异于某些基因型的谱带。2.2 相似指数及聚类

2、分析表3 IRAP引物扩增信息Table 3 Summary of PCR amplification corresponding toindividual primer in IRAP analysis 引物 Primer 总带数 Total number of bands 多态性带数 The number of polymorphic bands 多态性带比例 The percent of polymorphic bands rtdk14-f1 5 0 0% rtdk12-f2 10 9 90% rtdk20-f3 3 0 0% rtdk13-f5 9 7 77.78% rtdk15-f6

3、 5 0 0% rtdk7-f7 4 0 0% rtdk16-f8 5 0 0% rtdk14-r1 7 0 0% rtdk13-r5 3 0 0% rtdk13-r6 8 6 75.00% rtdk16-r9 6 4 66.67% rtdk16-r10 8 5 62.50% rtdk11-p1 6 5 83.33% rtdk4-p3 7 5 71.43% rtdk10-p4 2 0 0% rtdk5-p5 6 3 50.00% rtdk2-p6 10 6 60.00% rtdk5-p7 11 8 72.73% rtdk17-p8 8 5 62.50% Total 123 63 51.22%

4、Average 7 4 图1 4个引物在12份磨盘柿基因型中的IRAP扩增电泳图谱A: rtdk4-p3; B: rtdk2-p6; C: rtdk13-f5; D: rtdk16-r10M：DL2000marker; 1. BZMP；212. MP2MP12。黑色箭头表示特异于某一基因型的条带Fig.1 IRAP profiles of four different retrotransposonprimers on 12 Mopanshi genotypesA: rtdk4-p3; B: rtdk2-p6; C: rtdk13-f5; D: rtdk16-r10M: DL2000marke

5、r. 1. BZMP; 2-12. MP2-MP12. Black arrowsindicated special bands in a certain genotypes表4 可用于鉴别12份磨盘柿基因型的有效IRAP标记Table 4 Useful inter-retrotransposon amplified polymorphic (IRAP) markers, inbasepairs (bp), for the identification of 12 Mopanshi genotypes 引物 Primer 样品 Materials 可识别基因型Identifiable genot

6、ypes BZMP MP2 MP3 MP4 MP5 MP6 MP7 MP8 MP9 MP10 MP11 MP12 rtdk4-P3 875 A A A A A A A A A A A BZMP, MP3, MP9, MP10 580 580 580 580 580 580 580 580 A 580 580 750 A 750 750 750 750 750 750 750 750 A 720 rtdk2-P6 720 A 720 720 A 720 720 720 MP4, MP7 A 440 rtdk13-f5 750 A 750 A A A MP2, MP6, MP8, MP11 A 6

7、30 A A 875 A 740 A 740 rtdk16-10 375 A MP5, MP12 注：A：供试基因型中缺失的标记Note: A: marker absent in tested genotypes图2 基于IRAP分子标记的20份磨盘柿基因型UPGMA聚类图Fig.2 Dendrogram of 20 genotypes revealed by UPGMAcluster analysis based on IRAP molecular data由UPGMA 聚类所得的树状图图2可见，在相似指数0.92水平上亲缘关系，12份磨盘柿基因型紧密相聚，之后与同为中国原产的完全甜柿品种罗

8、田甜柿相聚形成Cluster1。供试日本原产柿品种相聚为Cluster2，且次郎与其芽变前川次郎直接相聚，然后与其子代阳丰聚为一支；富有与其芽变松本早生先相聚，再与松本早生芽变上西早生聚为一支。君迁子作为外类群，与柿Cluster1、Cluster2明显分开，形成单独一支。3 讨论据北京市房山区林业局在成熟期、果实形态、耐贮性及植株抗性等方面的调查结果初步说明，供试变异单株与磨盘柿有别亲缘关系，且性状表现根本稳定数据未发表站大全。本试验通过逆转座子分子标记技术，检测到它们与标准品种间存在不同程度的遗传差异，说明其遗传物质已发生改变，并非饰变。这些变异单株的发现和鉴定可能为磨盘柿芽变新品种选育提

9、供了理论依据。本试验试材代表了柿属不同程度的变异，包括种间、种下品种间、亲本与子代间，以及直接芽变和间接芽变品种的变异。君迁子作为与柿的亲缘关系较近，二者间平均相似指数0.68；不同柿品种间的平均相似指数低于0.89；芽变品种间的平均相似指数大于0.89。供试的11个变异单株与其标准品种间的平均相似性为0.91，大于种间和普通品种间的相似水平亲缘关系，与芽变品种间的相似水平接近；从聚类图上看，变异单株间及其与标准品种间的分支模式也与种间和普通品种间明显不同，结合DNA分析结果和形态学特征可初步判断供试变异单株应该属于磨盘柿的芽变。逆转座子变异可能是植物芽变的机制之一，本研究结果也说明逆转座子可

10、能参与了柿芽变的形成，而不同试材间的相似指数的差异，可能反映基因组变异的局部细节。如前川次郎和次郎、富有和松本早生、DMP2和BZDMP以及DMP10和DMP7、DMP8间，可能说明逆转座子插入只涉及很少位点或少数基因的突变；如富有和上西早生，DMP3、DMP10、DMP11和BZDMP间那么可能涉及基因组中较大的结构变化，包括染色体畸变以及由此所致的基因或DNA分子的线性顺序变化。参考文献References【1】KALENDAR R, GROBT, REGINA M, SUONIEMI A, SCHULMAN A. IRAP and REMAP: two newretrotransposo

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