基于IRAP技術(shù)的‘磨盤(pán)柿’變異單株鑒定

1、基于IRAP技術(shù)的磨盤(pán)柿變異單株鑒定論文導(dǎo)讀:：君遷子作為與柿的親緣關(guān)系較近。許多主栽柿品種其中51.2263條具多態(tài)性。19個(gè)引物中，11個(gè)無(wú)多態(tài)性，其它引物多態(tài)性介于50.0090.00。利用此11個(gè)多態(tài)引物可將20份試材全部區(qū)分。19個(gè)逆轉(zhuǎn)座子引物的詳細(xì)擴(kuò)增情況見(jiàn)表3。供試12份磨盤(pán)柿中，19個(gè)引物共擴(kuò)增109條帶，多態(tài)性帶21條，多態(tài)比例19.27。其中，4個(gè)引物rtdk4-p3親緣關(guān)系，rtdk2-p6，rtdk13-f5和rtdk16-r10具多態(tài)性。利用多態(tài)引物能很好地對(duì)12份磨盤(pán)柿基因型鑒別區(qū)分表4站大全。圖1箭頭所指為可用于鑒別的特異于某些基因型的譜帶。2.2 相似指數(shù)及聚類

2、分析表3 IRAP引物擴(kuò)增信息Table 3 Summary of PCR amplification corresponding toindividual primer in IRAP analysis 引物 Primer 總帶數(shù) Total number of bands 多態(tài)性帶數(shù) The number of polymorphic bands 多態(tài)性帶比例 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 圖1 4個(gè)引物在12份磨盤(pán)柿基因型中的IRAP擴(kuò)增電泳圖譜A: rtdk4-p3; B: rtdk2-p6; C: rtdk13-f5; D: rtdk16-r10M：DL2000marker; 1. BZMP；212. MP2MP12。黑色箭頭表示特異于某一基因型的條帶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 可用于鑒別12份磨盤(pán)柿基因型的有效IRAP標(biāo)記Table 4 Useful inter-retrotransposon amplified polymorphic (IRAP) markers, inbasepairs (bp), for the identification of 12 Mopanshi genotypes 引物 Primer 樣品 Materials 可識(shí)別基因型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：供試基因型中缺失的標(biāo)記Note: A: marker absent in tested genotypes圖2 基于IRAP分子標(biāo)記的20份磨盤(pán)柿基因型UPGMA聚類圖Fig.2 Dendrogram of 20 genotypes revealed by UPGMAcluster analysis based on IRAP molecular data由UPGMA 聚類所得的樹(shù)狀圖圖2可見(jiàn)，在相似指數(shù)0.92水平上親緣關(guān)系，12份磨盤(pán)柿基因型緊密相聚，之后與同為中國(guó)原產(chǎn)的完全甜柿品種羅

8、田甜柿相聚形成Cluster1。供試日本原產(chǎn)柿品種相聚為Cluster2，且次郎與其芽變前川次郎直接相聚，然后與其子代陽(yáng)豐聚為一支；富有與其芽變松本早生先相聚，再與松本早生芽變上西早生聚為一支。君遷子作為外類群，與柿Cluster1、Cluster2明顯分開(kāi)，形成單獨(dú)一支。3 討論據(jù)北京市房山區(qū)林業(yè)局在成熟期、果實(shí)形態(tài)、耐貯性及植株抗性等方面的調(diào)查結(jié)果初步說(shuō)明，供試變異單株與磨盤(pán)柿有別親緣關(guān)系，且性狀表現(xiàn)根本穩(wěn)定數(shù)據(jù)未發(fā)表站大全。本試驗(yàn)通過(guò)逆轉(zhuǎn)座子分子標(biāo)記技術(shù)，檢測(cè)到它們與標(biāo)準(zhǔn)品種間存在不同程度的遺傳差異，說(shuō)明其遺傳物質(zhì)已發(fā)生改變，并非飾變。這些變異單株的發(fā)現(xiàn)和鑒定可能為磨盤(pán)柿芽變新品種選育提

9、供了理論依據(jù)。本試驗(yàn)試材代表了柿屬不同程度的變異，包括種間、種下品種間、親本與子代間，以及直接芽變和間接芽變品種的變異。君遷子作為與柿的親緣關(guān)系較近，二者間平均相似指數(shù)0.68；不同柿品種間的平均相似指數(shù)低于0.89；芽變品種間的平均相似指數(shù)大于0.89。供試的11個(gè)變異單株與其標(biāo)準(zhǔn)品種間的平均相似性為0.91，大于種間和普通品種間的相似水平親緣關(guān)系，與芽變品種間的相似水平接近；從聚類圖上看，變異單株間及其與標(biāo)準(zhǔn)品種間的分支模式也與種間和普通品種間明顯不同，結(jié)合DNA分析結(jié)果和形態(tài)學(xué)特征可初步判斷供試變異單株應(yīng)該屬于磨盤(pán)柿的芽變。逆轉(zhuǎn)座子變異可能是植物芽變的機(jī)制之一，本研究結(jié)果也說(shuō)明逆轉(zhuǎn)座子可

10、能參與了柿芽變的形成，而不同試材間的相似指數(shù)的差異，可能反映基因組變異的局部細(xì)節(jié)。如前川次郎和次郎、富有和松本早生、DMP2和BZDMP以及DMP10和DMP7、DMP8間，可能說(shuō)明逆轉(zhuǎn)座子插入只涉及很少位點(diǎn)或少數(shù)基因的突變；如富有和上西早生，DMP3、DMP10、DMP11和BZDMP間那么可能涉及基因組中較大的結(jié)構(gòu)變化，包括染色體畸變以及由此所致的基因或DNA分子的線性順序變化。參考文獻(xiàn)References【1】KALENDAR R, GROBT, REGINA M, SUONIEMI A, SCHULMAN A. IRAP and REMAP: two newretrotransposo

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