部分柿品種葉綠體基因組 trnS-trnfM區(qū)域的序列變異

1、 部分柿品種葉綠體基因組trn S-trnf M區(qū)域的序列變異1胡德昌,張青林,羅正榮*華中農(nóng)業(yè)大學(xué)園藝植物生物學(xué)教育部重點(diǎn)實(shí)驗(yàn)室,湖北武漢(430070摘要:應(yīng)用通用引物擴(kuò)增10個(gè)柿屬基因型(包括柿品種9個(gè)和近緣種1個(gè)葉綠體基因組trn S-trnf M區(qū)域,克隆測序得長度為1155bp1160bp的核苷酸序列,包含trn S,psb Z,trn G 和trnf M基因編碼序列及其基因間隔區(qū)序列。共檢測出出17個(gè)序列突變位點(diǎn),包括11個(gè)替換,3個(gè)插入和3個(gè)缺失。供試中國原產(chǎn)柿品種突變位點(diǎn)2個(gè)、日本原產(chǎn)柿品種為14個(gè),差異顯著。相對(duì)古老的柿品種序列保守且高度同源,而近期選育出的陽豐變異水平最高

2、,部分變異位點(diǎn)與母本富有相同。鄰接法分析結(jié)果表明原產(chǎn)中國的柿種質(zhì)與日本的分別聚類,推測兩者具有不同起源。序列比對(duì)和系統(tǒng)發(fā)育分析結(jié)果均表明中國原產(chǎn)完全澀柿磨盤柿與完全甜柿寶蓋甜柿和鄂柿1號(hào)起源親本親緣關(guān)系較近。關(guān)鍵詞:柿,君遷子,葉綠體DNA,trn S-trnf M,序列變異1 前言柿(D.kaki Thunb.隸屬于柿屬植物(Diospyros spp.,是重要的東亞果樹。多數(shù)柿品種為六倍體(2n=6X=90,但近來發(fā)現(xiàn)部分品種為九倍體(2n=9X=135(Zhuang et al., 1990。前人曾采用RAPD(Yamagishi et al., 2005,AFLP(Kanzaki et

3、 al., 2000,SRAP(Guo et al., 2006,IRAP和REMAP(Guo et al., 2006等分子標(biāo)記進(jìn)行過總基因組DNA遺傳多樣性檢測和聚類分析,但由于柿核基因組較大且倍性復(fù)雜,無法確認(rèn)其品種間的遺傳關(guān)系。Yonemori et al.(1996采用PCR-RFLP技術(shù)分析了柿屬植物葉綠體基因組DNA的擴(kuò)增片段長度多樣性,但未檢測到不同柿品種間的差異。Ino et al.(2002進(jìn)而對(duì)cpDNA mat K區(qū)域進(jìn)行過序列分析,也發(fā)現(xiàn)供試柿品種完全一致。葉綠體基因組一般為母系遺傳,結(jié)構(gòu)和序列比較保守,較核基因組變異頻率低。因此, cpDNA分析常被用作植物種上的遺

4、傳多樣性和系統(tǒng)進(jìn)化研究(Palmer, 1987; Badenes et al., 1995; Xu et al., 2001 。trn S-trnf M區(qū)域位于高等植物葉綠體基因組的大拷貝區(qū)(LSC,large single copy regions,包含trn S-UGA (tRNA-Ser,psb Z(ycf9, trn G-GCC(tRNA-Gly和trnf M-CAU四個(gè)基因序列編碼區(qū)域,而基因間隔區(qū)(Intergenic spacer,IGS為非編碼序列,進(jìn)化速率較快,可用于探討植物種間甚至種內(nèi)不同基因型間的遺傳關(guān)系(Wu et al., 2005; King et al., 19

5、98。本文分析了10個(gè)染色體倍性、分布及果實(shí)脫澀性狀不同的柿屬植物cpDNA trn S-trnf M區(qū)域的序列變異,以期為探討核基因組復(fù)雜的多倍體物種的種下遺傳關(guān)系提供科學(xué)依據(jù)。2 材料與方法2.1試材試材為柿屬10個(gè)基因型(表1。其中,君遷子(D. lotus L.1個(gè)單株和柿(D. kaki Thunb. 9個(gè)品種,包括原產(chǎn)中國的3個(gè)甜柿、1個(gè)澀柿品種,以及原產(chǎn)日本的3個(gè)完全甜柿、1個(gè)不1基金項(xiàng)目:國家自然科學(xué)基金(No. 30471203和高等學(xué)校博士學(xué)科點(diǎn)專項(xiàng)科研基金(No.20030504014資助。*通訊作者Author for correspondence ( E-mail:

6、luozhr 完全甜柿和1個(gè)不完全澀柿品種。在供試材料中,陽豐為富有×次郎的F1代。所有試材均取自華中農(nóng)業(yè)大學(xué)柿圃。2.2DNA提取取鮮嫩葉片57g,參考Doyle等(1987CTAB法提取總DNA。2.3trnS-trnfM 區(qū)域PCR擴(kuò)增與測序引物序列:正向引物(53GAGAGAGAGGGATTCGAACC,反向引物(53 CATAACCTTGAGGTCACGGG(Demesure et al., 1995。反應(yīng)體系:1×PCR Buffer, 1.5 mM Mg2+, 0.2 mM dNTPs, 0.2 µM primers, 1U Taq DNA poly

7、merase (TaKaRa,Japan,DNA 30 ng,反應(yīng)體積25 µl,模板DNA 30 ng。擴(kuò)增程序:94 4 min 1個(gè)循環(huán),94 1 min,55 1 min, 72 2 min 35個(gè)循環(huán),72 10min 1個(gè)循環(huán),4保溫。所有DNA擴(kuò)增反應(yīng)均在Whatman Biotrate PCR儀上進(jìn)行。擴(kuò)增產(chǎn)物通過溴化乙錠(EB染色的2%瓊脂糖凝膠電泳檢測。SYNGENE凝膠成像系統(tǒng)觀察并記錄譜帶。利用UniQ柱型回收試劑盒(上海生物工程有限公司回收純化擴(kuò)增產(chǎn)物,以PMD-18T作載體進(jìn)行TA克隆,應(yīng)用質(zhì)粒電泳和酶切法確定陽性克隆。所得陽性克隆由北京奧科生物技術(shù)有限公

8、司在3730型號(hào)自動(dòng)測序儀上進(jìn)行雙向測序。表1 本試驗(yàn)供試的柿屬植物基因型及其一般生物學(xué)信息Table1 The genotypes, sources and cp DNA trn S-trnf M lengths of the genus Diospyros in this study編號(hào)No. 基因型Genotype學(xué)名Scientific name倍性Ploidy level脫澀類型Astringent type原產(chǎn)地Source序列長度Length (bp1 君遷子Date plum D. lotus L. 2n=2x=30 - China11552 磨盤柿Mopanshi D.kak

9、i Thunb.2n=6x=90 PCA China 11563 羅田甜柿Luotiantianshi D.kaki Thunb. 2n=6x=90 PCNA China 11564 寶蓋甜柿Baogaitianshi D.kaki Thunb. 2n=6x=90 PCNA China 11565 鄂柿1號(hào)Eshi No.1 D.kaki Thunb. 2n=6x=90 PCNA China 11566 禪寺丸Zenjimaru D.kaki Thunb. 2n=6x=90 PVNA Japan 11557 富有Fuyuu D.kaki Thunb. 2n=6x=90 PCNA Japan 1

10、1578 陽豐Youhou D.kaki Thunb. 2n=6x=90 PCNA Japan 11609 次郎Jirou D.kaki Thunb. 2n=6x=90 PCNA Japan 115610 平核無Hiratanenashi D.kaki Thunb. 2n=9x=135PV A Japan1156 注:PCNA,完全甜柿;PVNA ,不完全甜柿;PVA ,不完全澀柿;PCA,完全澀柿。Note: PCNA, pollination constant non-astringent; PVNA, pollination variant non-astringent; PVA,pol

11、lination variant astringent; PCA, pollination constant astringent.2.4序列比對(duì)和聚類分析以磨盤柿作為對(duì)照,應(yīng)用Clustal X 軟件(Thompson et al.,1997對(duì)所獲的cpDNAtrn S-trnf M 區(qū)域序列進(jìn)行比對(duì),統(tǒng)計(jì)供試材料的堿基突變,插入和缺失位點(diǎn)。應(yīng)用MEGA2 (Molecular Evolutionary Genetics Analysis軟件(Kumar et al., 2001進(jìn)行鄰接法分析(Neighbor- joining Method和構(gòu)建系統(tǒng)發(fā)育樹。3結(jié)果與分析 3.1柿屬植物cp

12、DNA trn S-trnf M區(qū)域的通用引物擴(kuò)增及其序列分析擴(kuò)增產(chǎn)物電泳檢測結(jié)果表明君遷子和柿及柿品種間cpDNA trn S-trnf M區(qū)域核苷酸序列無明顯長度差異(圖1?；厥諗U(kuò)增片段,克隆測序結(jié)果顯示cpDNA trn S-trnf M區(qū)域包括psb Z 和trn G兩個(gè)基因編碼序列,trn S 和trnf M基因的部分編碼序列以及三個(gè)基因間隔區(qū)。君遷子為1155bp;9個(gè)柿品種相應(yīng)序列長度介于1155 bp1160 bp之間,其中禪寺丸序列最短(1155bp,陽豐序列最長(1160bp(表1。以磨盤柿為例,trn S-trnf M區(qū)域A,T,C和G堿基的含量分別為32.96%,29.

13、84%,18.08%和19.12%,A+T含量為62.8%,G+C含量為37.2%。編碼區(qū)域?yàn)?91bp,基因間隔區(qū)域?yàn)?65bp。應(yīng)用BLAST軟件與煙草(Nicotiana tabacum, No.Z00044; Shinozaki et al., 1986、柑橘(Citrus sinensis, No.DQ864733; Bausher et al., 2006和葡萄(Vitis vinifera, No.DQ424856; Jansen et al., 2006GenBank中登錄序列進(jìn)行比對(duì),同源性為69%77%,變異主要發(fā)生在非編碼區(qū)域序列。應(yīng)用Clustal X軟件對(duì)6個(gè)完全甜柿

14、品種,2個(gè)不完全甜柿品種和近緣野生種君遷子與磨盤柿葉綠體基因組trn S-trnf M區(qū)域序列進(jìn)行比較,共存在17個(gè)突變位點(diǎn),包括11個(gè)替換,3個(gè)插入和3個(gè)缺失,未發(fā)現(xiàn)大片段插入和缺失(表2。在四個(gè)編碼區(qū)域中,僅trn S區(qū)域檢測到1個(gè)堿基變異位點(diǎn);而非編碼區(qū)域序列的變化豐富,IGS I,II和III 區(qū)域分別發(fā)生3個(gè), 6個(gè)和7個(gè)變異位點(diǎn)。本實(shí)驗(yàn)中,中國部分柿品種間存在2個(gè)堿基變異位點(diǎn),未發(fā)現(xiàn)插入和缺失;而日本柿品種存在14個(gè)突變位點(diǎn),包括3個(gè)插入和2個(gè)缺失(表2。結(jié)果表明中國和日本的不同品種突變差異顯著。中國完全甜柿種質(zhì)寶蓋甜柿和鄂柿1號(hào)與澀柿磨盤柿序列完全一致,結(jié)果表明寶蓋甜柿和鄂柿1號(hào)

15、起源母本與澀柿磨盤柿親緣關(guān)系相近。羅田甜柿僅在89bp(T-C和1050bp(C-T兩個(gè)位點(diǎn)發(fā)生核苷酸突變。日本完全甜柿次郎和不完全澀柿平核無在此區(qū)域DNA序列完全相同;不完全甜柿禪寺丸在268bp,907bp兩個(gè)位點(diǎn)發(fā)生堿基變異和1063bp 發(fā)生單個(gè)堿基A缺失;完全甜柿富有只在791bp一個(gè)位點(diǎn)發(fā)生單個(gè)核苷酸A插入;富有×次郎雜交近期選育品種陽豐此區(qū)域DNA序列高度變異,共發(fā)生13個(gè)變異位點(diǎn)包括9個(gè)堿基變異和4個(gè)核苷酸插入。陽豐與富有在此區(qū)域791bp處變異位點(diǎn)相同,均發(fā)生單個(gè)堿基A的插入,而與父本次郎明顯不同,支持cpDNA母系遺傳規(guī)律。 圖1 君遷子和9個(gè)柿品種cpDNA t

16、rn S-trnf M區(qū)域擴(kuò)增產(chǎn)物2%瓊脂糖凝膠電泳圖譜。編號(hào)110如表1。M:DL2000 marker.Fig.1 Amplification products of cpDNA trn S-trnf M region from Date plum and 9 persimmon varieties detected by 2%agorose gel. M:DL2000 marker. 110 refer to the numbers listed in Table 1. 注:a 磨盤柿相應(yīng)序列長度;b 磨盤柿序列5端起相應(yīng)位置;c 未檢測到變異位點(diǎn)。Note: a Aligned len

17、gth referred to Mpanshis sequence; b Mutation site was denoted the position of Mopanshis sequence from the 5-end; c no mutation was observed in corresponding region.3.2 基于cpDNA trnS-trnfM 區(qū)域的序列變異的柿屬植物系統(tǒng)發(fā)育分析根據(jù)trn S-trnf M 區(qū)域序列變異位點(diǎn),應(yīng)用MAGE 軟件鄰接法分析,構(gòu)建系統(tǒng)發(fā)育樹(圖2。君遷子作為柿近緣野生種,形成外類群。在日本甜柿種質(zhì)中,完全甜柿品種陽豐與母本富有具有共同

18、的變異位點(diǎn),聚為一組。不完全澀柿平核無和不完全甜柿禪寺丸相聚,然后與完全甜柿次郎相聚,表明不同倍性和脫澀類型的非完全甜柿起源親本存在較近的親緣關(guān)系,不完全甜柿和不完全澀柿母本均為完全甜柿種質(zhì)。原產(chǎn)中國的完全甜柿鄂柿1號(hào)單獨(dú)成組。中國原產(chǎn)的兩個(gè)古老品種-完全甜柿羅田甜柿與完全澀柿磨盤柿聚類,然后與近期發(fā)現(xiàn)的中國原產(chǎn)甜柿新種質(zhì)寶蓋甜柿聚為一組,與Guo et al.(2006應(yīng)用IRAP 和REMAP 分子標(biāo)記技術(shù)分析聚類結(jié)果一致。根據(jù)樹枝長度和分歧時(shí)間推斷磨盤柿和羅田甜柿起源親本分化時(shí)間與富有和陽豐,以及禪寺丸和平核無一致。在應(yīng)用鄰接法構(gòu)建系統(tǒng)發(fā)育樹圖中,原產(chǎn)中國柿種質(zhì)與日本柿種質(zhì)分別聚類,表明

19、兩者具有不同的起源,與我們前期的推論一致(Luo et al., 1999;Guo et al., 2006;Hu et al., 2006。cpDNA 區(qū)域cpDNAregions序列長度 Aligned length a (bp 變異位點(diǎn)編號(hào) Mutation number 變異位點(diǎn) Mutation site b (bp 變異基因型 Genotype 變異類型 Comment trn S91bp 1 89 Luotiantianshi base change from T to C 2138 Youhou base change from C toT 3157 Youhou base c

20、hange from T to C IGS I 352bp 4268 Zenjimaru base change from C to T psb Z 189bp-c - - - 5661 Youhou Insertion A 6 704 Youhou base change from A to C 7 751 D. lotus deletion A8 791 Fuyuu Youhouinsertion A9904 Youhou base change from T to A IGS II 275bp 10907 Zenjimaru base change from A to G trn G 7

21、1bp- - - - 111002 Youhou base change from A to T 121046 Youhou base change from T to C 13 1050 Luotiantianshi base change from C to T14 1054 Youhou deletion C15 1063 Zenjimaru deletion A16 1064 Youhou insertion GGGIGS III 138bp 17 1100 Youhou base change from G to Ttrnf M 40bp - - - -表2 君遷子和9個(gè)柿品種cpD

22、NA trn S -trnf M 區(qū)域的序列比較Table2 the sequences characterizations of cpDNA trn S-trnf M region in Date plum and 9 persimmon varieties 圖2 基于葉綠體基因組trn S-trnf M區(qū)域序列變異位點(diǎn)的9個(gè)柿品種與近緣野生種君遷子的系統(tǒng)發(fā)育樹。分枝旁數(shù)值為500次自展檢驗(yàn)所獲得自展值。Fig.2 Phylogenetic tree among 9 persimmon varieties and related species D. lotus based on the m

23、utation sites in cpDNA trn S-trnf M regions using MAGE Neighbor-Joining method. Numbers next to the branches indicate thebootstrap values of 500 replicates.4討論應(yīng)用RFLP(Nakamura et al., 1994和PCR-RFLP(Yonemori et al., 1996分子標(biāo)記進(jìn)行遺傳多樣性研究結(jié)果表明,柿葉綠體基因組檢測位點(diǎn)變異程度較低,而mat K區(qū)域測序結(jié)果顯示三個(gè)供試柿品種cpDNA序列相同(Ino et al., 200

24、2。在其他物種中,cpDNA trn S-trnf M區(qū)域酶切位點(diǎn)種內(nèi)材料呈現(xiàn)多態(tài)性表明此區(qū)域序列存在豐富變異(Wu et al., 2005; King et al., 1998。本實(shí)驗(yàn)對(duì)9個(gè)柿品種cpDNA trn S-trnf M區(qū)域特異擴(kuò)增并克隆測序,序列比對(duì)結(jié)果表明部分柿品種存在多個(gè)堿基突變,插入和缺失位點(diǎn)。相對(duì)于限制性內(nèi)切酶酶切技術(shù),cpDNA相應(yīng)區(qū)域擴(kuò)增片段測序得到的變異位點(diǎn)信息更為豐富有效。君遷子(2n=2X=30染色體數(shù)目與柿(2n=6X, 9X=90, 135差異顯著,但與磨盤柿相應(yīng)區(qū)域序列比較僅存在一個(gè)位點(diǎn)變異,表明兩者起源親本關(guān)系較近,與染色體原位雜交技術(shù)(Chio e

25、t al., 2003b,Ty1-copia反轉(zhuǎn)錄轉(zhuǎn)座子(Nakasuka et al., 2002,SRAP(Guo et al., 2006a分析結(jié)果一致,支持Yonemori et al.(1998基于cpDNA PCR-RFLP分析結(jié)果中關(guān)于柿與君遷子有共同起源親本的推論。供試材料中,原產(chǎn)中國柿品種磨盤柿(公元1116年記載和羅田甜柿(公元1034年前記載以及日本品種禪寺丸(1214年發(fā)現(xiàn)、富有(1844年發(fā)現(xiàn),次郎(1844年發(fā)現(xiàn)均為相對(duì)古老的柿品種,在本實(shí)驗(yàn)所分析區(qū)域的序列同源性較高,表明其起源親本親緣關(guān)系相近。寶蓋甜柿和鄂柿1號(hào)為在湖北省大別山區(qū)發(fā)現(xiàn)的甜柿種質(zhì)(潘德森等,1994

26、,但與澀柿品種磨盤柿序列相同,鄰接法系統(tǒng)發(fā)育分析結(jié)果也表明兩者親本與磨盤柿存在較近的親緣關(guān)系,與Guo et al.(2006b應(yīng)用IRAP和REMAP分子標(biāo)記聚類結(jié)果和Hu et al.(2006線粒體DNA(Mitochondrial DNA,mtDNA分析結(jié)果一致。陽豐于1967年由富有×次郎雜交育成,本實(shí)驗(yàn)結(jié)果顯示其與母本存在共同變異位點(diǎn),支持cpDNA母系遺傳方式。日本原產(chǎn)柿種質(zhì)間自然雜交和人工選擇頻繁,可能是其比中國原產(chǎn)柿種質(zhì)變異頻率較高的原因。缺乏完整序列信息是葉綠體基因組應(yīng)用于作物育種研究主要障礙。迄今,已有23個(gè)高等植物種類包括柑桔、葡萄等園藝植物的葉綠體基因組測序

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