史培瑤 陳麗娟 孫昊杰 程夢(mèng)豪 肖 進(jìn) 袁春霞 王秀娥 王海燕

頂芒山羊草特異寡核苷酸探針開(kāi)發(fā)和oligo-FISH核型構(gòu)建

史培瑤 陳麗娟 孫昊杰 程夢(mèng)豪 肖 進(jìn) 袁春霞 王秀娥 王海燕*

南京農(nóng)業(yè)大學(xué)作物遺傳與種質(zhì)創(chuàng)新國(guó)家重點(diǎn)實(shí)驗(yàn)室 / 細(xì)胞遺傳研究所/ 現(xiàn)代作物生產(chǎn)省部共建協(xié)同創(chuàng)新中心，江蘇南京 210095

栽培小麥近緣物種頂芒山羊草(, 2=2=14, MM)是小麥改良的三級(jí)基因庫(kù)。為準(zhǔn)確鑒定頂芒山羊草M基因組染色體或染色體區(qū)段, 本研究利用二代測(cè)序獲得頂芒山羊草M基因組序列信息, 從中鑒定出16條可能的特異衛(wèi)星重復(fù)序列。根據(jù)這些序列設(shè)計(jì)12個(gè)寡核苷酸(oligo)探針進(jìn)行oligo-FISH, 結(jié)果表明, 其中10個(gè)探針可在頂芒山羊草染色體上產(chǎn)生明顯的雜交信號(hào)。對(duì)探針特異性分析發(fā)現(xiàn), 5個(gè)探針僅在頂芒山羊草染色體上產(chǎn)生雜交信號(hào), 在小麥染色體上未觀察明顯雜交信號(hào), 可作為頂芒山羊草特異探針鑒定小麥背景中的頂芒山羊草染色體。選擇在頂芒山羊草染色體上信號(hào)分布豐富的3個(gè)探針(oligo-pAc89、oligo-pAc148、oligo-pAc225)組成探針套ONPS#AC1, 結(jié)合利用本實(shí)驗(yàn)室根據(jù)小麥D亞基因組開(kāi)發(fā)的寡核苷酸探針庫(kù), 構(gòu)建了頂芒山羊草的oligo-FISH核型。本研究構(gòu)建的FISH核型可以準(zhǔn)確識(shí)別頂芒山羊草各條染色體, 為挖掘、轉(zhuǎn)移和利用頂芒山羊草優(yōu)異基因提供了快速準(zhǔn)確的鑒定手段。

頂芒山羊草; 二代測(cè)序; 衛(wèi)星重復(fù)序列; 寡聚核苷酸探針; 熒光原位雜交

頂芒山羊草(, 2=2=14, MM)是山羊草屬二倍體物種, 是小麥的三級(jí)基因庫(kù)。已有研究表明, 普通小麥D基因組遺傳多樣性最低, 而M基因組和D基因組遺傳關(guān)系較近, M基因組導(dǎo)入小麥可以豐富小麥D基因組的遺傳多樣性[1-2]。頂芒山羊草具有抗小麥條銹病[3]、葉銹病和白粉病[4]、抗小麥孢囊線蟲(chóng)病[5]、小麥癭蚊病、小麥蚜蟲(chóng)[4]以及耐鹽等優(yōu)良性狀[6], 是小麥遺傳改良的重要基因資源。利用遠(yuǎn)緣雜交和染色體工程可以將頂芒山羊草的優(yōu)異基因?qū)氲狡胀ㄐ←淸7]。已有研究報(bào)道將M基因組抗稈銹病、條銹病等基因?qū)肓嗽耘嘈←淸3]。Zuo等[8]利用四倍體小麥-頂芒山羊草雙二倍體STM4與中國(guó)春(CS)、博紫1313 (BZ1313)、川農(nóng)16 (CN16, 含1BL/1RS易位)進(jìn)行復(fù)合雜交, 選育了兩個(gè)高抗白粉病的普通小麥-頂芒山羊草7M(7A)二體代換系, 推測(cè)7M染色體攜帶抗小麥白粉病基因。Liu等[7]利用創(chuàng)制的一套小麥-頂芒山羊草的染色體系(其中包括6個(gè)2M-7M小麥-頂芒山羊草二體異附加系DA2M-DA7M, 1個(gè)小麥-頂芒山羊草二體異代換系DS6M(6A)), 推測(cè)頂芒山羊草的2M和7M染色體可能分別攜帶抗小麥條銹病和白粉病基因。

利用染色體工程轉(zhuǎn)移近緣物種優(yōu)異基因過(guò)程中,快速準(zhǔn)確的鑒定外源染色質(zhì)是種質(zhì)創(chuàng)新的關(guān)鍵手段。隨著麥類植物等基因組學(xué)的快速發(fā)展, 已經(jīng)快速發(fā)展出基因組原位雜交(genomichybridization, GISH) 、熒光原位雜交(fluorescencehybridization, FISH)和基于簡(jiǎn)單重復(fù)序列、單核苷酸多態(tài)性等分子標(biāo)記的外源染色質(zhì)鑒定技術(shù)[9-13]。例如, 宮文萍等[14]利用oligo-FISH分別對(duì)頂芒山羊草、無(wú)芒山羊草與普通小麥的雙二倍體進(jìn)行分析, 結(jié)果發(fā)現(xiàn), (GAA)8重復(fù)序列探針可用于鑒定小麥背景中的頂芒山羊草各染色體。Parisod和Badaeva[15]利用重復(fù)序列探針pAs1、pSc119.2、pTa535等對(duì)部分山羊草屬物種進(jìn)行分子細(xì)胞遺傳學(xué)核型分析, 結(jié)果表明, pAs1探針可以在頂芒山羊草染色體上產(chǎn)生豐富的信號(hào)。Song等[16]利用寡核苷酸探針pSc119.2、pTa71結(jié)合(AAC)5, (ACT)7和(CTT)12構(gòu)建了頂芒山羊草的FISH核型并進(jìn)行了染色體結(jié)構(gòu)多樣性的分析。目前, 在頂芒山羊草染色體鑒定研究中, 利用的重復(fù)序列探針來(lái)源于其他物種, 對(duì)頂芒山羊草染色體無(wú)特異性, 而且目前基于重復(fù)序列信息開(kāi)發(fā)的FISH探針只在染色體一些特定區(qū)域如長(zhǎng)短臂的端部或著絲粒區(qū)域產(chǎn)生較為局限的點(diǎn)狀信號(hào), 不能覆蓋整條染色體, 因此, 對(duì)染色體重排等的鑒定具有一定局限性[17]。因此, 迫切需要基于頂芒山羊草基因組信息, 開(kāi)發(fā)頂芒山羊草特異的oligo探針, 提高頂芒山羊草染色質(zhì)的鑒定精度、特異性和效率。

近年來(lái), 基于單拷貝探針庫(kù)的染色體涂染(oligo- painting)技術(shù)大大提高了鑒定的效率、準(zhǔn)確度和精度。目前, 該技術(shù)已成功應(yīng)用于特定染色體的識(shí)別、核型構(gòu)建等方面[18-20]。Han等[21]基于單拷貝基因序列開(kāi)發(fā)了黃瓜3號(hào)染色體長(zhǎng)臂和短臂特異的寡核苷酸探針庫(kù), 利用該套探針對(duì)黃瓜及其近緣種的特定染色體進(jìn)行了涂染。該項(xiàng)技術(shù)也在小麥族物種中已經(jīng)得到應(yīng)用。本實(shí)驗(yàn)室Song等[22]根據(jù)已完成測(cè)序的小麥品種中國(guó)春的參考基因組序列, 開(kāi)發(fā)了4D染色體特異的寡核苷酸探針庫(kù), 首次在小麥上建立了染色體涂染技術(shù), 同時(shí)發(fā)現(xiàn)4D染色體特異的寡核苷酸探針庫(kù)也可以在頂芒山羊草的4M染色體產(chǎn)生信號(hào)。Li等[23]基于小麥A、B、D基因組和大麥H基因組序列比較分析(序列相似性>96%), 開(kāi)發(fā)了一套1～7部分同源群的oligo探針, 可用于小麥族不同屬種間染色體的識(shí)別和比較基因組學(xué)探究。

本研究利用TAREAN軟件在頂芒山羊草二代測(cè)序基因組中, 鑒定特異重復(fù)序列, 根據(jù)重復(fù)序列基序開(kāi)發(fā)寡核苷酸(oligo)探針, 通過(guò)oligo-FISH篩選頂芒山羊草特異oligo-探針; 利用特異oligo-探針和前期開(kāi)發(fā)的普通小麥D亞基因組特異oligo-painting探針, 構(gòu)建頂芒山羊草M基因組染色體FISH核型, 完善頂芒山羊草染色體鑒定體系, 為挖掘和利用頂芒山羊草的優(yōu)異基因提供有效的細(xì)胞學(xué)手段。

1 材料與方法

1.1 試驗(yàn)材料

頂芒山羊草(, 2=14, 基因組MM, 登錄號(hào)為PI551063、PI542176); 小傘山羊草(, 2=14, 基因組UU, 登錄號(hào)為PI276994); 單芒山羊草(, 2=14, 基因組NN, 登錄號(hào)為PI554418); 節(jié)節(jié)麥(, 2=14, 基因組DD, 登錄號(hào)為PI511362); 擬斯卑爾脫山羊草(, 2=14, 基因組SS, 登錄號(hào)為PI560750), 大麥(, 2=14, 基因組HH, 登錄號(hào)為PI542176); 黑麥(, 2=2=14, 基因組RR, 登錄號(hào)為PI542176); 以上小麥野生近緣物種引自美國(guó)國(guó)家植物種質(zhì)資源庫(kù)(NPGSUSA)。簇毛麥(, 2=14, 基因組VV, 登錄號(hào)為91C43), 引自英國(guó)劍橋植物園; 普通小麥中國(guó)春(Chinese Spring, CS)由南京農(nóng)業(yè)大學(xué)細(xì)胞遺傳研究所(CINAU)保存。

1.2 二倍體頂芒山羊草的基因組測(cè)序

采用CTAB法提取頂芒山羊草(PI551063)幼葉基因組DNA[24]。通過(guò)DNA片段化、末端修復(fù)、連接和PCR擴(kuò)增構(gòu)建DNA文庫(kù), 利用Agilent 2100進(jìn)行DNA文庫(kù)質(zhì)量控制后, 利用雙末端測(cè)序法進(jìn)行BGISEQ-500測(cè)序(委托中國(guó)深圳BGI公司完成), 獲得的原始序列去除低質(zhì)量、低復(fù)雜度、短于35 bp的reads序列, 得到高質(zhì)量測(cè)序數(shù)據(jù)用于進(jìn)一步分析。

1.3 重復(fù)序列鑒定和oligo探針開(kāi)發(fā)

重復(fù)序列鑒定參照程夢(mèng)豪等[25]的方法。利用重復(fù)序列分析軟件RepeatExplorer 2 (http://www. repeatexplorer.org/)對(duì)所有reads進(jìn)行串聯(lián)重復(fù)序列分析, 經(jīng)序列聚類和拼接, 得到M基因組重復(fù)序列信息。利用oligo 7寡聚核苷酸探針設(shè)計(jì)軟件[26], 將鑒定出的上述衛(wèi)星DNA序列設(shè)計(jì)成(55±4) bp的寡聚核苷酸探針, 每個(gè)重復(fù)序列開(kāi)發(fā)1個(gè)oligo探針, 設(shè)計(jì)好的探針序列由擎科生物公司合成, 在探針的5￠末端用6-FAM (綠色熒光)或TAMRA (紅色熒光)熒光基團(tuán)對(duì)設(shè)計(jì)的寡聚核苷酸探針進(jìn)行熒光修飾。

衛(wèi)星重復(fù)序列和探針命名: 本研究重復(fù)序列命名為pAc (p代表探針, Ac代表頂芒山羊草的屬名和種名的首字母)+重復(fù)序列聚類簇的編號(hào)。例如, 來(lái)自M基因組的重復(fù)序列cluster89的命名為pAc89; 基于該重復(fù)序列開(kāi)發(fā)的oligo探針相應(yīng)命名為oligo- pAc89; 用于核型構(gòu)建的3個(gè)探針oligo-pAc89、oligo-pAc148、oligo-pAc225所組成的探針套命名為ONPS#AC1 (oligonucleotide probe set of), 其中3個(gè)探針的用量比例為1︰1︰1。

1.4 有絲分裂中期染色體制片

染色體中期同步化處理參照Song等[22]的方法。根尖細(xì)胞中期染色體制片使用的滴片法參照Lei等[27]的方法。

1.5 寡核苷酸探針的熒光原位雜交(oligo-FISH)

oligo-FISH在Lei等[27]的基礎(chǔ)上稍作修改。制片首先在紫外交聯(lián)儀中交聯(lián)1～4 min, 強(qiáng)度為0.125 J cm–2。然后將交聯(lián)后的染色體制片在含0.15 mol L–1NaOH的70%酒精溶液中變性5 min, 隨后依次在70%、90%和100%的酒精中梯度脫水5 min, 晾干備用。將15mL的雜交液滴加在制片上, 雜交液組成: 7.5 μL甲酰胺(Formamide, FA), 1.5 μL緩沖液(20× SSC), 0.5 μL鮭魚(yú)精DNA (Salmon sperm DNA, ssDNA), 10 pmol oligo探針, 然后用ddH2O補(bǔ)足至15 μL。蓋上蓋玻片, 置于濕盒, 37℃雜交6 h以上; 雜交后的制片在ddH2O中洗10 min (42℃水浴), 氣干; 滴加7mL含DAPI的H1200 (VECTA)防熒光淬滅劑, 蓋上蓋玻片; 將制片放于Olympus BX51型熒光顯微鏡下暗環(huán)境進(jìn)行鏡檢, 并用Olympus DP72型CCD相機(jī)照相。

1.6 染色體涂染熒光原位雜交(oligo-painting FISH)

染色體涂染實(shí)驗(yàn)所用的探針是本實(shí)驗(yàn)室利用中國(guó)春的參考基因組序列開(kāi)發(fā)的中國(guó)春D亞基因組染色體特異oligo-painting探針。oligo-painting參照Song等的方法[22]。交聯(lián)后的染色體制片每張滴加100mL的胃蛋白酶稀釋液, 放置于濕盒中處理1 h, 之后用2×SSC緩沖液在室溫下洗滌3次, 每次5 min;接下來(lái)分別移至70%、90%和100%的酒精中梯度脫水3 min; 滴加100 μL 70%甲酰胺(dFA)于制片上, 蓋上蓋玻片, 迅速放入88℃雜交儀中變性4 min; 變性后, 分別在–20℃預(yù)冷的70%、90%和100%酒精中梯度脫水, 每次5 min, 氣干; 雜交液的配制: 10 μL去離子甲酰胺(100% dFA), 2 μL 20×SSC, 4 μL 50%硫酸葡聚糖(50% DS), 6 μL寡核苷酸探針(200 ng μL–1); 將雜交液滴加在氣干的染色體制片上, 蓋上蓋玻片, Elmers膠封邊緣, 37℃雜交2 d左右。將雜交后的制片放置于2×SSC緩沖液中室溫下洗滌5 min, 移至42℃預(yù)熱的2×SSC中洗滌10 min, 之后在1×PBS緩沖液中室溫下洗滌5 min; 配制檢測(cè)液: 每張片子準(zhǔn)備2 μL Anti-Dig-羅丹明+80 μL TNB用于地高辛標(biāo)記的紅色探針的信號(hào)檢測(cè); 將配好的檢測(cè)液充分混勻, 滴加在氣干的染色體制片上, 蓋上蓋片, 37℃孵育2 h, 之后在1×PBS中洗滌3次, 每次5 min, 氣干。滴加6.5 μL每張含DAPI的H1200 (VECTA)防熒光淬滅劑, 蓋上潔凈的蓋玻片, 在Olympus BX53型熒光顯微鏡下鏡檢, Olympus DP81型CCD相機(jī)照相。

2 結(jié)果與分析

2.1 頂芒山羊草M基因組特異重復(fù)序列篩選

對(duì)利用RepeatExplorer2 (http://www.repeatexplorer.org/)鑒定出頂芒山羊草的串聯(lián)重復(fù)序列進(jìn)行聚類和拼接, 共獲得可能的M基因組特異衛(wèi)星重復(fù)序列16條, 其中8條高置信度重復(fù)序列, 8條低置信度重復(fù)序列(表1)。在鑒定的重復(fù)序列中, 基序最長(zhǎng)的是CL149, 為663 bp, 占M基因組0.1%; 基序最短的是CL225, 為46 bp, 占M基因組0.028%; 其余14條序列的基序長(zhǎng)度范圍49～637 bp, 占M基因組的0.03%～0.28%。

將上述重復(fù)序列的基序在NCBI數(shù)據(jù)庫(kù)進(jìn)行BLASTN (Percent Identity≥80%, Query Coverage≥80%)序列比對(duì)發(fā)現(xiàn), 其中4條(CL103、18S、CL133和CL115)基序與已知的重復(fù)序列相同, 推測(cè)它們是不同物種中較保守的序列; 6條序列可以比對(duì)到已知的同源序列, 其中, CL301與小麥的重復(fù)序列pTa713、pTa885、pTa551、pTa779相似, CL238與節(jié)節(jié)麥的重復(fù)序列4P6-14、4P6-9和4P6-2相似, 另外4條序列(CL89、CL149、CL198、CL225)分別與小麥中的1740-J17、1716-E15、190H5、醇溶蛋白相關(guān)、醇溶蛋白相關(guān)3B BAC和同源, 均不是典型的串聯(lián)重復(fù)序列; 6條序列(CL105、CL146、CL148、CL217、CL259、CL263)未比對(duì)到已知同源序列, 推測(cè)可能是M基因組特異重復(fù)序列(表1)。

2.2 oligo探針開(kāi)發(fā)及其基因組特異性分析

利用oligo 7寡聚核苷酸設(shè)計(jì)軟件[27]開(kāi)發(fā)了基于上述12條衛(wèi)星DNA序列的oligo探針: oligo-pAc89、oligo-pAc105、oligo-pAc146、oligo-pAc148、oligo- pAc149、oligo-pAc198、oligo-pAc217、oligo-pAc225、oligo-pAc238、oligo-pAc259、oligo-pAc263和oligo- pAc301, 基序介于51～59 bp (表1)。

表1 開(kāi)發(fā)的12個(gè)oligo探針的序列信息

利用開(kāi)發(fā)的探針在頂芒山羊草和小麥品種中國(guó)春中進(jìn)行oligo-FISH, 結(jié)果表明, oligo-pAc146和oligo-pAc198在2個(gè)材料中均未觀察到明顯雜交信號(hào)。oligo-pAc105、oligo-pAc148、oligo-pAc149、oligo-pAc217和oligo-pAc259可在頂芒山羊草染色體上產(chǎn)生明顯雜交信號(hào), 而在中國(guó)春染色體上未觀察到明顯信號(hào), 推測(cè)這5個(gè)探針可作為M基因組特異探針識(shí)別小麥背景中的M基因組染色體(圖1)。oligo-pAc149、oligo-pAc217和oligo-pAc259分別在頂芒山羊草的1對(duì)染色體上產(chǎn)生雜交信號(hào); oligo- pAc148可在頂芒山羊草的12條染色體上產(chǎn)生雜交信號(hào)。oligo-pAc89、oligo-pAc225、oligo-pAc238、oligo-pAc263、oligo-pAc301在頂芒山羊草和中國(guó)春染色體上均可產(chǎn)生雜交信號(hào)(圖2), 但信號(hào)分布和數(shù)量存在差異, oligo-pAc89、oligo-pAc301、oligo- pAc225、oligo-pAc238和oligo-pAc263分別在頂芒山羊草的12條、8條、6條、4條、2條染色體上有雜交信號(hào), oligo-pAc301、oligo-pAc89、oligo-pAc238、oligo-pAc225、oligo-pAc263分別在中國(guó)春的28條、12條、12條、10條和6條染色體上有雜交信號(hào)。oligo-pAc89、oligo-pAc148、oligo-pAc225和oligo- pAc301在頂芒山羊草染色體上產(chǎn)生的雜交信號(hào)較豐富, oligo-pAc238在頂芒山羊草的6條染色體上產(chǎn)生信號(hào), 而oligo-pAc105、oligo-pAc149、oligo-pAc217、oligo-pAc263和oligo-pAc259只在頂芒山羊草的2條染色體上產(chǎn)生雜交信號(hào)。

圖1 5個(gè)oligo探針在頂芒山羊草(PI542176)和中國(guó)春有絲分裂中期染色體的oligo-FISH

A1, A2: 探針oligo-pAc105; B1, B2: 探針oligo-pAc259; C1, C2: 探針oligo-pAc149; D1, D2: 探針oligo-pAc217; E1, E2: 探針oligo- pAc148。染色體用DAPI套染(藍(lán)色); 5個(gè)寡聚核苷酸探針用5′TAMRA修飾(紅色)。標(biāo)尺為10 μm。

A1, A2: probe oligo-pAc105; B1, B2: probe oligo-pAc259; C1, C2: probe oligo-pAc149; D1, D2: probe oligo-pAc217; E1, E2: probe oligo-pAc148. Chromosomes were counterstained with DAPI (blue); five oligo probes were modified with 5′TAMRA (red). Bar: 10 μm.

A1, A2: 探針oligo-pAc263; B1, B2: 探針oligo-pAc225; C1, C2: 探針oligo-pAc238; D1, D2: 探針oligo-pAc301; E1, E2: 探針oligo- pAc89。染色體用DAPI套染(藍(lán)色); 探針oligo-pAc225、oligo-pAc238和oligo-pAc263用5′TAMRA修飾(紅色); 探針oligo-pAc89和oligo-pAc301用5′FAM修飾(綠色)。標(biāo)尺為10 μm。

A1, A2: probe oligo-pAc263; B1, B2: probe oligo-pAc225; C1, C2: probe oligo-pAc238; D1, D2: probe oligo-pAc301; E1, E2: probe oligo-pAc89. Chromosomes were counterstained with DAPI (blue); oligo probes oligo-pAc225, oligo-pAc238, and oligo-pAc263 were modified with 5′TAMRA (red), and oligo-pAc89 and oligo-pAc301 were modified with 5′FAM (green). Bar: 10 μm.

為驗(yàn)證這5個(gè)oligo探針(oligo-pAc105、oligo- pAc148、oligo-pAc149、oligo-pAc217和oligo-pAc259)對(duì)于頂芒山羊草的特異性, 利用B2DSC[28](= 85,= 80)分析了探針序列在中國(guó)春參考基因組中的拷貝數(shù)和物理位置。分析發(fā)現(xiàn)oligo-pAc105未能搜索到同源序列, 其余4個(gè)探針序列可以比對(duì)到同源序列, 但數(shù)量和位置分布不同。oligo-pAc149和oligo-pAc217同源序列在中國(guó)春基因組的不同區(qū)域預(yù)測(cè)的拷貝數(shù)很少, 且散在分布在各條染色體上; oligo-pAc148同源序列僅局限于中國(guó)春基因組的小麥4A染色體和4B染色體端部的1 Mb之內(nèi); oligo- pAc259同源序列或僅局限于中國(guó)春基因組染色體的1 Mb之內(nèi), 或雖是集中分布、但平均在每1 Mb內(nèi)的同源序列數(shù)很少。由此可見(jiàn), 這些探針在中國(guó)春基因組中或散在分布且拷貝數(shù)較低, 或僅集中分布于1 Mb基因組區(qū)間, 推測(cè)由此導(dǎo)致這些探針無(wú)法在中國(guó)春染色體上產(chǎn)生FISH信號(hào)。

利用探針oligo-pAc105、oligo-pAc148、oligo- pAc149、oligo-pAc217和oligo-pAc259在7個(gè)二倍體小麥近緣物種進(jìn)行oligo-FISH, 結(jié)果發(fā)現(xiàn), oligo- pAc217在7個(gè)物種中均未產(chǎn)生雜交信號(hào); oligo- pAc148、oligo-pAc105、oligo-pAc149和oligo-pAc259在小傘山羊草U基因組、擬斯卑爾脫山羊草S基因組和大麥H基因組上均無(wú)雜交信號(hào), 但可以在其他基因組上產(chǎn)生雜交信號(hào); oligo-pAc148在單芒山羊草N基因組、簇毛麥V基因組、黑麥R基因組產(chǎn)生雜交信號(hào); oligo-pAc105在單芒山羊草N基因組上產(chǎn)生雜交信號(hào); oligo-pAc149和oligo-pAc259在節(jié)節(jié)麥D基因組上產(chǎn)生雜交信號(hào)(圖3)。比較5個(gè)探針在7個(gè)二倍體小麥近緣物種中的oligo-FISH分析結(jié)果發(fā)現(xiàn), 探針oligo-pAc105和oligo-pAc148可在單芒山羊草N基因組的部分染色體產(chǎn)生雜交信號(hào); 探針oligo- pAc149和oligo-pAc259可在節(jié)節(jié)麥D基因組的部分染色體產(chǎn)生雜交信號(hào)。據(jù)此我們推測(cè)頂芒山羊草M基因組與單芒山羊草的N基因組和節(jié)節(jié)麥的D基因組親緣關(guān)系較近, 這與已知的頂芒山羊草與節(jié)節(jié)麥和單芒山羊草親緣關(guān)系相符合。但我們也發(fā)現(xiàn), 探針oligo-pAc148在簇毛麥V基因組、黑麥R基因組的部分染色體也產(chǎn)生雜交信號(hào), 這與已知的頂芒山羊草與簇毛麥和黑麥的親緣關(guān)系不一致。oligo- pAc217探針的特異性最好, 其余4個(gè)探針不僅能鑒定頂芒山羊草M基因組染色體, 也在鑒定單芒山羊草、黑麥、簇毛麥和節(jié)節(jié)麥染色體上有應(yīng)用潛力。

2.3 頂芒山羊草的oligo-FISH核型構(gòu)建和M基因組染色體識(shí)別

利用本實(shí)驗(yàn)室開(kāi)發(fā)的中國(guó)春D亞基因組染色體特異oligo-painting探針對(duì)頂芒山羊草進(jìn)行oligo- painting, 根據(jù)1D～7D各探針在M基因組雜交信號(hào), 可以區(qū)分1M～7M染色體。利用本研究開(kāi)發(fā)的3個(gè)oligo (oligo-pAc89、oligo-pAc148和oligo-pAc225)探針組合, 用FAM標(biāo)記組成為綠色熒光探針套ONPS#AC1, 對(duì)頂芒山羊草進(jìn)行oligo-FISH, 獲得1M～7M 7對(duì)染色體FISH信號(hào)特征, 構(gòu)建了頂芒山羊草染色體oligo-FISH核型(圖4)。結(jié)果表明, 該探針套可以在7對(duì)M基因組染色體上分別產(chǎn)生可分辨的FISH信號(hào)特征, 可彼此區(qū)分開(kāi)來(lái)。其中, 1M的著絲粒區(qū)域有較強(qiáng)的FISH信號(hào), 長(zhǎng)臂頂端有一較弱的FISH信號(hào); 2M的長(zhǎng)臂頂端和中部區(qū)域分別有一較弱的和中等強(qiáng)度FISH信號(hào); 3M的短臂和長(zhǎng)臂頂端各有一個(gè)較弱的FISH信號(hào), 其著絲粒區(qū)域有中等強(qiáng)度FISH信號(hào); 4M的短臂頂端和近頂端、長(zhǎng)臂頂端各有較弱的FISH信號(hào), 短臂近著絲粒區(qū)域FISH信號(hào)較強(qiáng); 5M的FISH信號(hào)較弱, 僅在長(zhǎng)臂近著絲粒區(qū)域和中部分別有較弱的FISH信號(hào); 6M的FISH信號(hào)也較弱, 在短臂頂端有較弱的FISH信號(hào), 著絲粒區(qū)域和長(zhǎng)臂近著絲粒區(qū)域信號(hào)更弱; 7M的短臂近著絲粒區(qū)域的FISH信號(hào)強(qiáng)度最大, 著絲粒區(qū)域、長(zhǎng)臂近著絲粒區(qū)域和長(zhǎng)臂與短臂末端各有較弱的FISH信號(hào)。

(圖3)

A1–A7: 探針oligo-pAc105; B1–B7: 探針oligo-pAc148; C1–C7: 探針oligo-pAc149; D1–D7: 探針oligo-pAc217; E1–E7: 探針oligo- pAc259。染色體用DAPI套染(藍(lán)色); 5個(gè)寡聚核苷酸探針用5′TAMRA修飾(紅色)。標(biāo)尺為10 μm。

A1–A7: probe oligo-pAc105; B1–B7: probe oligo-pAc148; C1–C7: probe oligo-pAc149; D1–D7: probe oligo-pAc217; E1–E7: probe oligo-pAc259. Chromosomes were counterstained with DAPI (blue); five oligo probes were modified with 5′TAMRA (red). Bar: 10 μm.

(圖4)

A: 1M～7M信號(hào)組合圖, 紅色為中國(guó)春D亞基因組染色體特異寡核苷酸探針涂染信號(hào), 綠色為ONPS#AC1信號(hào), 白色箭頭示寡核苷酸探針涂染的染色體; B: 圖A中剪切的1M～7M每條染色體的組合圖; C: 從圖B中分離出的1M～7M各染色體的中國(guó)春D亞基因組染色體特異寡核苷酸探針涂染信號(hào)圖; D: 從圖B中分離出的1M～7M每條染色體的ONPS#AC1信號(hào)圖, 標(biāo)尺為10 μm; E: 頂芒山羊草的核型模式圖。

A: the merged signals of chromosome 1M–7M using oligo-painting of sub-genome D chromosome specific oligo-painting probes (red) and oligo-FISH using ONPS#AC1 (green), white arrow points to the painted chromosome with oligonucleotide probe; B: the merged figures of 1 M–7M cutting from (A); C: the oligo-painting FISH signals digitally separated from (B); D: the oligo-FISH using ONPS#AC1 signals digitally separated from (B); E: the oligo-FISH karyotype of. Bars: 10 μm.

進(jìn)一步分析了5個(gè)探針(oligo-pAc105、oligo- pAc148、oligo-pAc149、oligo-pAc217和oligo-pAc259)在頂芒山羊草各染色體上的分布特征。將探針套ONPS#AC1分別與上述5個(gè)探針組合進(jìn)行oligo- FISH (圖5), 結(jié)果發(fā)現(xiàn), oligo-pAc105和oligo-pAc259探針產(chǎn)生的信號(hào)較強(qiáng), 都位于4M染色體的短臂頂端, 它們可用于特異識(shí)別4M染色體; oligo-pAc148在除2M染色體之外的12條染色體長(zhǎng)臂和短臂的頂端均產(chǎn)生較強(qiáng)的FISH信號(hào); oligo-pAc149在3M染色體長(zhǎng)臂近著絲粒區(qū)域有較強(qiáng)的FISH信號(hào), 在6M染色體短臂頂端信號(hào)較弱, 可用于特異的識(shí)別3M和6M染色體; oligo-pAc217在7M染色體短臂近著絲粒區(qū)域有較強(qiáng)的信號(hào), 可用于特異識(shí)別7M染色體。

3 討論

染色體分帶技術(shù)在經(jīng)典細(xì)胞遺傳學(xué)研究中發(fā)揮了重要作用, 但如果外源片段沒(méi)有特征帶型或不顯帶或帶型跟受體材料的帶型無(wú)明顯差異時(shí), 分帶技術(shù)在識(shí)別外源染色質(zhì)方面則很難發(fā)揮較大的作用。另外, 染色體分帶技術(shù)操作步驟繁瑣、需要較高的實(shí)驗(yàn)技能, 難以進(jìn)行高效的篩選和識(shí)別染色體的結(jié)構(gòu)變異。

基因組原位雜交和熒光原位雜交是鑒定特定染色體組來(lái)源或追蹤外源染色體片段的有效手段。近年來(lái)發(fā)展起來(lái)的基于寡核苷酸探針的FISH (oligo- FISH) 技術(shù)已經(jīng)成為染色體研究的重要手段。利用合成寡核苷酸探針的oligo-FISH, 不僅在探針開(kāi)發(fā)和合成方面減少了繁瑣的步驟, 降低了成本, 同時(shí)也簡(jiǎn)化了原位雜交的程序。在小麥中, 開(kāi)發(fā)了串聯(lián)序列重復(fù)(SSR)寡核苷酸探針, 例如oligo- pSc119.2、oligo-pTa535、oligo-k566、oligo-713和oligo-pAs1等常用探針, 用于FISH核型構(gòu)建和識(shí)別小麥染色體[29-33]。在小麥中常用的重復(fù)序列探針, 如oligo-pAs1, oligo-pSc119.2, Afa家族, (AAC)5、(GAA)n, oligo-pTa535和oligo-pTa71已被用于二倍體頂芒山羊草物種以及小麥-頂芒山羊草雙二倍體的FISH核型分析, 但這些探針在頂芒山羊草中的信號(hào)豐富度較為多變, oligo-pSc119.2、oligo-pTa535和oligo-pTa71均只在頂芒山羊草的某些染色體上產(chǎn)生信號(hào), 不能清楚的區(qū)分7對(duì)頂芒山羊草。對(duì)于涉及頂芒山羊草與小麥的異染色質(zhì)材料, (GAA)n不僅在背景小麥染色體上產(chǎn)生豐富的FISH信號(hào), 還可以在頂芒山羊草的染色體產(chǎn)生豐富且與小麥相似的FISH信號(hào), 降低對(duì)頂芒山羊草染色質(zhì)識(shí)別的準(zhǔn)確度。因此, 開(kāi)發(fā)頂芒山羊草特異的重復(fù)序列探針不但可以識(shí)別小麥背景中的外源染色體, 還可以區(qū)分頂芒山羊草的各條染色體; 同時(shí), 將開(kāi)發(fā)的頂芒山羊草特異探針與其他重復(fù)序列探針或基因組探針結(jié)合使用, 可大大提高對(duì)涉及頂芒山羊草的外源種質(zhì)材料的鑒定準(zhǔn)確性。

圖5 5個(gè)oligo探針?lè)謩e與綠色熒光探針套相結(jié)合分別在頂芒山羊草(PI542176)有絲分裂中期染色體的oligo-FISH

A: 探針oligo-pAc105; B: 探針oligo-pAc148; C: 探針oligo-pAc149; D: 探針oligo-pAc217; E: 探針oligo-pAc259; 染色體用DAPI套染(藍(lán)色)。5個(gè)寡聚核苷酸探針用5’TAMRA修飾(紅色); 綠色為ONPS#AC1信號(hào)。標(biāo)尺為10 μm。

A: probe oligo-pAc105; B: probe oligo-pAc148; C: probe oligo-pAc149; D: probe oligo-pAc217; E: probe oligo-pAc259. Chromosomes were counterstained with DAPI (blue); five oligo probes were modified with 5’TAMRA (red). ONPS#AC1 probewas modified with 5’FAM (green). Bars: 10 μm.

伴隨著基因組測(cè)序技術(shù)的發(fā)展和越來(lái)越多物種全基因組序列發(fā)布, 利用生物信息學(xué)分析篩選物種特異重復(fù)序列、開(kāi)發(fā)物種或基因組特異FISH探針成為可能。本研究利用TAREAN從頂芒山羊草的測(cè)序數(shù)據(jù)中鑒定出衛(wèi)星重復(fù)序列并開(kāi)發(fā)成oligo探針, 通過(guò)與小麥參考基因組的序列比對(duì)結(jié)合oligo-FISH,成功開(kāi)發(fā)了頂芒山羊草特異探針。本實(shí)驗(yàn)室Song等[22]根據(jù)普通小麥中國(guó)春的參考基因組序列開(kāi)發(fā)了4D染色體特異的寡核苷酸染色體涂染探針, 這個(gè)探針可以成功的識(shí)別頂芒山羊草的4M染色體。本實(shí)驗(yàn)室進(jìn)一步開(kāi)發(fā)了小麥D亞基因組各染色體特異的寡核苷酸涂染探針, 利用該套探針可以成功的識(shí)別頂芒山羊草的1M～7M染色體。因此, 將oligo- painting技術(shù)和oligo-FISH技術(shù)結(jié)合起來(lái), 發(fā)揮兩者各自的優(yōu)勢(shì), 可以準(zhǔn)確的構(gòu)建頂芒山羊草的FISH核型。本研究首先利用oligo-painting探針在染色體準(zhǔn)確識(shí)別方面的優(yōu)勢(shì), 先識(shí)別出頂芒山羊草的各染色體; 然后在此基礎(chǔ)上利用新開(kāi)發(fā)的頂芒山羊草特異的oligo-FISH探針來(lái)構(gòu)建其核型, 基于此方法構(gòu)建的核型, 可以提高頂芒山羊草染色體識(shí)別的精度, 可以彌補(bǔ)之前利用小麥核型模式圖來(lái)識(shí)別頂芒山羊草各染色體的缺陷。

4 結(jié)論

本研究基于頂芒山羊草的二代測(cè)序數(shù)據(jù)開(kāi)發(fā)出10個(gè)能在頂芒山羊草上產(chǎn)生信號(hào)的寡核苷酸探針, 其中5個(gè)探針可用于鑒定小麥背景中特定的頂芒山羊草染色體。選擇3個(gè)探針組成探針套與普通小麥D亞基因組特異的染色體涂染探針結(jié)合構(gòu)建了頂芒山羊草的FISH核型, 該核型可以準(zhǔn)確識(shí)別頂芒山羊草各條染色體。

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Development of specific oligonucleotide probe library ofand construction of oligo-FISH karyotype

SHI Pei-Yao, CHEN Li-Juan, SUN Hao-Jie, CHENG Meng-Hao, XIAO Jin, YUAN Chun-Xia, WANG Xiu-E, and WANG Hai-Yan*

National Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization / Cytogenetics Institute / Collaborative Innovation Center for Modern Crop Production Co-sponsored by Province and Ministry (CIC-MCP), Nanjing Agricultural University, Nanjing 210095, Jiangsu, China

(, 2=2=14, MM) is a tertiary gene bank for wheat improvement. In order to accurately identify the chromosomes ofM genome or the chromosome segments transferred into wheat, the next-generation sequencing information ofM genome were obtained. Based on the next-generation sequencing information ofM genome, 12 oligonucleotide probes were designed for oligo-FISH analysis according to the 16 possible specific satellite repeats identified. The oligo-FISH results showed that ten of the probes could produce obvious hybridization signals on the chromosomes of. The probe specificity analysis revealed that the five probes generated hybridization signals on the chromosomes of, but there was no obvious hybridization signal on the chromosomes in wheat, which used as the specific probes to identify the chromosomes or chromosome segments of M genome in wheat background. Three probes (oligo-pAc89, oligo-pAc148, and oligo-pAc225) with abundant signal distribution on the chromosomes ofwere selected to form a probe set named ONPS#AC1. Combined with the oligonucleotide probe library developed according to wheat D sub genome, the oligo-FISH karyotype ofwas constructed, which can accurately identify each chromosome of the M genome, providing an important molecular cytogenetic basis for mining, transferring, and utilizing the excellent genes of.

; second-generation sequencing; satellite repeats; Oligo nucleotide probe; fluorescencehybridization

10.3724/SP.J.1006.2023.21048

本研究由國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2020YFE0202900), 中央高校基本科研業(yè)務(wù)費(fèi)(KYZZ2022003), 江蘇省現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系(JATS[2021]463)和江蘇省種業(yè)振興項(xiàng)目(JBGS[2021]006, 013, 047)資助。

This study was supported by the National Key Research and Development Program of China (2020YFE0202900), the Fundamental Research Funds for the Central University (KYZZ2022003), the Jiangsu Province Modern Agricultural Industry Technology System (JATS[2021]463), and the Seed Industry Revitalization Project of Jiangsu Province (JBGS[2021]006, 013, 047).

王海燕, E-mail: hywang@njau.edu.cn

E-mail: 2019101115@njau.edu.cn

2022-07-06;

2022-10-10;

2022-10-21.

URL: https://kns.cnki.net/kcms/detail/11.1809.S.20221020.1828.002.html

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).