擬斯卑爾脫山羊草的FISH核型分析

董磊，董晴，張文利，胡曉龍，王洪剛，王玉海

（1棗莊學(xué)院，山東棗莊 277160；2山東農(nóng)業(yè)大學(xué)農(nóng)學(xué)院/山東農(nóng)業(yè)大學(xué)作物生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，山東泰安 271018）

擬斯卑爾脫山羊草的FISH核型分析

董磊1,2，董晴1，張文利1，胡曉龍1，王洪剛2，王玉海1

（1棗莊學(xué)院，山東棗莊 277160；2山東農(nóng)業(yè)大學(xué)農(nóng)學(xué)院/山東農(nóng)業(yè)大學(xué)作物生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，山東泰安 271018）

【目的】建立擬斯卑爾脫山羊草的FISH核型，分析明確不同來(lái)源擬斯卑爾脫山羊草的FISH核型特點(diǎn)，比較不同擬斯卑爾脫山羊草及其與普通小麥的FISH核型差異?！痉椒ā恳詿晒鈽?biāo)記的寡核苷酸Oligo-pTa535和Oligo-pSc119.2為探針，利用熒光原位雜交（fluorescence in situ hybridization，F(xiàn)ISH）技術(shù)分析pTa535和pSc119.2在不同擬斯卑爾脫山羊草、四倍體小麥和普通小麥染色體上的雜交信號(hào)分布特點(diǎn)；以禾本科植物著絲粒專化寡核苷酸Oligo-CCS1為探針，明確擬斯卑爾脫山羊草的著絲粒位置，測(cè)量擬斯卑爾脫山羊草染色體相關(guān)參數(shù)；通過(guò)FISH核型比較明確不同擬斯卑爾脫山羊草及其與小麥核型的多態(tài)性差異?！窘Y(jié)果】Oligo-pTa535主要分布在小麥的D和A組染色體上，在小麥的B組染色體上僅有零星分布，在5份擬斯卑爾脫山羊草的染色體中未顯示Oligo-pTa535雜交信號(hào)。Oligo-pSc119.2雜交信號(hào)主要分布在小麥的B組染色體上，在小麥的A、D組染色體中分布較少，但在5份擬斯卑爾脫山羊草染色體上均有廣泛分布。根據(jù)Oligo-pTa535和Oligo-pSc119.2雜交信號(hào)在小麥染色體上的分布特點(diǎn)，可以將小麥的不同染色體相互區(qū)分開來(lái)。Oligo-pSc119.2雜交信號(hào)在不同倍性、不同品種的小麥B組染色體上的分布特點(diǎn)基本相似，而不同來(lái)源擬斯卑爾脫山羊草的Oligo-pSc119.2的FISH核型差異較大，甚至在同一細(xì)胞內(nèi)的2條同源染色體上Oligo-pSc119.2雜交信號(hào)的分布也具有明顯差異。不同來(lái)源的擬斯卑爾脫山羊草與小麥B染色體組的FISH核型存在明顯差異。PI542238的7對(duì)染色體均為中間著絲粒染色體，核型公式為2n=14=14m。其余4份擬斯卑爾脫山羊草的4S染色體均為近中著絲粒染色體，其余染色體均為中間著絲粒染色體，核型公式皆為2n=14=12m+2sm?！窘Y(jié)論】擬斯卑爾脫山羊草染色體上含有豐富的與pSc119.2高度同源的重復(fù)序列，不含有與pTa535高度同源的重復(fù)序列。不同來(lái)源的擬斯卑爾脫山羊草之間以及同一來(lái)源的擬斯卑爾脫山羊草的個(gè)體間甚至同一個(gè)體內(nèi)的同源染色體間在 pSc119.2的分布上均具有遺傳多樣性。以O(shè)ligo-pSc119.2為探針建立的擬斯卑爾脫山羊草染色體FISH核型與小麥B組染色體的核型具有顯著差異。利用熒光標(biāo)記的Oligo-pTa535和Oligo-pSc119.2為探針進(jìn)行FISH分析，可以準(zhǔn)確區(qū)分?jǐn)M斯卑爾脫山羊草的不同染色體，并能將擬斯卑爾脫山羊草與小麥的染色體區(qū)分開來(lái)。

普通小麥；擬斯卑爾脫山羊草；核型分析；FISH；寡核苷酸

0 引言

【研究意義】小麥近緣物種是進(jìn)行小麥品種遺傳改良的重要基因資源[1-3]。擬斯卑爾脫山羊草（Aegilops speltoides，SS，2n=14）是山羊草屬擬斯卑爾脫山羊草組中的重要物種，是普通小麥B染色體組最可能的供體種[4-6]。擬斯卑爾脫山羊草遺傳變異豐富，在小麥的遺傳改良中具有重要利用價(jià)值，它的許多抗?。ㄈ鏛r28、Lr35、Lr36、Lr47、Lr51、Sr32、Sr39、Pm12、Pm32和Pm53等）、抗蟲（如Gb5與抗Hessianfly基因）和耐鹽基因已被轉(zhuǎn)入普通小麥[7-14]。擬斯卑爾脫山羊草在小麥的籽粒品質(zhì)性狀改良和耐熱育種中也具有重要價(jià)值[15-16]。植物染色體核型分析是研究植物染色體數(shù)量及結(jié)構(gòu)變異、形態(tài)結(jié)構(gòu)特征和物種起源與進(jìn)化關(guān)系的重要方法，在植物育種特別是植物遠(yuǎn)緣雜交育種中亦具有重要應(yīng)用價(jià)值[4,17-19]。利用FISH技術(shù)，對(duì)不同來(lái)源擬斯卑爾脫山羊草的染色體核型進(jìn)行分析，并與小麥的B染色體組核型進(jìn)行比較，可為擬斯卑爾脫山羊草基因組的遺傳多樣性以及擬斯卑爾脫山羊草染色體組與小麥B染色體組差異研究提供參考依據(jù)?！厩叭搜芯窟M(jìn)展】前人對(duì)山羊草多個(gè)物種的染色體核型進(jìn)行了分析。TEOH等[20]研究發(fā)現(xiàn)不同來(lái)源的擬斯卑爾脫山羊草的 C-分帶核型表現(xiàn)出明顯的多態(tài)性，并建立了4個(gè)不同居群的標(biāo)準(zhǔn)核型及相應(yīng)的核型模式圖。FRIEBE等[17]研究建立了不同來(lái)源的8份單芒山羊草、8份無(wú)芒山羊草和10份頂芒山羊草的標(biāo)準(zhǔn)核型，發(fā)現(xiàn)3種山羊草的C-分帶帶型在種內(nèi)均表現(xiàn)出較高的多態(tài)性。FRIEBE等[21]對(duì)分別來(lái)自前蘇聯(lián)、土耳其和希臘的19份尾狀山羊草的C-分帶帶型進(jìn)行了分析，結(jié)果表明，同一來(lái)源的尾狀山羊草帶型的一致性較高，而不同來(lái)源的尾狀山羊草之間的帶型差異較大。此后，F(xiàn)RIEBE等[22-23]又分別建立了西爾斯山羊草和小傘山羊草的C-帶核型圖及其模式圖。上述多個(gè)山羊草物種的染色體核型圖均是基于 C-帶技術(shù)建立的，信號(hào)及染色體背景顏色均為黑色，反差小，視覺(jué)效果較差。近年來(lái)，在植物染色體核型分析中越來(lái)越多的研究采用某些克隆重復(fù)序列或寡核苷酸序列為探針，利用FISH技術(shù)建立FISH核型圖。特別是同時(shí)利用多個(gè)探針建立的核型圖，探針信號(hào)與染色體背景反差大、條帶豐富、視覺(jué)效果明顯好于C-帶核型，有效提高了染色體核型分析的效果。BADAEVA等[24]以克隆重復(fù)序列pAs1和pSc119.2為探針，建立了4套單芒山羊草的標(biāo)準(zhǔn)核型。MIRZAGHADERI等[25]分別利用寡核苷酸pSc119.2和（CTT）10、pSc119.2和pTa535作為探針，建立了尾狀山羊草、小傘山羊草、柱穗山羊草和鉤刺山羊草的標(biāo)準(zhǔn)FISH核型。TANG等[26]開發(fā)了寡核苷酸序列pTa535和pSc119.2，前者主要分布于普通小麥的A、D組染色體上，在B組染色體上無(wú)分布或分布較少，后者主要分布于小麥B組染色體上，在A組和D組染色體上僅有零星分布。進(jìn)一步支持了小麥A-D基因組之間的同源性遠(yuǎn)高于A-B和D-B之間同源性的研究結(jié)果[27]。將熒光標(biāo)記的Oligo-pTa535和Oligo-pSc119.2用作探針進(jìn)行小麥染色體FISH分析，根據(jù)兩種探針?biāo)a(chǎn)生的 FISH核型可有效區(qū)分小麥的21對(duì)染色體，因此，在小麥的遺傳育種研究中得到廣泛應(yīng)用[28-35]。DELGADO等[36]發(fā)現(xiàn)pTa535在大麥染色體上也有較多分布，根據(jù) pTa535所產(chǎn)生的帶型可將大麥的7對(duì)染色體區(qū)分開。CUADRADO等[37]證明 pSc119.2在黑麥染色體上也有大量分布，并可用于區(qū)分黑麥的染色體?！颈狙芯壳腥朦c(diǎn)】關(guān)于pTa535和pSc119.2在擬斯卑爾脫山羊草染色體上的分布特點(diǎn)以及利用這兩種寡核苷酸序列構(gòu)建擬斯卑爾脫山羊草FISH核型的研究尚未見報(bào)道?！緮M解決的關(guān)鍵問(wèn)題】本研究利用pTa535和pSc119.2兩種寡核苷酸重復(fù)序列為探針，對(duì)5種不同來(lái)源的擬斯卑爾脫山羊草進(jìn)行FISH核型分析，建立FISH核型圖和模式圖，比較分析不同來(lái)源擬斯卑爾脫山羊草的染色體核型差異；并以四倍體小麥和普通小麥為對(duì)照，比較擬斯卑爾脫山羊草S組染色體FISH核型與小麥B組染色體的核型差異。

1 材料與方法

1.1 植物材料

5份擬斯卑爾脫山羊草（PI369594、PI422448、PI542244、PI542238和PI542264，SS，2n=14），由中國(guó)科學(xué)院遺傳與發(fā)育生物學(xué)研究所王道文博士實(shí)驗(yàn)室提供。PI542244主要分布于土耳其東南部的烏爾法地區(qū)，采集于土耳其哈蘭遺址與烏爾法市交界處東北7 km處，具體位置為北緯36.95°，東經(jīng)38.92°，海拔350 m。其主要性狀為：一年生，葉片無(wú)毛，每個(gè)小穗的最外側(cè)兩小花的外稃頂端各有一個(gè)長(zhǎng)芒。PI542238主要分布于土耳其東南部的迪亞巴克爾地區(qū)，采集于土耳其區(qū)域農(nóng)業(yè)研究站，具體位置為北緯37.97°，東經(jīng)40.33°，海拔820 m。其主要性狀為：一年生，葉片無(wú)毛，側(cè)生小穗外稃無(wú)芒。PI542264主要分布于土耳其的烏爾法地區(qū)，采集于土耳其烏爾法市中心以東48 km處，具體位置為北緯37.20°，東經(jīng)39.23°，海拔625 m。其主要性狀為：一年生，穗粗壯，側(cè)生小穗無(wú)芒，穗長(zhǎng)約20 cm，頂部小穗具兩根長(zhǎng)芒。PI369594和PI422448的具體來(lái)源和地理分布不詳，前者的主要性狀為一年生，葉片無(wú)毛，側(cè)生小穗含3—4朵小花，外稃具有細(xì)長(zhǎng)的芒；后者的主要性狀為一年生，葉片有毛，穗細(xì)長(zhǎng)微彎，每小穗含3—5朵小花，除頂端小穗外稃具長(zhǎng)芒外，側(cè)生小穗外稃無(wú)芒，每個(gè)小穗的護(hù)穎及頂端小穗的芒上具有較密集的毛狀刺。栽培二粒小麥DM4（AABB，2n=28）引自中國(guó)農(nóng)業(yè)科學(xué)院作物科學(xué)研究所；普通小麥品種煙農(nóng) 15和中國(guó)春（AABBDD，2n=42）由山東農(nóng)業(yè)大學(xué)小麥染色體工程育種實(shí)驗(yàn)室繁殖保存。

1.2 方法

染色體制片和原位雜交參照KATO等方法[38]進(jìn)行，擬斯卑爾脫山羊草核型制作參照MOLNáR等[39]的標(biāo)準(zhǔn)進(jìn)行。以Tamra（6-carboxytetramethyl -rhodamine）標(biāo)記的禾本科著絲粒專化寡核苷酸CCS1為探針，分析明確擬斯卑爾脫山羊草的著絲粒位置；每個(gè)材料隨機(jī)選取5個(gè)細(xì)胞輪廓清楚、染色體數(shù)目完整、分散良好、形態(tài)清晰的根尖細(xì)胞，利用 Micromeasure 3.3軟件測(cè)量并計(jì)算染色體的相對(duì)長(zhǎng)度和臂比。四倍體小麥和普通小麥的核型制作參照TANG等[26]的標(biāo)準(zhǔn)進(jìn)行。每份山羊草隨機(jī)選取25粒種子、四倍體小麥DM4和2個(gè)普通小麥品種均隨機(jī)選取 3粒種子，利用 Tamra（6-carboxytetramethylrhodamine）標(biāo)記的寡核苷酸pTa535和6-FAM（6-carboxy -fluorescein）標(biāo)記的寡核苷酸pSc119.2為探針，對(duì)其進(jìn)行FISH核型分析。pTa535和pSc119.2 2種重復(fù)序列最初分別來(lái)源于普通小麥和黑麥[26]。CCS1、pTa535和pSc119.2 3種寡核苷酸由上海英維捷基公司合成。

2 結(jié)果

2.1 擬斯卑爾脫山羊草和小麥的FISH分析

同時(shí)利用CCS1、pTa535和pSc119.2對(duì)5種不同來(lái)源的擬斯卑爾脫山羊草、栽培二粒小麥 DM4和 2個(gè)普通小麥品種的根尖細(xì)胞進(jìn)行了原位雜交分析（圖1），CCS1在5份擬斯卑爾脫山羊草所有染色體的著絲粒部位均顯示了明顯的紅色雜交信號(hào)。pTa535在5份擬斯卑爾脫山羊草的根尖細(xì)胞染色體上未顯示明顯的雜交信號(hào)，而pSc119.2在5份擬斯卑爾脫山羊草所有染色體上均顯示了豐富的綠色雜交信號(hào)，這些信號(hào)主要分布于擬斯卑爾脫山羊草染色體的兩端，在染色體的中部幾乎沒(méi)有信號(hào)（圖1-A—圖1-E）。與擬斯卑爾脫山羊草相似，pSc119.2在DM4的B組染色體上也顯示了豐富的雜交信號(hào)，這些信號(hào)主要分布在染色體長(zhǎng)臂的端部、近端部或中部及短臂的端部，在著絲粒附近未見分布。在DM4的A組染色體中，除了4A長(zhǎng)臂末端及 5A短臂末端產(chǎn)生了少量雜交信號(hào)外，A組其他染色體未發(fā)現(xiàn)pSc119.2的雜交信號(hào)。pTa535的信號(hào)主要分布在DM4的A組染色體上，在B組的7對(duì)染色體上未顯示明顯的信號(hào)。除了5B染色體的著絲粒信號(hào)較弱外，CCS1在DM4的其他染色體的著絲粒部位均顯示了強(qiáng)烈的紅色熒光信號(hào)（圖1-F）。

圖1 擬斯卑爾脫山羊草、栽培二粒小麥DM4、煙農(nóng)15和中國(guó)春的染色體FISH分析Fig. 1 FISH patterns of the chromosomes in Ae. speltoides, T. dicoccum S. DM4, T. aestivum Yannong 15 and Chinese Spring

在2個(gè)普通小麥中，pTa535的紅色雜交信號(hào)在D組染色體上的分布最廣泛，其次是A組染色體，而在B組染色體上幾乎沒(méi)有分布或分布極少。與DM4相似，pSc119.2的綠色雜交信號(hào)在普通小麥A組中的4A長(zhǎng)臂末端及5A短臂末端或長(zhǎng)臂中部有少量分布，在2D短臂末端也有少量分布。pSc119.2的雜交信號(hào)在B組染色體上的分布最豐富，且在每對(duì)染色體上分布的位置、數(shù)量均有明顯差異（圖1-G、圖1-H）。

圖2 擬斯卑爾脫山羊草與小麥B組FISH 核型比較Fig. 2 Karyotype comparison between Ae. speltoides and B genomes of wheat

以四倍體小麥DM4、普通小麥品種煙農(nóng)15和中國(guó)春的B組染色體核型為對(duì)照，對(duì)5份擬斯卑爾脫山羊草的FISH核型進(jìn)行了比對(duì)（圖2）。結(jié)果表明，在同一個(gè)部分同源群內(nèi)，DM4的FISH帶型與普通小麥相似，而不同來(lái)源的擬斯卑爾脫山羊草染色體的FISH帶型均與小麥中對(duì)應(yīng)的 B組染色體存在明顯差異。不同擬斯卑爾脫山羊草相同部分同源群內(nèi)的染色體，其FISH帶型也具有明顯差異，甚至同一個(gè)細(xì)胞內(nèi)的一對(duì)同源染色體的FISH帶型也不完全相同。例如，PI369594的2條6S染色體短臂末端的隨體信號(hào)差異明顯，一個(gè)隨體信號(hào)出現(xiàn)在隨體與短臂的交界處，另一個(gè)隨體信號(hào)則出現(xiàn)在隨體末端（白色箭頭所示）；PI422448的2條4S染色體，其中一條染色體長(zhǎng)臂近末端顯示pSc119.2熒光信號(hào)，而其同源染色體的對(duì)應(yīng)部位沒(méi)有雜交信號(hào)（紅色箭頭所示）；PI542238的2條 1S染色體中，一條染色體長(zhǎng)臂末端僅有較弱的熒光信號(hào)，而另一條染色體的對(duì)應(yīng)部位的熒光信號(hào)較強(qiáng)，5S的2條染色體中，一條染色體長(zhǎng)臂上3處顯示熒光信號(hào)，而另一條染色體的長(zhǎng)臂上僅有2處顯示熒光信號(hào)（黃色和綠色箭頭所示）；PI542244的2條1S染色體在長(zhǎng)臂末端存在明顯信號(hào)差異（藍(lán)色箭頭所示），其2條6S染色體的長(zhǎng)臂帶型也存在差異，其中一條染色體的長(zhǎng)臂有2處顯示pSc119.2的雜交信號(hào)，而另一條染色體的長(zhǎng)臂上有3處顯示雜交信號(hào)（灰色箭頭所示）。但是，無(wú)論在四倍體小麥DM4還是在2個(gè)普通小麥品種中，每一對(duì)同源染色體的pSc119.2熒光帶型都完全一致。

2.2 擬斯卑爾脫山羊草的FISH核型圖

在5份擬斯卑爾脫山羊草中分別隨機(jī)選取25粒種子萌發(fā)、采集根尖進(jìn)行染色體制片，獲得良好中期細(xì)胞分裂相的種子數(shù)分別為 8粒（PI369594）、8粒（PI422448）、9粒（PI542244）、9粒（PI542238）和7粒（PI542264）。為了更直觀準(zhǔn)確地描述pSc119.2雜交信號(hào)在5份擬斯卑爾脫山羊草7對(duì)染色體的分布特點(diǎn)，進(jìn)一步建立了它們的 FISH核型模式圖，對(duì)同一份擬斯卑爾脫山羊草不同籽粒來(lái)源的相同序號(hào)染色體存在的差異帶型及頻率進(jìn)行了統(tǒng)計(jì)，并將不同帶型的頻率標(biāo)注到相應(yīng)染色體的FISH模式圖上（圖3）。

2.3 擬斯卑爾脫山羊草的染色體核型參數(shù)

對(duì)5份擬斯卑爾脫山羊草染色體的短臂和長(zhǎng)臂長(zhǎng)度等參數(shù)進(jìn)行了測(cè)定（表1）。PI542238的7對(duì)染色體均為中間著絲粒染色體，其核型公式為2n=14=14m。其余4份山羊草的7對(duì)染色體，除4S為近中著絲粒染色體外，其他染色體均為中間著絲粒染色體，核型公式皆為2n=14=12m+2sm。

3 討論

建立植物 FISH核型圖，不僅在植物的分類及進(jìn)化研究中具有重要意義[40-41]，在植物育種特別是植物遠(yuǎn)緣雜交育種中也具有重要價(jià)值。LINC等[42]以克隆重復(fù)序列pSc119.2和Afa family為探針建立了二倍體長(zhǎng)穗偃麥草的 FISH核型，不僅能區(qū)分二倍體長(zhǎng)穗偃麥草的7對(duì)染色體，而且也能將偃麥草與普通小麥的染色體完全區(qū)分開，并根據(jù)核型特征進(jìn)一步對(duì)普通小麥-二倍體長(zhǎng)穗偃麥草一套附加系及11個(gè)普通小麥-二倍體長(zhǎng)穗偃麥草雙端體附加系進(jìn)行了鑒定，成功地識(shí)

別出其中的偃麥草染色體或染色體臂。DELGADO等[36]證明，依據(jù)寡核苷酸Oligo-pTa535和Oligo-pAs1在野生大麥染色體上的分布特點(diǎn)，可以將7對(duì)大麥染色體與小麥染色體區(qū)分開。本研究結(jié)果表明，通過(guò)FISH核型分析可以在染色體基組的水平上準(zhǔn)確區(qū)分?jǐn)M斯卑爾脫山羊草和小麥的染色體，建立的擬斯卑爾脫山羊草的 FISH核型，揭示了不同來(lái)源擬斯卑爾脫山羊草及其與小麥B染色體組的多樣性差異，它在區(qū)分?jǐn)M斯卑爾脫山羊草不同染色體及鑒定小麥遺傳背景中的擬斯卑爾脫山羊草染色體或染色體片段中具有利用價(jià)值。

表1 擬斯卑爾脫山羊草的染色體參數(shù)Table 1 Chromosome parameters of Ae. speltoides used in this study

圖3 擬斯卑爾脫山羊草的染色體FISH核型模式圖Fig. 3 FISH idiogram of the chromosomes of five accessions of Ae. speltoides

關(guān)于小麥屬B染色體組的起源問(wèn)題，至今尚未找到與其完全相同的供體。顏濟(jì)等[43]綜合分析了目前多方面研究結(jié)果認(rèn)為，小麥的B染色體組來(lái)源于擬斯卑爾脫山羊草S染色體組的可能性很大，S染色體組通過(guò)組間染色體的局部代換、重組及基因突變等重要變化而演化形成B染色體組。通過(guò)對(duì)擬斯卑爾脫山羊草S染色體組FISH核型與四倍體小麥DM4及2個(gè)普通小麥品種 B染色體組 FISH核型的比較分析發(fā)現(xiàn)，5份擬斯卑爾脫山羊草的pSc119.2雜交信號(hào)分布與四倍體小麥DM4和2個(gè)普通小麥品種的B組染色體確實(shí)存在明顯差異。這表明，擬斯卑爾脫山羊草的染色體組與小麥的B染色體組確實(shí)存在較大的遺傳分化。這些結(jié)果對(duì)于深入研究小麥B染色體組的起源與進(jìn)化及其與擬斯卑爾脫山羊草染色體組的遺傳關(guān)系具有參考價(jià)值。

由 U.S. National Plant Germplasm System網(wǎng)站（ https://npgsweb.ars-grin.gov/gringlobal/view2.aspx?d v=web_site_taxon_accessionlist¶ms=:taxonomyid= 1547;:siteid=19）信息可知，本研究的5份擬斯卑爾脫山羊草中，除PI369594和PI422448等2份擬斯卑爾脫山羊草的來(lái)源和和分布不詳外，其他3份山羊草均來(lái)自土耳其。其中，PI542244和PI542264主要分布于土耳其的烏爾法地區(qū)，PI542238主要分布于土耳其的迪亞巴克爾地區(qū)，而烏爾法和迪亞巴克爾均位于土耳其的東南部，地理位置相近。另外，PI542244、PI542238和PI542264的性狀差異明顯。這說(shuō)明，擬斯卑爾脫山羊草在土耳其東南部地區(qū)（特別是烏爾法和迪亞巴克爾）可能有著廣泛分布，且遺傳變異豐富，加之?dāng)M斯卑爾脫山羊草具有異花授粉習(xí)性[44]，具有遺傳差異的擬斯卑爾脫山羊草混生在一起，相互之間容易發(fā)生天然雜交，這可能是造成不同擬斯卑爾脫山羊草的FISH核型表現(xiàn)差異的原因。

4 結(jié)論

擬斯卑爾脫山羊草染色體上含有豐富的與pSc119.2高度同源的重復(fù)序列，不含有與pTa535高度同源的重復(fù)序列。不同來(lái)源擬斯卑爾脫山羊草之間以及同一來(lái)源的擬斯卑爾脫山羊草的單株間甚至單株內(nèi)的同源染色體間在pSc119.2的分布上均具有遺傳多樣性。以O(shè)ligo-pSc119.2建立的擬斯卑爾脫山羊草染色體FISH核型與小麥B組染色體的核型具有顯著差異。利用熒光標(biāo)記的Oligo-pTa535和Oligo-pSc119.2為探針進(jìn)行 FISH分析，可以準(zhǔn)確區(qū)分?jǐn)M斯卑爾脫山羊草的不同染色體，并能將擬斯卑爾脫山羊草與小麥的染色體區(qū)分開來(lái)。

[1] 周陽(yáng), 何中虎, 張改生, 夏蘭琴, 陳新民, 高永超, 井趙斌, 于廣軍. 1BL/1RS易位系在我國(guó)小麥育種中的應(yīng)用. 作物學(xué)報(bào), 2004, 30(6): 531-535.

ZHOU Y, HE Z H, ZHANG G S, XIA L Q, CHEN X M, GAO Y C, JING Z B, YU G J. Utilization of 1BL/1RS translocation in wheat breeding in China. Acta Agrinomica Sinica, 2004, 30(6): 531-535. (in Chinese)

[2] 張麗, 張懷渝, 任正隆, 羅培高. 小麥-黑麥 1BL/1RS易位系在小麥遺傳改良中的應(yīng)用現(xiàn)狀及前景分析. 分子植物育種, 2010, 8(14): 1-8.

ZHANG L, ZHANG H Y, REN Z L, LUO P G. The progress and prospect of 1BL/RS translocation line in wheat genetic improvement. Molecular Plant Breeding, 2010, 8(14): 1-8. (in Chinese)

[3] 鐘冠昌, 穆素梅, 張正斌. 麥類遠(yuǎn)緣雜交. 北京: 科學(xué)出版社, 2002: 92-97.

ZHONG G C, MU S M, ZHANG Z B. Wide Hybridization in Triticeae. Beijing: China Science Press, 2002: 92-97. (in Chinese)

[4] MAESTRA B, NARANJO T. Homoeologous relationships of Aegilops speltoides chromosomes to bread wheat. Theoretical and Applied Genetics, 1998, 97: 181-186.

[5] SALSE J, CHAGUé V, BOLOT S, MAGDELENAT G, HUNEAU C, PONT C, BELCRAM H, COULOUX A, GARDAIS S, EVRARD A, SEGURENS B, CHARLES M, RAVEL C, SAMAIN S, CHARMET G, BOUDET N, CHALHOUB B. New insights into the origin of the B genome of hexaploid wheat: Evolutionary relationships at the SPA genomic region with the S genome of the diploid relative Aegilops speltoides. BMC Genomics, 2008, 9: 555.

[6] PETERSEN G, SEBERG O, YDE M, BERTHELSEN K. Phylogenetic relationships of Triticum and Aegilops and evidence for the origin of the A, B, and D genomes of common wheat (Triticum aestivum). Molecular Phylogenetics and Evolution, 2006, 39(1): 70-82.

[7] MAGO R, VERLIN D, ZHANG P, BANSAL U, BARIANA H, JIN Y, ELLIS J, HOXHA S, DUNDAS I. Development of wheat-Aegilops speltoides recombinants and simple PCR-based markers for Sr32 and a new stem rust resistance gene on the 2S#1 chromosome. Theoretical and Applied Genetics, 2013, 126: 2943-2955.

[8] JIA J, DEVOS K M, CHAO S, MILLER T E, READER S M, GALE M D. RFLP-based maps of the homoeologous group-6 chromosomes of wheat and application in the tagging of Pm12, a powdery mildew resistance gene transferred from Aegilops speltoides to wheat. Theoretical and Applied Genetics, 1996, 92: 559-565.

[9] NAIK S, GILL K S, PRAKASA RAO V S, GUPTA V S, TAMHANKAR S A, PUJAR S, GILL B S, RANJEKAR P K. Identification of a STS marker linked to the Aegilops speltoidesderived leaf rust resistance gene Lr28 in wheat. Theoretical and Applied Genetics, 1998, 97: 535-540.

[10] PETERSEN S, LYERLY J H, WORTHINGTON M L, PARKS W R, COWGER C, MARSHALL D S, BROWN-GUEDIRA G, MURPHY J P. Mapping of powdery mildew resistance gene Pm53 introgressed from Aegilops speltoides into soft red winter wheat. Theoretical and Applied Genetics, 2015, 128: 303-312.

[11] YU G T, KLINDWORTH D L, FRIESEN T L, FARIS J D, ZHONG S B, RASMUSSEN J B, XU S S. Development of a diagnostic co-dominant marker for stem rust resistance gene Sr47 introgressed from Aegilops speltoides into durum wheat. Theoretical and Applied Genetics, 2015, 128: 2367-2374.

[12] DUBCOVSKY J, LUKASZEWSKI A J, ECHAIDE M, ANTONELLI E F, PORTER D R. Molecular characterization of two Triticum speltoides interstitial translocations carrying leaf rust and greenbug resistance genes. Crop Science, 1998, 38: 1655-1660.

[13] NOORI S A S. Assessment for salinity tolerance through intergeneric hybridisation: Triticum durum × Aegilops speltoides. Euphytica, 2005, 146: 149-155.

[14] YUDINA R S, LEONOVA I N, SALINA E A, KHLESTKINA E K. Change in salt tolerance of bread wheat as a result of the introgression of the genetic material of Aegilops speltoides and Triticum timopheevii. Russian Journal of Genetics: Applied Research, 2016,6(3): 244-248.

[15] PSHENICHNIKOVA T A, SIMONOV A V, ERMAKOVA M F, CHISTYAKOVA A K, SHCHUKINA L V, MOROZOVA E V. The effects on grain endosperm structure of an introgression from Aegilops speltoides Tausch. into chromosome 5A of bread wheat. Euphytica, 2010, 175: 315-322.

[16] AWLACHEW Z T, SINGH R, KAUR S, BAINS N S, CHHUNEJA P. Transfer and mapping of the heat tolerance component traits of Aegilops speltoides in tetraploid wheat Triticum durum. Molecular Breeding, 2016, 36: 78.

[17] FRIEBE B, BADAEVA E D, KAMMER K, GILL B S. Standard karyotypes of Aegilops uniaristata, Ae. mutica, Ae. comosa subspecies comosa and heldreichii (Poaceae). P1ant Systematics Evolution, 1996, 202: 199-210.

[18] FERNáNDEZ-CALVIN B, ORELLANA J. Metaphase-I bound-arm frequency and genome analysis in wheat-Aegilops hybrids. 2. Cytogenetical evidence for excluding Ae. sharonensis as the donor of the B genome of polyploid wheats. Theoretical and Applied Genetics, 1993, 85(5): 587-592.

[19] FRIEBE B, QI L L, NASUDA S, ZHANG P, TULEEN N A, GILL B S. Development of a complete set of Triticum aestivum-Aegilops speltoides chromosome addition lines. Theoretical and Applied Genetics, 2000, 101: 51-58.

[20] TEOH S B, MILLER T E, READER S M. Intraspecific variation in C-banded chromosomes of Aegilops comosa and Ae. Speltoides. Theoretical and Applied Genetics, 1983, 65: 343-348.

[21] FRIEBE B, SCHUBERT V, BLIITHNER W D, HAMMER K. C-banding pattern and polymorphism of Aegilops caudata and chromosomal constitutions of the amphiploid T. aestivum-Ae. caudata and six derived chromosome addition lines. Theoretical and Applied Genetics, 1992, 83: 589-596.

[22] FRIEBE B, JIANG J, TULEEN N, GILL B S. Standard karyotype of Triticum umbellulatum and the characterization of derived chromosome addition and translocation lines in common wheat. Theoretical and Applied Genetics, 1995, 90: 150-156.

[23] FRIEBE B, TULEEN N A, GILL B S. Standard karyotype of Triticum searsii and its relationship with other S-genome species and common wheat. Theoretical and Applied Genetics, 1995, 91: 248-254.

[24] BADAEVA E D, DEDKOVA O S, ZOSHCHUK S A, AMOSOVA A V, READER S M, BERNARD M, ZELENIN A V. Comparative analysis of the N-genome in diploid and polyploid Aegilops species. Chromosome Research, 2011, 19: 541-548.

[25] MIRZAGHADERI G, HOUBEN A, BADAEVA E D. Molecularcytogenetic analysis of Aegilops triuncialis and dentification of its chromosomes in the background of wheat. Molecular Cytogenetics, 2014, 7: 91.

[26] TANG Z X, YANG Z J, FU S L. Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis. Journal of Applied Genetics, 2014, 55: 313-318.

[27] FERNFINDEZ-CALVFN B, ORELLANA J. Metaphase I-bound arms frequency and genome analysis in wheat-Aegilops hybrids. 3. Similar relationships between the B genome of wheat and S or SLgenomes of Ae. speltoides, Ae. longissima and Ae. sharonensis. Theoretical and Applied Genetics, 1994, 88: 1043-1049.

[28] 王玉海, 何方, 鮑印廣, 明東風(fēng), 董磊, 韓慶典, 李瑩瑩, 王洪剛.高抗白粉病小麥-山羊草新種質(zhì)TA002的創(chuàng)制和遺傳研究. 中國(guó)農(nóng)業(yè)科學(xué), 2016, 49(3): 418-428.

WANG Y H, HE F, BAO Y G, MING D F, DONG L, HAN Q D, LI Y Y, WANG H G. Development and genetic analysis of a novel wheat-Aegilops germplasm TA002 resistant to powdery mildew. Scientia Agricultura Sinica, 2016, 49(3): 418-428. (in Chinese)

[29] WANG Y H, WANG H G. Characterization of three novel wheat-Thinopyrum intermedium addition lines with novel storage protein subunits and resistance to both powdery mildew and stripe rust. Journal of Genetics and Genomics, 2016, 43: 45-48.

[30] KRUPPA K, TüRK?SI E, MAYER M, TóTH V, VIDA G, SZAKáCS é, MOLNáR-LáNG M. McGISH identification and phenotypic description of leaf rust and yellow rust resistant partial amphiploids originating from a wheat × Thinopyrum synthetic hybrid cross. Journal of Applied Genetics, 2016, 57: 427-437.

[31] LI G R, WANG H J, LANG T, LI J B, LA S X, YANG E N, YANG Z J. New molecular markers and cytogenetic probes enable chromosome identification of wheat-Thinopyrum intermedium introgression lines for improving protein and gluten contents. Planta, 2016, 244: 865-876.

[32] 陳雷, 李萌, 王洋洋, 邱玲, 湯述堯, 唐宗祥, 符書蘭. 小麥-黑麥1BL/1RS 易位系中的染色體結(jié)構(gòu)變異. 麥類作物學(xué)報(bào), 2015, 35(8): 1038-1043.

CHEN L, LI M, WANG Y Y, QIU L, TANG S Y, TANG Z X, FU S L. Structural variation of chromosomes in wheat-rye 1BL/1RS translocation lines. Journal of Triticeae Crops, 2015, 35(8): 1038-1043. (in Chinese)

[33] SCHNEIDER A, RAKSZEGI M, MOLNáR-LáNG M, SZAKáCS é. Production and cytomolecular identification of new wheat-perennial rye (Secale cereanum) disomic addition lines with yellow rustresistance (6R) and increased arabinoxylan and protein content (1R, 4R, 6R). Theoretical and Applied Genetics, 2016, 129: 1045-1059.

[34] ZHUANG L F, LIU P, LIU Z Q, CHEN T T, WU N, SUN L, QI Z J. Multiple structural aberrations and physical mapping of rye chromosome 2R introgressed into wheat. Molecular Breeding, 2015, 35: 133.

[35] FU S L, REN Z L, CHEN X M, YAN B J, TAN F Q, FU T H, TANG Z X. New wheat-rye 5DS-4RS·4RL and 4RS-5DS·5DL translocation lines with powdery mildew resistance. Journal of Plant Research, 2014, 127: 743-753.

[36] DELGADO A, CARVALHO A, MARTíN A C, MARTíN A, LIMA-BRITO J. Use of the synthetic Oligo-pTa535 and Oligo-pAs1 probes for identification of Hordeum chilense-origin chromosomes in hexaploid Tritordeum. Genetic Resources and Crop Evolution, 2016, 63: 945-951.

[37] CUADRADO A, JOUVE N. Evolutionary trends of different repetitive DNA sequences during speciation in the genus secale. The Journal of Heredity, 2002, 93(5): 339-345.

[38] KATO A, JONATHAN C L, BIRCHLER J A. Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize. Proceedings of the National Academy of Sciences of the USA, 2004, 101(37): 13554-13559.

[39] MOLNáR I, KUBALáKOVá M, ?IMKOVá H, FARKAS A, CSEH A, MEGYERI M, VRáNA J, MOLNáR-LáNG M, DOLE?EL J. Flow cytometric chromosome sorting from diploid progenitors of bread wheat, T. urartu, Ae. speltoides and Ae. Tauschii. Theoretical and Applied Genetics, 2014, 127: 1091-1104.

[40] BADAEVA E D, AMOSOVA A V, MURAVENKO O V, SAMATADZE T E, CHIKIDA N N, ZELENIN A V, FRIEBE B, GILL B S. Genome differentiation in Aegilops. 3. Evolution of the D-genome cluster. Plant Systematics and Evolution, 2002, 231: 163-190.

[41] BADAEVA E D, AMOSOVA A V, SAMATADZE T E, ZOSHCHUK S A, SOSHTAK N G, CHIKIDA N N, ZELENIN A V, RAUPP W J, FRIEBE B, GILL B S. Genome differentiation in Aegilops. 4. Evolution of the U-genome cluster. Plant Systematics Evolution, 2004, 246: 45-76.

[42] LINC G, SEPSI A, MOLNáR-LáNG M. A FISH karyotype to study chromosome polymorphisms for the Elytrigia elongata E genome. Cytogenetic and Genome Research, 2012, 136: 138-144.

[43] 顏濟(jì), 楊俊良. 小麥族生物系統(tǒng)學(xué)（第一卷第二版）, 小麥-山羊草復(fù)合群. 北京: 中國(guó)農(nóng)業(yè)出版社, 2013: 43-53.

YAN J, YANG J L. Triticeae Systematics (volume 1 2ndedition), Triticum- Aegilops complx. Beijing: China Agriculture Press, 2013: 43-53. (in Chinese)

[44] 董玉琛, 鄭殿升. 中國(guó)小麥遺傳資源. 北京: 中國(guó)農(nóng)業(yè)出版社, 2000: 152.

DONG Y C, ZHENG D S. The Wheat Genetic Resource in China. Beijing: China Agriculture Press, 2000: 152. (in Chinese)

（責(zé)任編輯 李莉）

Karyotypic Analysis of Aegilops speltoides Revealed by FISH

DONG Lei1,2, DONG Qing1, ZHANG WenLi1, HU XiaoLong1, WANG HongGang2, WANG YuHai1
(1Zaozhuang University, Zaozhuang 277160, Shandong;2Agronomy College, Shandong Agricultural University/State Key Laboratory of Crop Biology of Shandong Agricultural University, Tai’an 271018, Shandong)

【Objective】The objective of this study is to reveal the karyotypic polymorphism of Aegilops speltoides (Aegilops short for Ae. hereafter) and the karyotypic difference between common wheat and Ae. speltoides via the establishment of FISH karyotype of Ae. speltoides.【Method】Multicolor fluorescence in situ hybridization (mc-FISH) was employed to detect the distribution of Oligo-pSc119.2 and Oligo-pTa535 in chromosomes of Ae. speltoides; centromere-specific oligonucleotide CCS1 wasused to identify the location of centromeres on chromosomes; FISH karyotype comparison was conducted to show the karyotypic differences between Ae. speltoides and wheat.【Result】In wheat, oligo-pTa535 signals were observed mainly on chromosomes in A and D genomes, and only very sporadic signals were found in B genome. However, oligo-pTa535 signals were absent in Ae. speltoides of five accessions. Oligo-pSc119.2, compared to a little distribution in A and D genomes, shined plentiful fluorescence in the whole genome B in wheat, and especially in S genomes in Ae. speltoides of five accessions used in this study. Different pairs of chromosomes in wheat could be distinguished from each other according to the distribution of Oligo-pSc119.2 and Oligo-pTa535 on chromosomes of wheat. FISH patterns produced by Oligo-pSc119.2 in wheat showed similarity among wheat materials whether of different ploidy or of different varieties of same ploidy, and that in Ae. speltoides varied depending on accessions. Even homologous chromosomes in one cell in Ae. speltoides exhibited differences in FISH pattern. Oligo-pSc119.2 FISH patterns of five accessions each showed obvious differences from that of B genomes in wheat. Four of five Ae. speltoides accessions possess six pairs of metacentric chromosomes except for homologous pairs 4S of submetacentric chromosomes, which were involved in the karyotype formula 2n = 14 = 12m + 2sm, and the rest one, PI542238, however, houses seven pairs of metacentric chromosomes which resulted in the karyotype formula 2n = 14 = 14m. 【Conclusion】 Chromosomes of Ae. speltoides house rich repetitive DNA sequences highly homologous to pSc119.2 and lack that highly homologous to pTa53. The distribution of pSc119.2 on chromosomes of Ae. speltoides showed differences between accessions, between plants of one accession and even between homologous chromosomes in one plant. FISH patterns produced by Oligo-pSc119.2 on Ae. speltoides chromosomes exhibited significant difference from that on chromosomes of B genomes in wheat. FISH analysis, using Oligo-pSc119.2 and Oligo-pTa535 as probes, not only could differentiate the chromosomes in wheat from that in Ae. speltoides, but also could dishtinguish the chromosomes from each other whether in wheat or in Ae. speltoides.

Triticum aestivum; Aegilops speltoides; karyotypic analysis; FISH; oligonucleotides

2017-01-03；接受日期：2017-02-17

國(guó)家“十三五”重點(diǎn)研發(fā)計(jì)劃（2016YFD0102004）、山東省博士基金（BS2011SW053）、作物生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室開放課題（2015KF06）、棗莊學(xué)院國(guó)家基金預(yù)研究項(xiàng)目（2016YY14）

聯(lián)系方式：董磊，E-mail：dong2012lei@163.com。通信作者王洪剛，E-mail：hgwang@edu.sdau.cn。通信作者王玉海，E-mail：yhwang92@163.com