陳方永,王引,倪海枝,張啟,顏幫國(guó)

(1.浙江省柑橘研究所,浙江黃巖318026;2.浙江省蘭溪市農(nóng)業(yè)局經(jīng)濟(jì)特產(chǎn)技術(shù)推廣站,浙江蘭溪321100)

浙江2個(gè)地產(chǎn)白沙枇杷種質(zhì)資源親緣關(guān)系鑒定

陳方永1*,王引1,倪海枝1,張啟2,顏幫國(guó)1

(1.浙江省柑橘研究所,浙江黃巖318026;2.浙江省蘭溪市農(nóng)業(yè)局經(jīng)濟(jì)特產(chǎn)技術(shù)推廣站,浙江蘭溪321100)

摘要在綜合觀察浙江2個(gè)白沙枇杷(黃巖軟條白沙和蘭溪白沙)種質(zhì)形態(tài)特征的基礎(chǔ)上,對(duì)兩者進(jìn)行倍性、氣孔檢測(cè)和基于分子標(biāo)記的親緣關(guān)系研究.結(jié)果表明:黃巖軟條白沙單果質(zhì)量是蘭溪白沙的1.38倍,而蘭溪白沙的種子數(shù)、果皮厚度、果實(shí)硬度分別是黃巖軟條白沙的1.36倍、1.096倍、1.22倍;流式細(xì)胞儀倍性檢測(cè)表明,兩者均為二倍體;氣孔檢測(cè)表明,兩者的氣孔密度和面積大小差異顯著,黃巖軟條白沙氣孔面積比蘭溪白沙大43.4%,氣孔密度比蘭溪白沙小17.35%;基于簡(jiǎn)單序列重復(fù)標(biāo)記的遺傳聚類分析表明,黃巖軟條白沙的遺傳相似系數(shù)為0.40,蘭溪白沙為0.45.由此可見,2個(gè)品種在成熟期的差異主要受種性和地域因素的影響,可認(rèn)為是不同的栽培種質(zhì).

關(guān)鍵詞白沙枇杷;同類種質(zhì);親緣關(guān)系;差異

浙江省是中國(guó)最重要的白沙枇杷即白色果肉枇杷產(chǎn)區(qū).全省現(xiàn)有白沙枇杷種植面積1.2×104hm2左右,年產(chǎn)量約6.5×104t,年產(chǎn)值25億元左右.浙江省臺(tái)州市的白沙枇杷主產(chǎn)區(qū)在原黃巖縣種植區(qū)域(現(xiàn)黃巖區(qū)、路橋區(qū)及椒江區(qū))與臨海、玉環(huán)、溫嶺等縣市;寧波市的主產(chǎn)區(qū)在寧海、象山、鄞州;杭州市的主產(chǎn)區(qū)在余杭塘棲、淳安、建德;金華市的主產(chǎn)區(qū)在蘭溪;麗水市的主產(chǎn)區(qū)在蓮都區(qū)及青田、松陽(yáng)等地;溫州市的主產(chǎn)區(qū)在樂清、蒼南、瑞安等地.各地種植的白沙枇杷品種如下:臺(tái)州以“黃巖軟條白沙”“槐梵”“溪上白沙”為主;寧波以“寧海白”為主;蘭溪以當(dāng)?shù)匚疵陌咨宠凌似贩N為主;麗水、溫州除地方資源外,主要為“黃巖軟條白沙”“寧海白”;杭州市的主栽品種為“余杭軟條白沙”“余杭硬條白沙”,其他各縣都是混合種植各類白沙品種.

白沙枇杷市場(chǎng)價(jià)格潛力大,尤其在沿海經(jīng)濟(jì)發(fā)達(dá)地區(qū),其價(jià)格是紅沙枇杷的5～10倍及以上;因此,在考慮優(yōu)生區(qū)的前提下,發(fā)展白沙(也稱白肉)枇杷具有十分重要的現(xiàn)實(shí)意義.然而,白沙枇杷存在裂果、腐爛率較高的問題,要推動(dòng)白沙枇杷產(chǎn)業(yè)可持續(xù)發(fā)展的突破口在于選育厚皮、抗逆性較強(qiáng)的種質(zhì)資源.梁國(guó)魯?shù)萚1]對(duì)四川7個(gè)紅沙枇杷品種和1個(gè)白沙枇杷品種的染色體進(jìn)行研究表明,白沙品種是紅沙品種的突變體類型,其染色體帶不具橙紅帶,核型是3B類型,而紅沙染色體帶具鮮明的橙紅帶,核型是2A類型.周坤杰等[2]對(duì)蘇州地區(qū)9個(gè)白沙枇杷種植情況調(diào)查表明,“豐玉”相對(duì)為優(yōu).任國(guó)慧等[3]用SPSS 16.0軟件對(duì)江蘇省20份白沙枇杷種質(zhì)的果實(shí)性狀進(jìn)行了多因子聚類分析,其分值最高、優(yōu)先推薦種植的品種為“冠玉”,分值最低的是“早黃”.袁衛(wèi)明等[4]在蘇州選育出了雜交新種質(zhì)“常綠5號(hào)”.該品種具早熟、避雨、豐產(chǎn)、矮化、抗凍的特點(diǎn),與母本(父本“甜種”,母本“白玉”)相比,其單果質(zhì)量增加22.60%,植株矮化率達(dá)31.14%,冠幅增加25.58%～30.76%,凍害率下降10%左右:可見,其綜合性狀偏向高值親本,有適度栽培價(jià)值.LI等[5]采用基于表達(dá)序列標(biāo)簽(expressed sequence tags,EST)的簡(jiǎn)單序列重復(fù)(simple sequence repeats,SSR)標(biāo)記技術(shù)研究了來(lái)自不同地區(qū)的47份枇杷材料的親緣關(guān)系,證實(shí)了來(lái)自同一地區(qū)的枇杷材料親緣關(guān)系相近.綜上所述,雖然前人對(duì)于白沙枇杷的品種選育進(jìn)行了相關(guān)研究,但目前還沒有選育出厚皮、抗逆性較強(qiáng)的新種質(zhì).筆者在多年的研究中發(fā)現(xiàn),同是白沙枇杷類的品種(材料),“黃巖軟條白沙”在自然栽培條件下常年裂果且腐爛現(xiàn)象嚴(yán)重,而浙江省蘭溪市主栽的“蘭溪白沙枇杷”(未命名,筆者暫定名)在成熟期裂果腐爛現(xiàn)象并不顯著;因此,本文在綜合觀察這2個(gè)品種(材料)形態(tài)特征的基礎(chǔ)上,對(duì)它們進(jìn)行了倍性、氣孔特征和基于分子標(biāo)記的親緣關(guān)系研究,旨在為新種質(zhì)的挖掘推廣提供技術(shù)參考.

1　材料與方法

1.1材料

以10年生為主的黃巖軟條白沙枇杷265株(0.33 hm2)、蘭溪白沙高接5株(浙江省臺(tái)州市黃巖區(qū)嶼頭鄉(xiāng)上鳳村浙江省柑橘研究所山地試驗(yàn)點(diǎn));以10年生為主的蘭溪白沙枇杷園239株(0.33 hm2)(浙江省蘭溪市女埠街道虹霓山枇杷專業(yè)合作社).

SSR分子標(biāo)記檢測(cè)除上述材料外,還包括試驗(yàn)點(diǎn)內(nèi)的溪上白沙、傀梵白沙、白荔枝、軟條白沙變異(永路)、福建紅白沙枇杷5個(gè)材料.

1.2方法

1.2.1黃巖軟條白沙和蘭溪白沙種性特點(diǎn)調(diào)查

在黃巖、蘭溪2地隨機(jī)采集5株白沙枇杷,每株隨機(jī)選取20片成年葉和2 kg成熟果實(shí),測(cè)定葉片縱橫徑、單果質(zhì)量、果實(shí)種子數(shù)、果皮厚度等;調(diào)查當(dāng)?shù)貧夂蛱攸c(diǎn).

1.2.2黃巖軟條白沙和蘭溪白沙的倍性檢測(cè)

參照陳方永[6]的方法用流式細(xì)胞儀(Cy Flow?Space,德國(guó)Partec公司)檢測(cè).

1.2.3黃巖軟條白沙和蘭溪白沙的葉片氣孔檢測(cè)

參照陳方永[6]的方法用光學(xué)顯微鏡(Leica DMI3000)檢測(cè).

1.2.4基于SSR分子標(biāo)記的黃巖軟條白沙和蘭溪白沙的親緣關(guān)系檢測(cè)

1.2.4.1DNA提取

參照陳方永等[7]和何橋[8]的方法提取葉片總DNA,檢測(cè)DNA純度與濃度,結(jié)果見表1.

表1　DNA提取質(zhì)量Table 1 Mass of extracted DNA

1.2.4.2聚合酶鏈?zhǔn)椒磻?yīng)體系建立

SSR-聚合酶鏈?zhǔn)椒磻?yīng)(polymerase chain reaction,PCR)擴(kuò)增體系總體積為30μL:10×PCR緩沖液3.0μL,50×d NTP混合物0.5μL,引物F (5′端摻入熒光)0.5μL和引物R 0.5μL(表2), Ta KaRa Taq酶0.4μL,50 ng/μL DNA模板1.0 μL,加dd H2O補(bǔ)至30μL.反應(yīng)程序:94℃預(yù)變性5 min;94℃變性15 s,55℃退火30 s,先72℃延伸60 s,再72℃延伸7 min,40個(gè)循環(huán);最后4℃保存.上樣于1%瓊脂糖凝膠孔中,120 V恒壓電泳15 min,在紫外透射光下觀察并拍照.

1.2.4.3短串聯(lián)重復(fù)序列檢測(cè)

取96孔反應(yīng)板,用記號(hào)筆標(biāo)明板名、實(shí)驗(yàn)日期,制作電子版短串聯(lián)重復(fù)序列(short tandem repeat, STR)檢測(cè)表,并自動(dòng)生成上機(jī)表.使用連續(xù)加樣器,吸取990μL HIDI和ROX500混合物,加入到96孔反應(yīng)板中,每孔10μL,將96孔板置于平板離心機(jī)中,離心500 g即停;使用12排10μL排槍,對(duì)照STR檢測(cè)表,在96孔板對(duì)應(yīng)的孔中加入50 pg樣品,將96孔板置于平板離心機(jī)中,離心500 g即停;使用封板膜密封96孔板,振蕩,將96孔板置于平板離心機(jī)中,離心500 g即停;然后置于PCR儀上,變性程序?yàn)?8℃,5 min,不加熱熱蓋,程序結(jié)束后立即將96孔板置于冰水混合物上急速冷卻;將96孔板置于平板離心機(jī)中,離心2 000 g即停.使用3730XL測(cè)序儀檢測(cè)STR樣本.

1.2.4.4引物擴(kuò)增

根據(jù)已發(fā)布的蘋果SSR引物擴(kuò)增信息及對(duì)應(yīng)的聚丙烯酰胺凝膠電泳結(jié)果[9],選取表2中的3對(duì)候選引物擴(kuò)增枇杷基因組DNA.

表2　供試的3對(duì)蘋果SSR引物的遺傳學(xué)參數(shù)表Table 2 Genetic parameters of three pairs of apple SSR primers

2結(jié)果與分析

2.1種性特征比較

從表3和圖1可以看出:蘭溪白沙比黃巖軟條白沙枇杷平均單果質(zhì)量低27.33%;黃巖軟條白沙裂果腐爛率、單果質(zhì)量分別是未命名的蘭溪白沙的2.72倍、1.38倍;反之,蘭溪白沙的種子數(shù)、果皮厚度、果實(shí)硬度分別是黃巖軟條白沙的1.36倍、1.096倍、1.22倍;此外,兩者的可溶性固形物(total soluble solid,TSS)含量也存在顯著差異,蘭溪白沙是黃巖軟條白沙的1.14倍;蘭溪白沙新葉縱橫徑為20.4 cm×5.6 cm,葉尖彎銳尖,葉緣淺鋸齒狀,主脈淺溝狀,左右脈不對(duì)稱(左大右小),脈徑沿葉尖方向變小,葉色淺綠,葉片不平整;黃巖軟條白沙新葉縱橫徑為20.8 cm×5.2 cm,葉尖彎尖,葉緣鋸齒狀明顯,主脈直線狀,左右脈較對(duì)稱,脈徑沿葉尖方向變小,葉色墨綠,葉片較平整.

表3　2個(gè)材料的差異比較Table 3 Difference comparison between two experimental materials

圖1　2個(gè)白沙枇杷種質(zhì)葉片和果實(shí)對(duì)比Fig.1 Contrast of leaf and fruit for two kinds of white-flesh loquat

2.2流式細(xì)胞儀檢測(cè)結(jié)果

流式細(xì)胞儀檢測(cè)結(jié)果(圖2)證實(shí),黃巖軟條白沙與蘭溪白沙均為二倍體.開機(jī)后檢測(cè)到出峰時(shí)間為1.0～1.5 min,在DNA相對(duì)含量為50～100處可見二倍體峰值均在70～75左右.

2.3氣孔檢測(cè)結(jié)果

從圖3可以看出,2個(gè)不同品種的枇杷材料氣孔形態(tài)相似,均屬于平行型氣孔,但氣孔大小和氣孔密度差別顯著.黃巖軟條白沙氣孔密度為431個(gè)/mm2,比蘭溪白沙氣孔密度小17.35%;黃巖軟條白沙氣孔面積平均為76μm2,而蘭溪白沙為53μm2.

2.4基于SSR標(biāo)記的親緣關(guān)系檢測(cè)結(jié)果

2.4.1SSR位點(diǎn)遺傳參數(shù)分析結(jié)果

從表4得知:7個(gè)供試材料的有效等位基因數(shù)在2.279 1到4.454 5之間,標(biāo)準(zhǔn)差為1.101 8; Shannon信息指數(shù)在0.898 2到1.611 5之間,標(biāo)準(zhǔn)差為0.357 4;期望雜合度在0.604 4到0.835 2之間,標(biāo)準(zhǔn)差為0.115 4.可見,選取的3個(gè)SSR位點(diǎn)遺傳多樣性相對(duì)較高.

圖2　2個(gè)材料的流式細(xì)胞儀倍性檢測(cè)結(jié)果Fig.2 Ploidy of two experimental materials detected by flow cytometer

圖3　2個(gè)材料的氣孔形態(tài)Fig.3 Stomatal morphology of two experimental materials

表4　SSR位點(diǎn)遺傳參數(shù)統(tǒng)計(jì)Table 4 Genetic parameters of SSR locus

2.4.2基于遺傳參數(shù)的分析結(jié)果

將7份材料進(jìn)行UPGMA聚類分析,繪制聚類樹狀圖(圖4).在遺傳相似系數(shù)(genetic similarity coefficient,GS)≈1.0處,可以把7份白沙枇杷種質(zhì)劃分為6類:第1類,軟條白沙變異(少籽卵圓型) (RTBY)與白荔枝(BL)首先聚在一起,兩者親緣關(guān)系最近;第2類,傀梵白沙(KF);第3類,溪上白沙(XS);第4類,軟條白沙(RTBS);第5類,蘭溪白沙(LX);第6類,福建紅白沙(FJ),與前6份種質(zhì)材料親緣關(guān)系最遠(yuǎn).

3　討論

圖4　7份枇杷種質(zhì)的遺傳聚類圖Fig.4 Genetic dendrogram of seven loquat germplasms

2個(gè)植物貌似相同,但又無(wú)法確定,傳統(tǒng)經(jīng)典的方法是進(jìn)行形態(tài)比較鑒定,主要是對(duì)花、葉、果3大器官組織進(jìn)行比較,然后確定是否為同一品種(種質(zhì)).這種方法雖然簡(jiǎn)便快捷,但也往往存在不同種植地、不同栽培水平造成的非遺傳因子變化,稱為飾變.本研究黃巖軟條白沙和蘭溪白沙果實(shí)綜合指標(biāo)差異較大,前者單果質(zhì)量與種子數(shù)分別為30.62 g/果、2.38粒/果,后者分別為22.25 g/果、3.24粒/果;從果皮厚度、果實(shí)硬度2項(xiàng)指標(biāo)衡量也明顯不同,后者的果皮厚度為0.375 mm、果實(shí)硬度為6.17 N,與前者的0.342 mm、5.04 N差異顯著.由于2個(gè)品種(種質(zhì))種植地不同,黃巖軟條白沙處于浙江東南部的海洋性氣候帶,蘭溪白沙處于浙西盆地的季風(fēng)氣候帶,后者提前5 d左右進(jìn)入夏季,晚5 d左右退出冬季.本研究從分子標(biāo)記、倍性檢測(cè)及葉片氣孔特征等方面綜合分析判定兩者是否存在親緣關(guān)系.結(jié)果表明,它們雖然都是二倍體植物,但兩者之間親緣關(guān)系較遠(yuǎn).基于SSR標(biāo)記的親緣關(guān)系聚類結(jié)果顯示,黃巖軟條白沙的GS為0.40,而蘭溪白沙為0.45.為此,筆者觀察了自有資源圃內(nèi)從原產(chǎn)地采穗高接種植的5株蘭溪白沙,它們除了成熟期較常年延后2～3 d外,花、葉、果等各個(gè)器官組織的基本特征沒有改變,顯示了該種質(zhì)遺傳的穩(wěn)定性.需要指出的是,雖然本研究分析遺傳相似性的聚類材料只有7個(gè),但筆者的前期研究[6]已經(jīng)有了較好的基礎(chǔ),用相關(guān)序列擴(kuò)增多態(tài)性(sequence-related amplified polymorphism,SRAP)分子標(biāo)記檢測(cè),已基本明確了現(xiàn)有的浙江省8份、福建省21份及新西蘭、日本2個(gè)國(guó)家各1份(共31份)白沙枇杷品種(種質(zhì))的親緣關(guān)系,故從綜合因素考慮,沒有重復(fù)研究相同材料.氣孔檢測(cè)結(jié)果表明:黃巖軟條白沙氣孔密度為431個(gè)/mm2,蘭溪白沙為521.5個(gè)/mm2,小于后者17.35%;氣孔面積前者平均為76μm2,后者為53 μm2,前者比后者大43.4%.目前,國(guó)內(nèi)的類似研究多為倍性檢測(cè)或基于分子標(biāo)記的親緣關(guān)系分析,而從表型特征檢測(cè)到親緣關(guān)系的相關(guān)系列研究還不多見.筆者前期研究表明,雖然白沙枇杷品種相同或相近,但在不同栽培地和不同栽培方法下,果實(shí)大小、色澤、綜合品質(zhì)明顯不同[10].傳統(tǒng)的形態(tài)鑒定無(wú)法準(zhǔn)確判定一個(gè)種質(zhì)的親緣關(guān)系,尤其是對(duì)在芽變或相似遺傳背景條件下不同栽培地種植的種質(zhì)鑒定;因此,需要進(jìn)行分子標(biāo)記、氣孔及其轉(zhuǎn)錄組技術(shù)的綜合配套檢測(cè)分析,也是體現(xiàn)果樹種質(zhì)遺傳關(guān)系的重要指標(biāo)[1113].對(duì)多倍體枇杷目標(biāo)起始密碼子多態(tài)性(start condon targeted polymorphism,SCo T)、紅皮沙梨品種SSR分子標(biāo)記親緣關(guān)系并結(jié)合配套技術(shù)研究取得了較好的效果[1213].CHEN等[13]用SCo T、引物結(jié)合位點(diǎn)(inter-primer binding site, IPBS)2個(gè)分子標(biāo)記聯(lián)合對(duì)浙江省31個(gè)楊梅資源品種進(jìn)行了親緣關(guān)系研究,排除了1個(gè)同物異名種質(zhì),區(qū)分出了2個(gè)突變體與野生型的差異.在本研究中黃巖軟條白沙果實(shí)顯著大于蘭溪白沙,而其氣孔面積比后者大43.4%,氣孔密度比后者小17.35%.通常,氣孔越大抗逆性可能較差.這與筆者前期對(duì)楊梅氣孔的研究結(jié)果吻合,大果型東魁楊梅的氣孔密度顯著小于小果型的地方品種,研究表明這是由其種性特點(diǎn)決定的[14].對(duì)于芽變或倍性變異的種質(zhì),需要分子標(biāo)記與轉(zhuǎn)錄組組合鑒定才能更加精準(zhǔn).如潘志勇利用RNA-Seq組學(xué)技術(shù),對(duì)廣西甜橙變異材料暗柳橙進(jìn)行了芽變研究[15],FENG等利用RNASeq技術(shù)進(jìn)行了楊梅色澤變化的機(jī)制研究[16],均取得了較好的效果.筆者在對(duì)楊梅的親緣關(guān)系研究中,把氣孔分析、分子標(biāo)記檢測(cè)、RNA-Seq檢測(cè)3項(xiàng)內(nèi)容作為倍性變化研究的技術(shù)關(guān)鍵,明確了2個(gè)突變體與野生型植株的倍性及其變異機(jī)制[1314,17].下一步我們將對(duì)本研究的2個(gè)種質(zhì)進(jìn)行深入分析,并利用新的轉(zhuǎn)錄組學(xué)技術(shù),明確產(chǎn)生種質(zhì)差異的候選基因,探討形成差異的機(jī)制;同時(shí),在面上適度開展高接換種與異地栽培,擴(kuò)大對(duì)這2個(gè)種質(zhì)的特征和特性觀察,將優(yōu)良品種和優(yōu)良栽培技術(shù)結(jié)合,培育出消費(fèi)者期望的品種.

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CHEN Fangyong1*,WANG Yin1,NI Haizhi1,ZHANG Qi2,YAN Bangguo1
(1.Citrus Research Institute of Zhejiang,Huangyan 318026,Zhejiang,China;2.Economic Specialty Technology Extension Station, Agricultural Bureau of Lanxi,Lanxi 321100,Zhejiang,China)

Summary Loquat,Eriobotrya japonica(Thunb.)Lindl.,is indigenous in southeast of China,which belongs to the family Rosaceae and subfamily Maloideae.Based on the color of fruit flesh,loquat can be sorted into redand white-flesh cultivars.Zhejiang Province is the major producing area of loquat in China,with an area of about 1.2×104hm2;the annual production of loquat is about 6.5×104t,with a value of about 2.5 billion Chinese Yuan. Therefore,germplasm resources of loquat are abundant in Zhejiang Province.However,there are two white-flesh loquat germplasms(Ruantiao white-flesh loquat from Huangyan,white-flesh loquat from Lanxi)which are different in leaf morphology and fruit shape from two producing areas of Zhejiang Province.Controversy on whether the two varieties are synonyms has existed for a long time,and no conclusive evidence was presented yet.

In this paper,morphological characteristics of the two varieties were examined,and their ploidy and stomata were analyzed,and their genetic relationships were identified using simple sequence repeat(SSR)marker.

Results showed that the single fruit mass of Ruantiao white-flesh loquat from Huangyan area was 1.38 times heavier than that of white-flesh loquat from Lanxi area.However,the number of seeds,pericarp thickness,fruithardness of white-flesh fruit from Lanxi were 1.36,1.096 and 1.22 times higher than those of Ruantiao whiteflesh loquat from Huangyan,respectively.Flow cytometry analysis indicated that the two varieties were both diploid.Stomatal density of white-flesh loquat from Lanxi was significantly higher than Ruantiao white-flesh loquat from Huangyan,while conversely,the stomatal area from Lanxi was significantly smaller than that from Huangyan.The cluster analysis using SSR marker showed that the genetic similarity coefficient(GS)of the loquat from Huangyan was 0.40,and 0.45 from Lanxi,indicating these two kinds of white-flesh loquats had a distant genetic relationship.The difference of the two varieties in maturation periods was mainly affected by variety characteristics and different geographical regions.

In conclusion,the two experimental materials have a distant genetic relationship and belong to different germplasms.The creation of the novel germplasm has positive significance in enriching market supply and industrial development of white-flesh loquat.

Key wordswhite-flesh loquat;similar germplasm;genetic relationship;difference

Genetic relationship identification on germplasm resources of two white-flesh loquats from two different producing areas of Zhejiang Province.Journal of Zhejiang University(Agric.&Life Sci.), 2016,42(6):739- 746

DOI:10.3785/j.issn.1008-9209.2016.01.211

中圖分類號(hào)S 667.3;S 602

文獻(xiàn)標(biāo)志碼A

基金項(xiàng)目:浙江省臺(tái)州市科技計(jì)劃(15NY15).

*通信作者(Corresponding author):陳方永(http://orcid.org/0000-0002-3134-9470),E-mail:cfy17266@126.com

收稿日期(Received):2016 01 21;接受日期(Accepted):2016 05 03;網(wǎng)絡(luò)出版日期(Published online):2016 08 10

URL:http://www.cnki.net/kcms/detail/33.1247.S.20160810.1234.002.html