基于微衛(wèi)星標(biāo)記的刺參群體遺傳結(jié)構(gòu)分析及與經(jīng)濟(jì)性狀的相關(guān)性研究

李云霞,李嬌,丁君,常亞青

(大連海洋大學(xué)農(nóng)業(yè)部北方海水增養(yǎng)殖重點(diǎn)開(kāi)放實(shí)驗(yàn)室,遼寧大連116023)

基于微衛(wèi)星標(biāo)記的刺參群體遺傳結(jié)構(gòu)分析及與經(jīng)濟(jì)性狀的相關(guān)性研究

李云霞,李嬌,丁君,常亞青

(大連海洋大學(xué)農(nóng)業(yè)部北方海水增養(yǎng)殖重點(diǎn)開(kāi)放實(shí)驗(yàn)室,遼寧大連116023)

采用微衛(wèi)星分子標(biāo)記技術(shù)對(duì)7個(gè)刺參Apostichopus japonicus(Selenka)群體:遼寧葫蘆島養(yǎng)殖群體、山東煙臺(tái)野生群體、山東萊州養(yǎng)殖群體、大連旅順底播群體、丹東東港養(yǎng)殖群體、大連廣鹿島底播群體、大連黑石礁野生群體的遺傳多樣性進(jìn)行分析,并隨機(jī)抽取7個(gè)群體中的180頭刺參進(jìn)行體長(zhǎng)、體寬、體積、體質(zhì)量和總棘數(shù)與17個(gè)微衛(wèi)星標(biāo)記之間的相關(guān)性研究。結(jié)果表明:用12個(gè)微衛(wèi)星標(biāo)記共擴(kuò)增獲得83個(gè)等位基因,每個(gè)位點(diǎn)的等位基因數(shù)為3～12個(gè),長(zhǎng)度為102～368 bp。刺參群體位點(diǎn)多樣性分析顯示,各刺參群體等位基因數(shù)為5.083 3～5.416 7,平均值為5.261 9;有效等位基因數(shù)為3.205 0～3.701 1,平均值為3.446 8;觀測(cè)雜合度為0.547 5～0.686 9,平均值為0.614 9;期望雜合度為0.649 1～0.697 1,平均值為0.674 1;多態(tài)性信息含量為0.598 9～0.638 6,平均值為0.620 0。對(duì)各群體的Hardy-Weinber平衡檢測(cè)顯示,7個(gè)刺參群體均存在不同程度的平衡偏離。刺參群體遺傳分化與基因流分析顯示,刺參群體間遺傳分化系數(shù)平均值為0.078 2,變異主要來(lái)源于群體內(nèi);基因流平均值為5.301 9,刺參群體間存在一定程度的基因交流。刺參群體間遺傳相似性系數(shù)為0.731 8～0.886 0,UPGMA聚類顯示,7個(gè)群體中山東煙臺(tái)野生群體單獨(dú)為一類,其他6個(gè)群體聚為一類。刺參微衛(wèi)星標(biāo)記與經(jīng)濟(jì)性狀相關(guān)性分析顯示,HC312、EAJ07位點(diǎn)與刺參體長(zhǎng)、體積、體質(zhì)量顯著相關(guān) (P＜0.05);IS45、EAJ03、EAJ04位點(diǎn)與刺參體長(zhǎng)、體積、體寬、體質(zhì)顯著相關(guān) (P＜0.05);SJ04位點(diǎn)僅與刺參總棘數(shù)顯著相關(guān) (P＜0.05);SJ09位點(diǎn)與刺參體積、體寬、體質(zhì)量顯著相關(guān) (P＜0.05);SJ18位點(diǎn)僅與刺參體寬顯著相關(guān) (P＜0.05);SJ19位點(diǎn)與刺參體積、體寬、體質(zhì)量、總棘數(shù)顯著相關(guān) (P＜0.05);SP072位點(diǎn)與刺參體積、體質(zhì)量顯著相關(guān) (P＜0.05)。本研究結(jié)果可為刺參群體結(jié)構(gòu)優(yōu)化、經(jīng)濟(jì)性狀的QTL定位、養(yǎng)殖和選育提供參考。

刺參;微衛(wèi)星;遺傳多樣性;經(jīng)濟(jì)性狀

刺參Apostichopus japonicus(Selenka)自然分布于中國(guó)北方沿海、朝鮮半島、日本和俄羅斯遠(yuǎn)東地區(qū)。近年來(lái)刺參養(yǎng)殖在中國(guó)北方沿海蓬勃開(kāi)展, 2011年產(chǎn)量達(dá)到13.7萬(wàn)t,刺參養(yǎng)殖已成為具有北方特色的海水養(yǎng)殖支柱產(chǎn)業(yè)之一,但隨著養(yǎng)殖規(guī)模的不斷擴(kuò)大,刺參良種的缺乏和養(yǎng)殖環(huán)境的惡化和病害的頻發(fā)等均影響著刺參產(chǎn)業(yè)的發(fā)展[1],其中刺參良種的缺乏是制約刺參養(yǎng)殖業(yè)發(fā)展的瓶頸。進(jìn)行刺參種質(zhì)改良首先應(yīng)對(duì)其種質(zhì)資源、遺傳結(jié)構(gòu)特征進(jìn)行分析;此外,在水產(chǎn)經(jīng)濟(jì)動(dòng)物育種中,由于對(duì)目標(biāo)性狀缺乏準(zhǔn)確可靠的早期預(yù)選手段,育種盲目性大、效率低也是急需解決的問(wèn)題。DNA分子標(biāo)記技術(shù)是進(jìn)行物種群體結(jié)構(gòu)分析和資源調(diào)查的有效手段,與經(jīng)濟(jì)性狀相關(guān)的DNA標(biāo)記可實(shí)現(xiàn)水產(chǎn)經(jīng)濟(jì)動(dòng)物育種目標(biāo)性狀的早期選擇。

微衛(wèi)星標(biāo)記技術(shù) (SSR)廣泛應(yīng)用于物種鑒定與遺傳變異分析、群體結(jié)構(gòu)與親緣關(guān)系分析、分子標(biāo)記輔助選擇等領(lǐng)域[2]。目前,海洋生物微衛(wèi)星標(biāo)記日益豐富[3-6],但有關(guān)刺參微衛(wèi)星標(biāo)記的報(bào)道有限[7-9],Kang等[10]、 Kim 等[11]、 Chang等[12]分別用微衛(wèi)星標(biāo)記對(duì)不同刺參群體進(jìn)行了遺傳差異、遺傳結(jié)構(gòu)和遺傳多樣性的分析。本研究中應(yīng)用微衛(wèi)星標(biāo)記對(duì)山東煙臺(tái)、萊州,以及遼寧葫蘆島、大連、丹東的7個(gè)刺參群體進(jìn)行遺傳多樣性分析及微衛(wèi)星標(biāo)記與刺參主要經(jīng)濟(jì)性狀的相關(guān)性研究,以期為刺參的養(yǎng)殖及選育提供理論基礎(chǔ)。

1 材料與方法

1.1 材料

7個(gè)刺參群體分別為山東煙臺(tái)野生群體(SDYS)、大連黑石礁野生群體 (DLYS)、丹東東港養(yǎng)殖群體 (DD)、葫蘆島養(yǎng)殖群體 (HLD)、旅順底播群體 (LS)、山東萊州養(yǎng)殖群體 (SDYZ)、廣鹿島底播群體 (GLD)。每個(gè)群體取30頭,分別取其管足存放于冰箱 (-80℃)中保存?zhèn)溆?。隨機(jī)取其中180頭刺參,測(cè)量其體長(zhǎng)、體寬、體積、體質(zhì)量和總棘數(shù)5個(gè)經(jīng)濟(jì)性狀,用于微衛(wèi)星與刺參經(jīng)濟(jì)性狀的相關(guān)性研究。

1.2 方法

1.2.1 基因組DNA的提取 刺參基因組DNA的提取參照文獻(xiàn) [13],用10 g/L瓊脂糖凝膠電泳進(jìn)行檢測(cè)。

1.2.2 PCR擴(kuò)增 微衛(wèi)星引物序列和退火溫度見(jiàn)表1,引物由上海生工生物技術(shù)有限公司合成。微衛(wèi)星反應(yīng)體系共20μL,包括DNA模板3μL,10× Buffer 2μL,上、下游引物各0.8μL,2.5 mmol/L dNTP 1.6μL,25 mmol/L MgCl21.2μL,5 U/μLTaq酶0.1μL,用雙蒸水補(bǔ)至20μL。反應(yīng)程序: 94℃下預(yù)變性5 min;94℃下變性30 s,退火30 s,72℃下延伸30 s,共進(jìn)行30個(gè)循環(huán);最后在72℃下再延伸10min。PCR擴(kuò)增產(chǎn)物采用8%的非變性聚丙烯酰胺凝膠電泳,銀染檢測(cè)。

表1 刺參微衛(wèi)星引物信息Tab.1 Information ofm icrostaellitemarkers used in sea cucumber Apostichopus japonicus(Selenka)

1.3 數(shù)據(jù)處理

1.3.1 刺參群體遺傳參數(shù)的計(jì)算 利用PopGene 1.32軟件計(jì)算各刺參群體的等位基因數(shù) (Na)、有效等位基因數(shù) (Ne)、觀測(cè)雜合度 (Ho)、期望雜合度 (He);單個(gè)群體水平近交系數(shù) (Fis)、總?cè)后w水平近交系數(shù) (Fit)、群體間分化系數(shù) (Fst)和基因流 (Nm);群體間遺傳相似系數(shù)、遺傳距離。同時(shí)利用該軟件檢測(cè)7個(gè)刺參群體在各個(gè)位點(diǎn)的Hardy-Weinberg平衡,并計(jì)算7個(gè)群體在各個(gè)微衛(wèi)星位點(diǎn)上的多態(tài)性信息含量 (PIC)。

1.3.2 微衛(wèi)星分子標(biāo)記與經(jīng)濟(jì)性狀間的相關(guān)分析

用SPSS 13.0軟件進(jìn)行刺參經(jīng)濟(jì)性狀與17個(gè)微衛(wèi)星位點(diǎn)的最小二乘法分析,確定二者的相關(guān)性。

2 結(jié)果

2.1 刺參群體微衛(wèi)星PCR擴(kuò)增

墾利油田群中某待開(kāi)發(fā)油田A所產(chǎn)原油黏度較高，50℃下黏度為2700 mPa·s，擬采用依托周邊油田開(kāi)發(fā)。選取周邊可能被依托開(kāi)發(fā)的3個(gè)不同黏度梯隊(duì)的稀油油田原油(稱為稀油a、稀油b和稀油c)作為組分油。按照各油田產(chǎn)油量配比，分別與墾利油田A原油摻混，利用HAAKE MARS高溫高壓流變儀及配套溫控器對(duì)混合原油進(jìn)行測(cè)量，各組分油黏溫特性如圖1～圖2所示。

利用表1中1～12號(hào)微衛(wèi)星引物對(duì)7個(gè)刺參群體進(jìn)行PCR擴(kuò)增,結(jié)果表明,12個(gè)微衛(wèi)星引物均能在7個(gè)群體中穩(wěn)定、重復(fù)地?cái)U(kuò)增出相應(yīng)條帶,部分?jǐn)U增結(jié)果見(jiàn)圖1。12個(gè)微衛(wèi)星位點(diǎn)共獲得83個(gè)等位基因,每個(gè)位點(diǎn)的等位基因數(shù)為3～12。

圖1 引物SP033在7個(gè)刺參群體中的擴(kuò)增產(chǎn)物電泳圖Fig.1 The electrophoresis of PCR amplification of primer SP0337 in 7 sea cucumber populations

2.2 刺參群體位點(diǎn)的遺傳多樣性分析

從表2可見(jiàn):7個(gè)刺參群體的Na為5.083 3～5.416 7,平均值為 5.261 9;Ne為 3.205 0～3.701 1,平均值為 3.446 8;Ho為 0.547 5～0.686 9,平均值為 0.614 9;He為 0.649 1～0.697 1,平均值為 0.674 1;PIC為 0.598 9～0.638 6,平均值為0.620 0。對(duì)各群體的Hardy-Weinber平衡檢測(cè)顯示,7個(gè)刺參群體均存在不同程度的平衡偏離。

2.3 刺參群體的遺傳分化與基因流

從表3可見(jiàn):7個(gè)刺參群體間Fst為0.013 7～0.180 5,平均值為 0.078 2;Nm為 1.141 3～18.055 8,平均值為5.301 9。

2.4 刺參群體的變異分析

從表4可見(jiàn):7個(gè)刺參群體中SDYZ與LS群體的遺傳相似度最高,相似系數(shù)為0.886 0,遺傳距離為0.121 1;HLD與SDYS群體的遺傳相似度最低,相似系數(shù)為0.731 8,遺傳距離為0.312 2。7個(gè)刺參群體UPMGA聚類圖見(jiàn)圖2,SDYS群體單獨(dú)為一類,SDYZ群體先與LS群體聚為一類,再與GLD、DD、HLD和DLYS群體聚在一起。

2.5 刺參經(jīng)濟(jì)性狀與微衛(wèi)星位點(diǎn)的相關(guān)性分析

測(cè)定的180頭刺參主要經(jīng)濟(jì)性狀均表現(xiàn)出連續(xù)變異的特點(diǎn),且符合正態(tài)分布。采用最小二乘法進(jìn)行17個(gè)微衛(wèi)星位點(diǎn) (表1)與刺參體長(zhǎng)、體寬、體積、體質(zhì)量和總棘數(shù)5個(gè)性狀的相關(guān)性分析,結(jié)果表明:HC312、EAJ07位點(diǎn)與刺參體長(zhǎng)、體積、體質(zhì)量顯著相關(guān) (P＜0.05);IS45、EAJ03、EAJ04位點(diǎn)與刺參體長(zhǎng)、體積、體寬、體質(zhì)量顯著相關(guān)(P＜0.05);SJ04位點(diǎn)僅與刺參總棘數(shù)顯著相關(guān)(P＜0.05);SJ09位點(diǎn)與刺參體積、體寬、體質(zhì)量顯著相關(guān) (P＜0.05);SJ18位點(diǎn)僅與刺參體寬顯著相關(guān) (P＜0.05);SJ19位點(diǎn)與刺參體積、體寬、體質(zhì)量、總棘數(shù)顯著相關(guān) (P＜0.05);SP072位點(diǎn)與刺參體積、體質(zhì)量顯著相關(guān) (P＜0.05)。具體計(jì)算結(jié)果限于篇幅未一一列出。此外,本研究中所獲得的這10個(gè)與刺參經(jīng)濟(jì)性狀相關(guān)的分子標(biāo)記中,存在一因多效或多因一效現(xiàn)象。

表2 7個(gè)刺參群體在12個(gè)位點(diǎn)的遺傳多樣性分析Tab.2 The diversity analysis of 12 loci in 7 sea cucumber populations

續(xù)表2 7個(gè)刺參群體在12個(gè)位點(diǎn)的遺傳多樣性分析Cont.tab.2 The diversity analysis of 12 loci in 7 sea cucumber populations

表3 7個(gè)刺參群體的遺傳分化與基因流Tab.3 Genetic differentiation and gene flow in 7 sea cucumber popu lations

圖2 基于7個(gè)刺參群體標(biāo)準(zhǔn)遺傳距離的UPGMA聚類圖Fig.2 UPGMA of 7 sea cucumber populations based on standard genetic distance

3 討論

3.1 刺參群體的遺傳多樣性和遺傳變異分析

本研究中發(fā)現(xiàn):7個(gè)刺參群體的Ho和He平均值分別為0.614 9、0.674 1,與Kim等[11]報(bào)道的韓國(guó)5個(gè)刺參群體的Ho和He接近,Ho高于Chang等[12]報(bào)道的5個(gè)刺參群體的Ho;12個(gè)微衛(wèi)星位點(diǎn)在7個(gè)刺參群體中的等位基因數(shù)為3～12個(gè),野生刺參群體多態(tài)性信息含量高于底播群體和養(yǎng)殖群體的結(jié)論與譚杰等[14]用微衛(wèi)星分析山東野生與養(yǎng)殖刺參群體所得的結(jié)論一致。

表4 7個(gè)刺參群體的遺傳相似系數(shù)(對(duì)角線以上)和Nei氏遺傳距離(對(duì)角線以下)Tab.4 Genetic identity(above diagonal)and Nei's genetic distance(below diagonal)in 7 sea cucumber populations

UPGMA聚類圖反映出山東野生群體單獨(dú)為一類,與其他6個(gè)群體的遺傳距離相對(duì)較遠(yuǎn),其他6個(gè)群體聚類順序與其地理分布有一定的相關(guān)性。7個(gè)刺參群體的遺傳分化系數(shù)平均值為0.078 2,表明有 7.82%的變異是由群體分化導(dǎo)致的,而92.18%的變異來(lái)源于群體內(nèi),群體間的遺傳分化水平較低,作為影響群體間遺傳分化的主要因素[15],Wright[16]認(rèn)為,基因流Nm大于1時(shí),能夠發(fā)揮其均質(zhì)化作用,從而抑制由遺傳漂變引起的群體間分化。本研究結(jié)果顯示,12個(gè)位點(diǎn)在7個(gè)刺參群體中的Nm值均大于 1,平均值達(dá)到5.301 9,群體間存在一定程度的基因交流,通過(guò)對(duì)研究結(jié)果與實(shí)際情況分析,推測(cè)不同地域刺參的交流是影響其群體間遺傳分化的主要因素。

3.2 刺參經(jīng)濟(jì)性狀與微衛(wèi)星位點(diǎn)的相關(guān)性分析

水產(chǎn)動(dòng)物的體長(zhǎng)、體寬等經(jīng)濟(jì)性狀表現(xiàn)為連續(xù)變異,受多基因控制,易受環(huán)境影響,借助與數(shù)量性狀連鎖的分子標(biāo)記可實(shí)現(xiàn)水產(chǎn)育種過(guò)程中的早期選擇,利用與經(jīng)濟(jì)性狀緊密相關(guān)的遺傳標(biāo)記還可提高選擇的準(zhǔn)確性、縮短育種周期。目前,已有微衛(wèi)星分子標(biāo)記與扇貝、大菱鲆、文蛤、牡蠣等海洋生物經(jīng)濟(jì)性狀連鎖分析的報(bào)道[17-21]。在刺參微衛(wèi)星標(biāo)記與經(jīng)濟(jì)性狀相關(guān)性研究方面,Wang等[22]利用10個(gè)微衛(wèi)星位點(diǎn)對(duì)大連2個(gè)刺參群體進(jìn)行了微衛(wèi)星標(biāo)記與刺參體質(zhì)量的相關(guān)性分析。孫國(guó)華等[23]進(jìn)行了10個(gè)微衛(wèi)星標(biāo)記與刺參體長(zhǎng)、體質(zhì)量的相關(guān)性分析,獲得了4個(gè)與刺參體長(zhǎng)、體質(zhì)量顯著相關(guān)的微衛(wèi)星標(biāo)記。本研究中共獲得10個(gè)與刺參主要經(jīng)濟(jì)性狀相關(guān)的標(biāo)記,經(jīng)濟(jì)性狀與標(biāo)記間存在一因多效或多因一效現(xiàn)象,這些與經(jīng)濟(jì)性狀相關(guān)的微衛(wèi)星標(biāo)記還可進(jìn)一步定位到刺參遺傳連鎖圖譜上,用于刺參經(jīng)濟(jì)性狀的QTL定位研究。

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Population genetic structure and relationship between microsatellite markers and economic traits in sea cucumber

LI Yun-xia,LI Jiao,DING Jun,CHANG Ya-qing
(Key Laboratory of Mariculture & Stock Enhancement in North China's Sea,Ministry of Agriculture,Dalian Ocean University,Dalian 116023,China)

Microsatellite marker technique was used to estimate extent of genetic diversity among 7 sea cucumber Apostichopus japonicus(Selenka) populations including farmed Huludao(HLD)in Laoning Province,wild Yantai (SDYS) in Shandong Province,farmed Liazhou (SDYZ) in Shandong Province,ranched Lüshun (LS) in Laoning Province,farmed Dandong (DD) in Laoning Province,ranched Guanglu (GLD) in Laoning Province,and wild Heishijiao(DLYS) in Laoning Province,and the relationship between economic characters of sea cucumbers and microsatellite markerswere analyze in 180 individuals sampled from 7 populations.The results showed that 83 allelic genes were amplified with 12 microsatellite markers and the number of alleles ranged from 3 to 12 per locus containing 102-368 bp.The locus diversity analysis showed that the alleles were found to be ranged from 5.083 3 to 5.416 7 in number in each sea cucumber population with an average of5.261 9,effective number of alleles ranged from 3.205 0 to 3.701 1 with an average of3.446 8,observed heterozygosity ranged from 0.547 5 to 0.686 9 with an average of 0.614 9,expected heterozygosity ranged from 0.649 1 to 0.697 1 with an average of 0.674 1,and polymorphic information content ranged from 0.598 9 to 0.638 6 with an average of0.620 0.Hardy-Weinber test indicated that there was a certain degree deviation from equilibrium in 7 sea cucumber populations.There was a certain degree of genetic exchange in sea cucumber populations,with genetic differentiation of0.078 2,largely derived from the variation within populations,and average gene flow of 5.301 9.Sea cucumber populations showed genetic similarity from 0.731 8 to 0.886 0.UPGMA showed that the SDYS population was clustered into one group alone.The relationship between economic characters of the sea cucumber and microsatellite markers revealed that HC312 and EAJ07 were significantly correlated with body length,body volume and body weight(P＜0.05),while IS45,EAJ03 and EAJ04 were significantly related with body length,body volume,bodywidth and body weight(P＜0.05);SJ04 had a significant relationship with total tubercle number(P＜0.05)only,while SJ09 was significantly correlated with body volume,body width and body weight(P＜0.05);SJ18 was significantly correlated only with body width(P＜0.05),while SJ19 was significantly related with body volume,body width,body weight and total tubercle number(P＜0.05);SP072 was significantly involved in body volume and body weight(P＜0.05). The findings provide some guidance for sea cucumber cultivation and breeding.

Apostichopus japonicus(Selenka);microsatellite;genetic diversity;economic trait

S917.4

A

2095-1388(2013)05-0438-07

2013-01-13

國(guó)家 “863”計(jì)劃項(xiàng)目 (2012AA10A412,2010AA10A401);遼寧省農(nóng)業(yè)攻關(guān)計(jì)劃重大項(xiàng)目 (2011203003)

李云霞 (1989-),女,碩士研究生。E-mail:ernan1989@163.com

丁君 (1973-),女,研究員。E-mail:dingjun1119@dlou.edu.cn