張成鋒蘇勝彥朱 健石連玉

(1. 中國水產(chǎn)科學(xué)研究院淡水漁業(yè)研究中心, 農(nóng)業(yè)部淡水漁業(yè)與種質(zhì)資源利用重點(diǎn)實(shí)驗(yàn)室, 無錫 214081; 2. 中國水產(chǎn)科學(xué)研究院黑龍江水產(chǎn)研究所, 農(nóng)業(yè)部淡水水產(chǎn)生物技術(shù)與遺傳育種重點(diǎn)實(shí)驗(yàn)室, 哈爾濱 150070)

富集優(yōu)勢基因型的后備親本篩選以及相關(guān)分子標(biāo)記的遺傳效應(yīng)分析

張成鋒1蘇勝彥1朱 健1石連玉2

(1. 中國水產(chǎn)科學(xué)研究院淡水漁業(yè)研究中心, 農(nóng)業(yè)部淡水漁業(yè)與種質(zhì)資源利用重點(diǎn)實(shí)驗(yàn)室, 無錫 214081; 2. 中國水產(chǎn)科學(xué)研究院黑龍江水產(chǎn)研究所, 農(nóng)業(yè)部淡水水產(chǎn)生物技術(shù)與遺傳育種重點(diǎn)實(shí)驗(yàn)室, 哈爾濱 150070)

選取黃河鯉新品系親本和子代共450尾, 5對(duì)微衛(wèi)星引物和4個(gè)基因區(qū)段擴(kuò)增引物開展基因型檢測, 并檢測它們與體重的關(guān)聯(lián)性以及分析相應(yīng)的遺傳效應(yīng)。結(jié)果顯示: Koi42和4個(gè)SNPs位點(diǎn)對(duì)體重有顯著的影響, 獲得具有超過富集2個(gè)優(yōu)勢基因型的候選親本共13尾, 富集優(yōu)勢分子標(biāo)記基因型的候選親本生長性能優(yōu)勢明顯。通過多元逐步回歸分析, 利用AIC最佳模型篩選到COⅠ626、D-Loop253和Koi42共3個(gè)位點(diǎn), 發(fā)現(xiàn)Koi42貢獻(xiàn)率較大, 經(jīng)Fisher精確性檢驗(yàn), 發(fā)現(xiàn)其與性別存在關(guān)聯(lián), 其加性效應(yīng)接近顯著水平(P＜0.05)。檢測的多個(gè)上位效應(yīng)組分中, 僅有D-Loop253和Koi42的加性效應(yīng)間的互作達(dá)到顯著水平。對(duì)遺傳方差組分進(jìn)行剖分發(fā)現(xiàn), 加性方差占11.4%, 兩個(gè)位點(diǎn)的加性效應(yīng)構(gòu)成的上位效應(yīng)占到77.5%, 因此這3個(gè)分子標(biāo)記對(duì)體重的影響主要以上位效應(yīng)為主, 而且是兩兩加性效應(yīng)的占比較大, 可以推斷出D-Loop 253和Koi42兩個(gè)分子標(biāo)記的上位效應(yīng)起主要作用。綜上所述結(jié)果提示Koi42及與其有互作的D-Loop253可用于黃河鯉新品系的標(biāo)記輔助選擇,可以開展多個(gè)分子標(biāo)記的富集選擇。

黃河鯉新品系; 基因型; 體重; 關(guān)聯(lián)分析; 上位效應(yīng)

鯉產(chǎn)業(yè)在我國水產(chǎn)養(yǎng)殖乃至世界淡水養(yǎng)殖中均占據(jù)舉足輕重的地位, 因而運(yùn)用現(xiàn)代育種技術(shù)提升我們鯉產(chǎn)業(yè)的科技水平, 發(fā)展鯉種業(yè)具有重要的意義, 這也是國家發(fā)展現(xiàn)代農(nóng)業(yè)和農(nóng)業(yè)產(chǎn)業(yè)升級(jí)的內(nèi)在要求。分子標(biāo)記輔助育種、分子育種、基因組育種、基因組關(guān)聯(lián)選擇、GBLUP (Genomic best linear unbiased prediction, GBLUP)選育等成為現(xiàn)代育種的重要內(nèi)容和主要手段(Jonas and de Koning)[1]。目前, 在鯉育種方面主要還是基于數(shù)量遺傳學(xué)的BLUP (Best linear unbiased prediction, BLUP)育種、分子輔助育種、分子育種。Vandeputte等[2]通過分子標(biāo)記和全因子交叉設(shè)計(jì), 成功的進(jìn)行了基于分子標(biāo)記的鯉配對(duì)繁殖技術(shù), 并估計(jì)了體重和體長的遺傳力, 均為0.33, 他們主要采用的是選育原始代親本, 然后再從它們的后代中增加留選親本的思路。使用同樣的方法, Kocour等[3]在鯉加工和肉質(zhì)性狀上同樣進(jìn)行了以分子育種為基礎(chǔ)的遺傳力估計(jì)。隨后, Vandeputte等[4]不僅估計(jì)了鯉實(shí)際分子育種的遺傳力, 還對(duì)選擇反應(yīng)作出評(píng)估: 配對(duì)方式采用全因子交叉設(shè)計(jì), 體重的遺傳力范圍為0.31—0.44, 體長的遺傳力范圍為0.21—0.33, 體重和體長的遺傳相關(guān)為0.97, 選育第3代時(shí)的遺傳進(jìn)展比較大。2011年, Vandeputte等[5]從整個(gè)育種流程和理論上分析總結(jié)(主要涉及理論推導(dǎo)、數(shù)據(jù)模擬到真實(shí)育種3個(gè)層次), 基于分子標(biāo)記的水產(chǎn)育種的育種效果, 證明了合適的標(biāo)記對(duì)于實(shí)際育種很有意義,也證實(shí)了此種方法在鯉育種中的可行性。

在現(xiàn)有分子標(biāo)記中, 微衛(wèi)星因其長度表現(xiàn)為高度多態(tài)性, 已成為真核生物基因組作圖中不可缺少的分子遺傳標(biāo)記。利用微衛(wèi)星的結(jié)構(gòu)特點(diǎn)和遺傳特性, 繪制高精度遺傳圖譜, 進(jìn)行個(gè)體、品種(系)鑒定是微衛(wèi)星的用途之一。Cheng等[6]和Zhang等[7]分別通過微衛(wèi)星構(gòu)建了鯉遺傳連鎖圖, 隨后與BAC文庫構(gòu)建的物理圖相結(jié)合, 產(chǎn)生了高密度圖譜[8]。SNP是指在染色體基因組水平上單個(gè)核苷酸的變異引起的DNA序列多態(tài)性, 主要包括單堿基的轉(zhuǎn)換、顛換、插入及缺失等形式。Xu等[9]從4個(gè)鯉品種的轉(zhuǎn)錄組測序結(jié)果中尋找SNPs位點(diǎn)。隨后, 他們?cè)?014年構(gòu)建了第一個(gè)高通量的SNP芯片, 其包含25萬個(gè)SNPs位點(diǎn), 在檢測的1072個(gè)樣本中約74.06%具有多態(tài)性[10]。此后, Zhang等[11]再次通過分子標(biāo)記構(gòu)建了鯉連鎖圖并研究了與鯉肌纖維相關(guān)的QTL (Quantitative trait locus, QTL)?？梢钥闯鲈邗幱N領(lǐng)域積累了大量的分子標(biāo)記和有效的檢測方法, 并做了相應(yīng)的分子育種工作。

研究報(bào)道表明相比傳統(tǒng)的BLUP選育, 基于高額成本的基因型檢測的育種可獲得較高的遺傳進(jìn)展[12]。如果遇到遺傳進(jìn)展不夠大或難以支持如此高的基因型檢測成本的情形, 結(jié)合傳統(tǒng)的BLUP估計(jì)、候選個(gè)體的預(yù)選擇和低密度SNP芯片的應(yīng)用是克服這些困難的好的選擇[1]。 因此, 基于實(shí)際情況本文通過篩選到的微衛(wèi)星和SNP分子標(biāo)記來研究它們富集后對(duì)鯉選育群體體重的影響。然而對(duì)于體重這種復(fù)雜的經(jīng)濟(jì)性狀, 表型和基因型之間并不存在嚴(yán)格的一一對(duì)應(yīng)關(guān)系, 而往往是參與表型形成的因素涉及同一位點(diǎn)上等位基因產(chǎn)生的加性效應(yīng)和顯性效應(yīng)、不同位點(diǎn)上非等位基因相互作用產(chǎn)生的上位效應(yīng)以及這些基因與環(huán)境的共同作用[13]。因此, 本文在獲得與體重顯著相關(guān)的分子標(biāo)記后,尤其是多個(gè)分子標(biāo)記后, 明確這些標(biāo)記通過哪幾種遺傳效應(yīng)影響該經(jīng)濟(jì)性狀是充分利用這些標(biāo)記進(jìn)行育種的重要參考和依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)材料

本實(shí)驗(yàn)選用的試驗(yàn)魚均養(yǎng)殖于中國水產(chǎn)科學(xué)研究院淡水漁業(yè)研究中心無錫南泉養(yǎng)殖試驗(yàn)基地,隨機(jī)選取450個(gè)黃河鯉選育候選個(gè)體, 其中親本168尾, 子代282尾。這些試驗(yàn)魚是中國水產(chǎn)科學(xué)研究院淡水漁業(yè)研究中心通過基于家系的BLUP選育和分子設(shè)計(jì)育種結(jié)合開展選育的黃河鯉核心群。具體養(yǎng)殖辦法是根據(jù)BLUP選育原理和分子設(shè)計(jì)育種結(jié)合的選配方案挑選出親魚進(jìn)行繁殖配對(duì), 繁殖的條件是水溫為18—20℃, 配對(duì)的雌魚和雄魚的近交系數(shù)低于2%; 雌魚和雄魚配對(duì)后放入置有棕櫚片制成的魚巢(水位高度為85—90 cm)1d后, 將魚巢轉(zhuǎn)入孵化網(wǎng)箱中進(jìn)行孵化3—4d后, 得到子代魚苗;要求將不同家系的魚苗隔離在不同的網(wǎng)箱中進(jìn)行早期培育, 溫度控制在20—28℃內(nèi), 水位高度設(shè)置為70—80 cm; 要求每半月加注1次新水; 孵出魚苗后的第2至第15天潑灑豆?jié){進(jìn)行喂養(yǎng), 每日潑灑2—3次, 之后用顆粒飼料喂養(yǎng), 每日投喂2—3次, 投喂量為魚苗體重的5%—6%; 當(dāng)魚苗長到7—15 g時(shí),在每個(gè)家系中各選50尾魚苗用注射器將無線射頻標(biāo)記注入魚腹腔, 進(jìn)行個(gè)體標(biāo)記, 從而識(shí)別每尾魚苗; 將標(biāo)記好的魚苗在室內(nèi)水泥池暫養(yǎng), 用顆粒飼料喂養(yǎng), 每日投喂2—3次, 投喂量為魚苗體重的3%—4%; 喂養(yǎng)5—7d后, 將標(biāo)記后的魚苗轉(zhuǎn)入室外土池中進(jìn)行常規(guī)養(yǎng)殖, 飼料為商用成魚料, 養(yǎng)殖到第3年的3月份測生長性能數(shù)據(jù)并鑒別雌雄。

本實(shí)驗(yàn)中的子代來自于親魚群體, 親魚的平均體重為1747.23 g, 子代的平均體重為946.49 g。要求親本對(duì)本文所使用引物(表 1)全部檢測, 子代則要求進(jìn)行Koi42微衛(wèi)星多態(tài)性檢測。在進(jìn)行體重稱重時(shí), 采用丁香油與乙醇的混合液將黃河鯉麻醉后,采用電子天平稱量體重(精確到1 g), 同時(shí)剪取的尾鰭立即于95%的酒精中固定, 采樣完畢, 樣品置于-20℃冰箱保存。按照TaKaRa DNA提取試劑盒說明書提取DNA。采樣的同時(shí)記錄采樣個(gè)體的體重,親本還需記錄性別。

1.2 分子標(biāo)記的多態(tài)性檢測

分子標(biāo)記的檢測分為2種情況進(jìn)行檢測, 微衛(wèi)星主要是通過聚丙烯酰胺凝膠電泳分離后統(tǒng)計(jì)位點(diǎn)的基因型, SNP通過PCR反應(yīng)后直接測序, 獲得的序列使用DNAMAN V6軟件進(jìn)行序列比對(duì), 并通過Chromas2.22軟件核查, 確定SNPs。

PCR反應(yīng)體系為25 μL, 包括10×buffer 15 μL, Mg2+(25 mmol/L) 1 μL, dNTPs (各2 mmol/L) 1 μL,上下游引物(10 mmol/L)各1 μL, 模板DNA 1 μL, Taq DNA聚合酶(Promega)1 U, dd H2O; 擴(kuò)增反應(yīng)均在TaKaRa公司PCR儀上完成。PCR反應(yīng)程序?yàn)? 94℃預(yù)變性3min; 94℃變性20s, 溫度56—66℃退火20s, 72℃延伸30s, 33個(gè)循環(huán); 72℃延伸10min。將反應(yīng)后的PCR產(chǎn)物用8%非變性聚丙烯酰胺凝膠電泳結(jié)合goldview顯色進(jìn)行檢測。如果是SNP位點(diǎn),送到Introvigen上海生物技術(shù)有限公司測序。

1.3 數(shù)據(jù)的統(tǒng)計(jì)分析

數(shù)據(jù)錄入到Office2010, 通過R3.1.14軟件的線性模型篩選多態(tài)位點(diǎn)。采用Natural and Orthogonal InterActions(NOIA)模型計(jì)算已篩選位點(diǎn)的上位效應(yīng)對(duì)體重的影響, 并對(duì)這些個(gè)位點(diǎn)影響體重的遺傳方差進(jìn)行剖分, 使用的是R軟件包noia[18—20]。

表 1 用于分子標(biāo)記篩選的基因及微衛(wèi)星引物序列Tab. 1 Primers of different genes and microsatellite used to explore the candidate molecular locus

2 結(jié)果

2.1 本文所使用多態(tài)性位點(diǎn)的篩選結(jié)果

將線粒體基因COⅠ、D-loop以及IGF (Insulin like growth factor, IGF)家族基因IGF2R、IGF2a基因進(jìn)行分段克隆、測序、尋找潛在的SNPs; 微衛(wèi)星引物是通過PCR擴(kuò)增、電泳分析并統(tǒng)計(jì)基因型, 然后將SNPs和微衛(wèi)星基因型與生長性狀進(jìn)行關(guān)聯(lián)統(tǒng)計(jì), 獲得對(duì)體重有顯著影響的多態(tài)位點(diǎn)(圖 1), 其描述的不同純合基因型之間存在顯著差異, 然后統(tǒng)計(jì)基因型頻率(表 2)。而測序的SNPs位點(diǎn)必須經(jīng)過測序峰圖進(jìn)行驗(yàn)證, 保證其準(zhǔn)確性。

2.2 HLJ13、MFW4、MFW7、MFW11、Koi42、IGF2a4#、IGF2R第一內(nèi)含子、COⅠ和D-Loop基因生長優(yōu)勢基因型的富集性個(gè)體篩選

對(duì)所有的具有生長優(yōu)勢的各個(gè)標(biāo)記基因型進(jìn)行親本的富集性檢測(表 3)。可以看出, 獲得具有超過2個(gè)優(yōu)勢位點(diǎn)的候選親本共13尾。然后對(duì)富集優(yōu)勢的候選親本進(jìn)行生長性能標(biāo)記效果檢測(圖2)?？梢钥闯? 富集優(yōu)勢分子標(biāo)記基因型的候選親本生長性能優(yōu)勢明顯。

2.3 所篩選位點(diǎn)對(duì)體重的貢獻(xiàn)率

為了研究這些個(gè)位點(diǎn)對(duì)體重的貢獻(xiàn)率, 通過多元逐步回歸分析, 利用AIC最佳模型篩選到COⅠ626、D-Loop 253和Koi42共3個(gè)分子標(biāo)記。首先, 把性別和這3個(gè)分子標(biāo)記作為自變量, 發(fā)現(xiàn)它們的貢獻(xiàn)率從大到小分別是性別(9.153381e-01)、D-Loop 253 (8.481305e-04)、Koi42 (9.883973e-05)、COⅠ626 (7.514281e-05), 而且3個(gè)分子標(biāo)記只有DLOOP253達(dá)到顯著水平 。當(dāng)去掉性別時(shí), 發(fā)現(xiàn)3個(gè)位點(diǎn)的貢獻(xiàn)率依大小順序?yàn)? Koi42 (0.105199624)、D-Loop (0.022124762)、COⅠ626 (0.005664277)。綜合這2個(gè)模型(有無性別作自變量)以及貢獻(xiàn)率的大小可以推斷Koi42位點(diǎn)可能與性別有一定的關(guān)聯(lián)。經(jīng)過Fisher精確性檢驗(yàn), 發(fā)現(xiàn)二者在所研究群體中確實(shí)存在關(guān)聯(lián)。

2.4 所篩選分子標(biāo)記的上位效應(yīng)和遺傳組分剖分

本文針對(duì)所篩選分子標(biāo)記作遺傳分析發(fā)現(xiàn): 3個(gè)分子標(biāo)記均存在加性效應(yīng), Koi42可以檢測到顯性效應(yīng), 檢測到多個(gè)上位效應(yīng)組分, 僅有D-Loop 253和Koi42的加性效應(yīng)間的互作達(dá)到顯著水平(表4)。對(duì)遺傳方差組分進(jìn)行剖分發(fā)現(xiàn), 加性方差占11.4%, 兩個(gè)位點(diǎn)的加性效應(yīng)構(gòu)成的上位效應(yīng)占到77.5%, 3個(gè)位點(diǎn)加性效應(yīng)構(gòu)成的上位效應(yīng)占到11.1%, 因此這3個(gè)分子標(biāo)記對(duì)體重的影響主要以上位效應(yīng)為主, 而且是兩兩加性效應(yīng)的占比較大, 可以推斷出D-Loop 253和Koi42兩個(gè)分子標(biāo)記的上位效應(yīng)起主要作用。

3 討論

3.1 富集分子標(biāo)記親本的選擇

如前所述, 分子輔助育種在鯉中已經(jīng)成功使用。對(duì)于富集SNPs的個(gè)體選擇, 李紅霞等[21]檢測了900尾建鯉的鳥氨酸脫羧酶(Ornithine decarboxylase, ODC) jlODC1a基因上6個(gè)和jlODC1b基因上4個(gè)SNP位點(diǎn), 發(fā)現(xiàn)了7個(gè)SNPs與建鯉增重顯性相關(guān), 富集4個(gè)的平均增重顯著快于富集0—3的個(gè)體增重, 且比0標(biāo)記的快約14%。這與本文中富集優(yōu)勢位點(diǎn)的親本較未進(jìn)行分子標(biāo)記篩選的親本具有較高的增重是一致的。

圖 1 供篩選微衛(wèi)星的引物和基因SNPs與體重的關(guān)聯(lián)分析Fig. 1 Correlated analysis between candidate genotype of microsatellite and functional gene SNPs and body weightA. 表示COⅠ基因的第626位置出現(xiàn)的SNP位點(diǎn)不同基因型之間體重差異顯著; B. 表示D-Loop基因的第253位置出現(xiàn)的SNP位點(diǎn)不同基因型之間體重差異顯著; C. 表示IGF2a基因的設(shè)計(jì)的4#引物擴(kuò)增出的SNP位點(diǎn)不同基因型之間體長體重比值差異顯著; D. 表示IGF2R基因的第1內(nèi)含子檢測到的SNP位點(diǎn)不同基因型之間體重差異顯著; 以上4個(gè)基因都是對(duì)親本群體的檢測效果, E. Koi42微衛(wèi)星引物是對(duì)子代的基因型檢測, 做分析時(shí), 做了對(duì)數(shù)轉(zhuǎn)換, 保證體重?cái)?shù)據(jù)服從正態(tài)分布A. the significant body weight difference between two genotypes of 626thposition in COⅠ; B. the significant body weight difference between two genotypes of 253thposition in D-Loop; C. the significant body length/body weight difference between two genotypes of IGF2a4#primers amplified product; D. the significant body weight difference between two genotypes of IGF2R intron 1 amplified product; Such four genes SNPs were used to observe the genotype of parent individuals, while Koi42 in this graph is used to explore the genotype of offspring. Vertical line showed the logical bodyweight in order to conform the Gaussian distribution

為了弄清楚這些分子標(biāo)記是通過什么樣的遺傳效應(yīng)影響目標(biāo)性狀的, 在本研究中, 通過多元逐步回歸進(jìn)一步篩選SNPs位點(diǎn), 得到3個(gè)多態(tài)位點(diǎn), 這提示此3個(gè)位點(diǎn)可用于黃河鯉新品系的選育。深入分析發(fā)現(xiàn)Koi42位點(diǎn)加性效應(yīng)接近顯著水平, 貢獻(xiàn)率最大, 因此該位點(diǎn)對(duì)于黃河鯉選育群體的育種值的計(jì)算有更為重要的作用, 可賦予較大的權(quán)重。有趣的是該位點(diǎn)與性別存在著一定的關(guān)聯(lián)(Fisher精確檢驗(yàn)), 這意味著對(duì)于性成熟較晚的鯉, 可用該位點(diǎn)進(jìn)行早期選擇(獲取雌魚信息)。盡管本文樣本量已經(jīng)達(dá)到450尾魚, 但是具體的關(guān)聯(lián)程度還需擴(kuò)大樣本量檢測。

表 2 所篩選的與生長相關(guān)不同基因或微衛(wèi)星引物所對(duì)應(yīng)的基因型及頻率Tab. 2 Genotypes and their frequency of different genes or microsatellite

表 3 黃河鯉親本的多態(tài)性位點(diǎn)富集情況Tab. 3 Polymorphism explored enrichment in the Huanghe carp candidate parents

3.2 不同位點(diǎn)間的上位效應(yīng)

圖 2 經(jīng)過分子標(biāo)記篩選到的親本具有顯著的體重差異(數(shù)據(jù)進(jìn)行了正態(tài)分布檢驗(yàn), 做了對(duì)數(shù)轉(zhuǎn)換)Fig. 2 Significant body weight difference was observed between candidate parents with selected loci and others without these loci (Vertical line showed the logical bodyweight in order to conform the Gaussian distribution)

表 4 所篩選3個(gè)位點(diǎn)的遺傳效應(yīng)分析Tab. 4 Genetic effect analysis of selected 3 loci

1918年Fisher從群體水平上提出上位效應(yīng)是指不同位點(diǎn)上基因的相互作用, 其效應(yīng)值是對(duì)單位點(diǎn)簡單加性效應(yīng)的偏離值[22]。隨后的研究表明上位效應(yīng)為功能基因間的表達(dá)調(diào)控網(wǎng)絡(luò)[23]。本研究發(fā)現(xiàn)加性效應(yīng)間、加性效應(yīng)和顯性效應(yīng)間的多種上位效應(yīng), 但僅有D-Loop 253和Koi42的加性效應(yīng)間的互作達(dá)到顯著水平, 因此, 二者富集型個(gè)體的留種也是對(duì)該上位效應(yīng)的選擇, 也可能是黃河鯉新品系體重基因調(diào)控網(wǎng)絡(luò)模式[24]之一。戶國等[25]研究發(fā)現(xiàn)對(duì)肉雞7周齡腹脂率有顯著影響的載脂蛋白B基因T123G位點(diǎn)和解偶聯(lián)蛋白基因C1197A位點(diǎn)存在基因間的上位效應(yīng)組分, 并認(rèn)為這種遺傳互作模式可能是影響脂肪性狀的重要因素。因此, 對(duì)目標(biāo)性狀有顯著影響的多個(gè)分子標(biāo)記的富集選擇是一種有效的育種手段。

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CANDIDATE PARENTS SELECTION CONTAINING MULTIPLE GENOTYPES WITH HIGHER PERFORMANCE AND CORRELATED MOLECULAR MARKERS’ GENETIC EFFECTS ANALYSIS

ZHANG Cheng-Feng1, SU Sheng-Yan1, ZHU Jian1and SHI Lian-Yu2
(1. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China; 2. Key Laboratory of Freshwater Aquatic Biotechnology and Breeding, Ministry of Agriculture, Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China)

Marker assistant breeding, molecular breeding and genome wide breeding can shorten the generation interval of germplasm improvement and creation, speed up the selection response. Contrast with traditional best linear unbiased prediction (BLUP), they can achieve the higher genetic gain with the higher cost. Practically, together with BLUP, pre-selection of candidate individuals, low density array, they will become an optional choice under the consideration of selection cost and efficiency. So, in the present article, we focus on the effect of limited markers enrichment on the candidate population growth performance.e.g. body weight. For the body weight phenotype can not only be determined by one genotype, but also by multiple loci additive effect, dominant effect and epitasis. Thus, multiple loci explored needed to do variance components analysis and identify the specific genetic effect of these loci. A total of 450 fish (parents and their offspring) were collected, which were from Nanquan farm in Freshwater Fisheries Research Center in Chinese Fishery Academy. A total of 5 microstallites primers and 4 primers used for functional gene regional amplification were selected to observe the corresponded genotypes of such individuals. Correlation analysis between effective molecular markers and body weight of Huanghe carp new strain and genetic effect analysis of such markers were examined too in the present paper. The result showed that, microstallite Koi42 and 4 SNPs from the functional genes have significant effect on the carp's body weight. 13 candidate parents which have over two genotypes with higher body weight were developed. These individuals have better growth performance compared to that without these perfect genotypes. Further analysis by multiple step-wise regressions was conducted in order to make sure which markers can be implicated in practical breeding together effectively. CO626, D-Loop 253 and Koi42 were left by both linear regression analysis and AIC criteria, where Koi42 has the highest contribution to the body weight variation among 3 markers. It is that Koi42 genotypes is related with sex by Fisher exact test. Genetic analysis supply the information Koi42 plays its role by additive effect (P＜0.1). Among many epitasis components, significant effect between D-Loop 253 additive effect and Koi42 additive effect was found. Through variance components partition analysis, additive variance account for 11.4%, and effect of epitasis between two loci had the higher percentage (77.5%). This told us 3 mar-kers listed above contribute to the body weight mainly by epitasis. All of these results illustrated that Koi42 with its interaction partner D-Loop 253 could be used to marker assistant breeding or multiple markers based co-selection.

Huanghe carp new strain; Genotype; Body weight; Correlation analysis; Epitasis

Q347; S962.1

A

1000-3207(2017)01-0079-07

10.7541/2017.10

2016-01-24;

2016-04-11

農(nóng)業(yè)部淡水水產(chǎn)生物技術(shù)與遺傳育種重點(diǎn)實(shí)驗(yàn)室開放課題(FBB201401); “十二五”科技支撐計(jì)劃“大宗淡水主養(yǎng)魚類新品種選育”(2012BAD26B02); 中國水產(chǎn)科學(xué)研究院基本科研業(yè)務(wù)費(fèi)(2016RC-LX03)資助 [Supported by the Open Project of Key Laboratory of Freshwater Aquatic Biotechnology and Breeding in Ministry of Agriculture (FBB201401); the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-Year Plan Period (2012BAD26B02); Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (2016RC-LX03)]

張成鋒(1979—), 男, 山東德州人; 博士研究生; 研究方向?yàn)樗a(chǎn)養(yǎng)殖。E-mail: zhangcf@ffrc.cn

石連玉(1960—), 男, 研究員; 研究方向?yàn)樗a(chǎn)育種。Tel: 0451-84861311, E-mail: sly2552@aliyun.com