本文選題：ENU誘變草魚　切入點：草魴雜交后代　出處：《上海海洋大學》2017年碩士論文　論文類型：學位論文

【摘要】：草魚(Cetpharyngodon idellus)為我國特有的淡水養(yǎng)殖品種,在養(yǎng)殖魚類中占有重要的經(jīng)濟地位。由于草魚繁殖周期長,親魚個體大等原因,給應用傳統(tǒng)選育方法育出優(yōu)良品種造成一定的難度,目前草魚尚無國家審定的良種。雌核發(fā)育為快速建立純系的有效途徑,利用微衛(wèi)星標記對雌核發(fā)育后代的遺傳多樣性進行評估,在生產(chǎn)性能優(yōu)良的長江水系草魚的基礎上進行良種選育可能是一種可行的方法�；诒緦嶒炇医�6齡ENU誘變草魚群體,挑選4對優(yōu)良親本建立家系,經(jīng)過生長性狀對比篩選出具有明顯生長差異的兩個家系進行mstn1、mstn2基因的SNP研究,以期篩選出ENU誘變草魚群體生長改良研究提供候選分子標記,進一步確定ENU誘變草魚群體分子標記輔助育種方案。具體研究如下:(1)以紫外線滅活的團頭魴(Megalobrama amblycephala)精子激活草魚卵子,冷休克抑制第二極體排出的方法誘導出長江水系優(yōu)良F2代草魚減數(shù)雌核發(fā)育子代。在后代中不僅存在雌核發(fā)育后代,還存在草魴雜交后代,雌核發(fā)育后代的體型與草魚一致,而草魴雜交后代的體型介于草魚與團頭魴之間。Partec CyFlow倍性分析儀測定結(jié)果顯示:普通草魚與雌核發(fā)育草魚的相對DNA含量分別為23.01和22.72,二者的DNA含量接近;而高體型子代的相對DNA含量為25.38,介于草魚與團頭魴(DNA含量28.21)之間,屬于草魴雜交后代。選取17個微衛(wèi)星標記對草魚群體、雌核發(fā)育草魚群體和草魴雜交后代的遺傳多樣性進行了檢測,共檢測出59個等位基因,其中43.18個有效等位基因。草魚對照群體、草魴雜交后代和雌核發(fā)育草魚群體的平均等位基因依次為3.57、2.86和2.79,平均有效等位基因依次為2.93、2.37和1.96,平均期望雜合度在依次為0.6502、0.5573和0.3775,多態(tài)信息含量(PIC)平均值依次為0.5738、0.4649和0.3791。與草魚對照群體相比,雌核發(fā)育草魚群體的遺傳多樣性顯著下降,表明通過減數(shù)雌核發(fā)育方法可獲得純合性較高的草魚個體。構(gòu)建了草魚后代不同群體的DNA指紋模式圖,篩選到不同群體的9個特異微衛(wèi)星標記,為草魚優(yōu)良群體的選育奠定基礎。(2)為了獲得雌核發(fā)育ENU誘變草魚群體的相關遺傳參數(shù),實驗采用Partec CyFlow倍性分析儀測定ENU誘變草魚群體(Q群體)和雌核發(fā)育ENU誘變草魚群體(E群體)相對DNA含量分別為24.02和23.80,二者的DNA含量接近,均為二倍體。選取28個微衛(wèi)星標記對Q群體和E群體多樣性進行了檢測。結(jié)果表明,E群體和Q群體的平均等位基因分別為3.7143、5.1786,平均有效等位基因分別為2.1857、4.0028,平均期望純合度分別為0.5122、0.2814,平均期望雜合度分別為0.4878、0.7186,多態(tài)信息含量(PIC)平均值分別為0.4282、0.6606。從個體在微衛(wèi)星位點的純合率分析,在E群體中,每個個體的純合度均小于1.00,說明沒有完全純合的個體。從每個微衛(wèi)星位點在群體的純合率分析,除了微衛(wèi)星位點5476,HLJC118和HLJC81外,其他位點的純合度以不同的速率得到明顯的提高。研究結(jié)果表明經(jīng)過減數(shù)雌核發(fā)育方法,ENU誘變草魚群體的各微衛(wèi)星位點的純合度以不同的速率得到提升,遺傳多樣性明顯降低,此方法可以獲得純合度較高的雌核發(fā)育ENU誘變草魚個體,為ENU誘變草魚良種選育提供重要遺傳數(shù)據(jù)資料。(3)對4個ENU誘變草魚家系進行生長對比,經(jīng)過175天養(yǎng)殖,從4個家系的生長對比發(fā)現(xiàn),從體重增長量的角度看,家系1和家系4相對于其他2個家系有明顯的優(yōu)勢,從體重增重率來看,家系4和家系2相對于其他2個家系存在明顯優(yōu)勢,綜合來看家系4的優(yōu)勢最明顯,家系3明顯弱于其他3個家系。家系4的8個性狀明顯大于其他3個家系;家系1的體高、頭長略大于家系2,其他的6個性狀明顯大于家系2和家系3;家系2的8個性狀明顯大于家系3。采用偏相關分析各個形態(tài)性狀與體重的相關程度得出:家系1中有全長、體長、尾柄長、體厚,相關系數(shù)依次為0.357、0.619、0.608、0.396;家系2中有全長、體長、頭長、體厚,相關系數(shù)依次為0.348、0.360、0.687、-0.384;家系3中有全長、體長、尾柄長、尾柄高,相關系數(shù)依次為0.529、0.449、-0.351、0.384;家系4中有全長、體長、體厚,相關系數(shù)依次為0.629、0.543、0.590,經(jīng)過因子分析可以明顯區(qū)分家系4與其他3個家系,以上統(tǒng)計方法可總結(jié)出家系4和家系3具有明顯的生長差異,運用雙向測序法對MSTN1、MSTN2基因在家系3、4中進行SNP位點篩選,對于MSTN1,家系3在465 nt C/G、467 nt G/A,而家系4在465 nt C/G均發(fā)生錯義突變;對于MSTN2,家系3和4在912 nt C/T均發(fā)生同義突變,而家系3在1027 nt G/A、家系4在366 nt A/G均產(chǎn)生錯義突變,位于非編碼區(qū)1390 nt A/T和1401 nt G/A在兩個家系均發(fā)現(xiàn)SNP位點。以上這些結(jié)果表明MSTN1、MSTN2基因的不同SNP位點與ENU誘變草魚的生長性狀存在緊密聯(lián)系。
[Abstract]:Grass carp (Cetpharyngodon idellus) is a freshwater aquaculture species endemic to China, occupies an important position in the economic fishes. Due to the grass carp long reproductive cycle broodstock individuals and other reasons, is difficult to breed fine varieties of application of traditional breeding method, at present there is no national certification grass carp seed. Gynogenesis is the effective way to the rapid establishment of pure lines, were evaluated using microsatellite markers on genetic diversity of gynogenetic offspring, breeding may be a feasible method based on the Yangtze River grass carp production of excellent performance. The laboratory was established at the age of 6 ENU by grass carp group based on the selection of 4 to establish excellent parent families the growth traits were selected for mstn1 with two families significantly differences in growth, SNP of the mstn2 gene, in order to find out the grass carp ENU mutation population growth improvement research For the candidate molecular markers, to further determine the grass carp group by ENU molecular marker assisted breeding program. The specific studies are as follows: (1) with UV inactivated bream (Megalobrama amblycephala) sperm activation grass carp eggs, cold shock suppression method of the second polar body emission induced by long river system generation grass carp F2 with excellent meiotic gynogenetic offspring gynogenetic offspring. Not only exist in the offspring, there is grass bream hybrids and gynogenetic progeny are consistent with grass carp, and between hybrid grass bream size between grass carp.Partec and bream CyFlow ploidy analyzer results show: Grass Carp and grass carp common gynogenetic relative DNA content were 23.01 and 22.72, close to the DNA content of the two; and relatively high content of DNA type offspring was 25.38, between grass carp and bream (DNA content 28.21), belonging to the grass bream hybrids. Select 17 micro Microsatellite markers on the grass carp group, grass carp group and gynogenetic grass bream hybrids genetic diversity of detection, detected a total of 59 alleles, of which 43.18 effective alleles. Grass carp control groups, hybrids and gynogenetic grass bream grass carp group's average alleles were 3.57,2.86 and 2.79, the average effective alleles were 2.93,2.37 and 1.96, the average expected heterozygosity were 0.6502,0.5573 and 0.3775, the polymorphism information content (PIC) the average values were 0.5738,0.4649 and 0.3791. control group compared with grass carp, grass carp group was activated by genetic diversity decreased significantly, showed that the meiotic gynogenesis method can be obtained homozygous high grass carp individuals. To construct the DNA fingerprint pattern of grass carp offspring of different groups, different groups of screened 9 specific microsatellite markers for the breeding of excellent foundation grass carp group The foundation. (2) in order to obtain the genetic parameters of gynogenesis induced by ENU of grass carp group, grass carp group experimental determination of ENU mutation by Partec CyFlow ploidy analyzer (Q group) and Mitogynogenesis group grass carp ENU mutagenesis (E) relative DNA content were 24.02 and 23.80, close to the DNA content of the two. All diploid. Selected 28 microsatellite markers in Q population and E population diversity were detected. The results showed that E group and Q group the average alleles were 3.7143,5.1786, the average effective allele was 2.1857,4.0028, the average expected homozygosity was 0.5122,0.2814, the average expected heterozygosity was 0.4878,0.7186. The polymorphism information content (PIC) were respectively 0.4282,0.6606. from individuals homozygous rate analysis in microsatellite loci, in the E group, each individual homozygosity was less than 1, indicating that there is no completely homozygous individuals. From each microsatellite locus in the homozygous rate analysis group, in addition to the 5476 microsatellite loci, HLJC118 and HLJC81, other sites of homozygosity were obviously increased at different rates. The results indicate that after meiotic gynogenesis method, ENU mutation grass carp group each microsatellite locus homozygosity at different rates improved genetic diversity decreased significantly, this method can obtain high homozygosity of gynogenetic ENU by grass carp individual, provide important data for ENU genetic mutation breeding. Grass carp (3) of the 4 ENU mutation in grass carp family growth comparison, after 175 days of culture, the growth of the contrast from 4 the family, from the perspective of weight increase, family 1 and 4 has obvious advantages compared with the other 2 families, the weight gain rate, family 4 and 2 compared with the other obvious advantages of 2 families, to comprehensive 鐪嬪緋，

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