【作者】 王伟继；

【导师】 王清印； 孔杰； Hanping Wang；

【作者基本信息】 中国海洋大学 ， 海洋生物， 2008， 博士

【摘要】 以中国对虾抗白斑综合症病毒病(White Spot Syndrome Virus, WSSV)选育群体第四代为母本,野生中国对虾为父本,采用单对亲本人工精荚移植的方式产生F1代,F1代个体家系内兄妹交产生F2代为作图群体。利用含WSSV的毒饵,采用口饲法进行作图个体的抗病感染实验,以个体摄食毒饵后的存活时间作为抗病指标,同时记录个体死亡时的体长、体重等参数,作为中国对虾抗病、生长性状的数量性状位点(Quantitative Traits Locus, QTL)定位分析的性状参数。实验共获得42尾个体作为作图群体。首先利用MAPMAKER/EXP3.0遗传连锁图谱分析软件构建了三个中国对虾的扩增片段限制性长度多态性(Amplified Fragment Length Polymorphism, AFLP)分子标记遗传连锁图谱。62对AFLP选择性引物组合共产生529个分离位点,其中符合1:1孟德尔分离比例的位点共253个,利用拟测交理论这将些位点分别构建了中国对虾F1亲本雌虾、雄虾的遗传连锁图谱。符合3:1孟德尔分离比例的位点共276个,利用F2自交模型和这些位点构建了中国对虾F1亲本共同的AFLP分子标记连锁图谱。三张连锁图上分别有31、25和44个连锁群,连锁的标记数量分别为94个、81个和129个。雌虾连锁图谱长度比雄虾的稍长。雌虾最大连锁群长度为82.9cM,连锁群最大标记数为6个;雄虾最大连锁群长度为103.8cM,连锁群最大标记数为8个;中国对虾共有图谱的连锁群最大长度80.6cM,连锁群最大标记数为8个。三张图谱的分辨率为分别为2.4cM、2.4cM和2.1cM。标记间隔距离分别为12.20cM、11.45cM和11.12cM。图谱密度已经达到中等密度连锁群的要求。各标记在连锁群上的分布比较均匀,没有出现成簇分布的现象。图谱覆盖率分别达到50.21%,51.93%和48.08%。分析了作图群体选择以及相应的作图策略在中国对虾中应用的可行性。在构建获得的中国对虾三张遗传连锁图谱的基础上,利用MAPMAKER/QTL1.1软件,以区间作图法(Interval mapping,IM)对中国对虾的抗病性和生长性状,包括体长、全长、体重进行了QTL定位分析。在三张连锁图上,共获得了11个QTL位点。其中四个和中国对虾全长相关,两个和体长相关,两个和体重相关,三个和抗病性相关。四个与中国对虾全长相关的QTL位点分别分布在四个不同的连锁群上,有两个与体长相关的QTL位点连锁,一个与体重相关的QTL位点连锁,另外一个单独分布在一个连锁群上。说明中国对虾全长是由多基因控制的性状。与抗病性相关的三个QTL位点分别分布在三个不同的连锁群上,其中有两个是和体重相关的QTL位点连锁的,说明抗病性和个体体重存在一定的程度的关联并且中国对虾抗病性状也是由多基因控制或影响的。11个QTL位点中,有4个位点的加性效应值为负,其余的均为正值。而显性效应只在中国对虾通用连锁图谱中获得定位的QTL位点上具有,并且均为正值。各位点的变异解释率从23.4%到66.9%不等,对导致高变异解释率的原因进行了分析和探讨。对某些QTL位点性状值差异的亲本来源进行了分析。利用性别特异性基因组池(sex-specific genomic DNA pool)策略,对分别由经过表型性征和组织解剖性腺确认的24尾雄性和24尾雌性蓝鳃太阳鱼(Bluegill sunfish, Lepomis macrochirus)组成的基因组池进行了AFLP分析,64对选择性引物组合共在两个性别特异性基因组中筛选获得7个与性别相关的特异性位点,其中6个位点与雌性相关,1个与雄性相关。在随后的个体性别特异性位点的检测中,7个位点在24尾个体中的检出频率从16.67%到41.67%不等。这些标记被作为与性别相关的候选标记纳入到随后的蓝鳃太阳鱼AFLP分子标记遗传连锁图谱的构建当中。在遗传连锁图谱的构建工作中,利用连续多代隔离培养的蓝鳃太阳鱼两个地理群体的个体分别作为父本和母本,采用单对亲本交配的方法获得F1代家系,作图群体包括两个亲本以及F1代共90尾孵化30天左右的仔鱼。以拟测交理论为构图策略,构建获得了蓝鳃太阳鱼雄鱼和雌鱼的AFLP分子标记遗传连锁图谱。64对AFLP选择性引物组合共产生1:1分离标记438个,其中包括一个雌性标记和9个共显性标记,这个性别标记与前期实验中获得的7个标记中的一个雌性相关标记对应。438个标记中,222个标记来自于母本,偏分离标记6个(P<0.01),作图标记数量为216个。母本连锁图包括39个连锁群(LOD≥4.0),其中大于4个标记的连锁群为21个。定位192个连锁标记,其中包括1个雌性性别标记和4个共显性标记。母本连锁群平均连锁标记数为4.92个,最大连锁群长度为122.9cM,连锁群最大标记数量为14个,标记间平均图距为11.29cM,连锁图谱实际长度为1727.8cM,母本图谱覆盖率为64.04%。其中,母本连锁图的8号连锁群定位了1个雌性性别标记,此连锁群被认为是和蓝鳃太阳鱼性别形成相关的。在母本连锁图的5、6、8、10、11号连锁上都发现由非常明显的标记成簇分布现象,除5号连锁群外,其余连锁群上标记成簇分布在连锁群的端部位置。父本分离标记219个,偏分离标记4个(P<0.01),作图标记数量为212个。父本连锁图包括40个连锁群(LOD≥4.0),其中大于4个标记的连锁群为21个。40个连锁群包括191个分离标记,其中包括4个共显性标记。父本连锁群平均连锁标记4.77个,最大连锁群长度为345.3cM,连锁群最大标记数量为19个,标记间平均图距为10.58cM,连锁图谱实际长度为1598.2cM,父本图谱覆盖率为66.26%。父本连锁图的11、31和32号连锁群存在明显的标记成簇分布现象,分布位置集中在连锁群的端部。成簇分布的标记位于连锁群的端部位置与蓝鳃太阳鱼的染色体结构为端部或近端部着丝粒构造相一致,暗示连锁群上标记成簇分布的位置可能对应染色体的着丝粒部位,不过,需要最后确定连锁群与染色体的物理定位关系后才能下结论。实验获得的蓝鳃太阳鱼母本39个连锁群与父本40个连锁群的数量都超过了该物种单倍体24条染色体的数目,说明目前获得连锁群有一些是分布在同一条染色体上的。以后的工作需要通过增加标记数量或标记种类以达到连锁群数目和染色单体数目的一致性。母本连锁图长度稍长于父本连锁图,大约相差129cM,这与其它高等生物以及鱼类中的虹鳟和斑马鱼的规律是一致的。父本连锁图与母本连锁图最大的差别在于父本拥有一个长达345.3cM的连锁群,而母本中缺乏与此长度相近的连锁群,表明蓝鳃太阳鱼雌性和雄性在染色体组成上有较大的差异。筛选获得的7个与性别相关的标记中,有6个是雌性相关的,而只有1个是雄性相关的,暗示蓝鳃太阳鱼雌性拥有异型性染色体或者拥有雄性不具备的染色体可能性比较高,即支持蓝鳃太阳鱼的性决定模式为ZZ/ZW型或XX/XO型。根据获得的父本连锁图谱在最大连锁群上与母本有较大的差异,结合与蓝鳃太阳鱼可以进行种间杂交的绿太阳鱼的性别决定机制为XX/XO型,初步认定蓝鳃太阳鱼的性染色体组成为XX/XO型。该研究结果首次报道了蓝鳃太阳鱼的AFLP分子标记遗传连锁图谱和性别标记以及其图谱上的定位。更多还原

【Abstract】 Amplified Fragment Length Polymorphism (AFLP) markers were used to construct the linkage map of Fenneropenaeus chinensis. We sampled maternal individual from the fourth successively selected generation of disease resistance population of F. chinensis and paternal individual from wild population, respectively. Artificial insemination was used to generate F1 family; F2 families totally 42 bodies came from sisterhood intercross between F1 families. The F2 progenies were firstly subjected to the WSSV challenge, the survival time were recorded as the index of disease resistance traits of F. chinensis, the data of its total length, body length and weight were recorded as well when the individual was found death. All the data recorded would be involved in the QTLs mapping. Sixty-two primer combinations produced 529 segregating bands; of which 253 segregate in a 1:1 model and 276 in a 3:1 model. The female and male 1:1 ratio makers were used to construct the respective linkage based on the pseudo-testcross strategy. The 3:1 ratio segregant markers were used to construct the common linkage map of F. chinensis using F2 intercross model. The female linkage map contains 94 segregating markers, which were linked in 31 linkage groups (including linkage couples), covering 51.39% of F. chinensis genome with the interval of 12.20cM. The length of maximum linkage group was 82.9 cM, the maximum number of markers in a linkage group was 6. The male linkage map contains 81 segregating markers, which were linked in 25 linkage groups, covering 50.21% of F. chinensis with the interval of 11.45cM. The length of maximum linkage group was 103.8 cM, the maximum number of markers in a linkage group was 8. The 276 markers in a 3:1 model were used to construct the common F. chinensis linkage map based on F2 intercross model strategy. The common linkage map consists of 44 linkage maps with 129 markers, covering 48.08% of F. chinensis genome with the interval of 11.12cM. The length of maximum linkage group was 80.6 cM, the maximum number of markers in a linkage group was 8. All the linked markers distributed evenly in linage groups, no cluster marker was found in any linkage group. The density and coverage of these three linkage groups showed that they matched the medium density linkage map standard. The linkage mapping strategies we applied in F. chinensis and mapping families were discussed. Based on the three linkage maps, QTLs (Quantitative Trait Loci) associated with body length, total length, weight and disease resistance were analyzed by MAPMAKER/QTL1.1 software using interval mapping (IM). There were totally 11 loci were detected on 6 linkage groups. Four of them were associated with body length, two with total length, two with weight and three with disease resistance. The four QTLs loci associated with body length distributed on four different linkage groups, two of them linked with QTLs of total length, one of them linked with QTLs of weight, and the last one distributed in a single linkage group, which suggested that the trait of total length was polygenic controlled. The three QTLs with disease resistance distributed on three different linkage groups, two of them linked with two QTLs of weight, which suggested that the individual’s disease resistance was related with the body’s weight and multi-genes controlled the disease resistance as well. Except four QTLs additive value were negative, the other seven were positive. The dominance value only owned by the QTLs that based on the common linkage groups of F. chinensis and they were all positive. The variance explained ratio of 11 QTLs was from 23.4% to 66.9%. Following with the QTLs additive and dominant values, the individual’s trait values of different genotypes were analyzed. The reasons that resulted in some very high variance explained were discussed based on the QTL mapping method, data recording and the size of mapping family. AFLP technique has been used in this study for detection the sex-specific markers of bluegill sunfish based on sex-type DNA pool strategy. The phenotypically female and male individuals that composed of sex-type genetic DNA pool were subjected to gonad anatomy to confirm their sexualities. Seven sex-specific markers were identified from 64 different primer combinations amplification across the female and male genomic DNA pools that composed of 24 female and male individuals, respectively. Six were female and one was male related marker. The sex-specific markers were subsequently analyzed individually: there were 16.67%-41.67% of 24 female or male individuals were detected sharing the sex-specific markers. In the linkage analysis, paternal and maternal bodies that selected from two different bluegill population that kept in South centers for several generations consisted in a single-pair parent. The single-pair parent and their 90 progenies were analyzed using as mapping family based on double-pseudo testcross strategy, sixty-four primer combinations that as same as in the sex-specific markers analysis produced totally 438 segregant loci, including 1 sex-specific and 9 co-dominant markers. In maternal linkage map, 192 markers distributed on 39 linkage groups (LOD≥4.0); There were 21 linkage groups that owned at least 4 markers, the maximum length and markers number of linkage groups were 122.9 cM and 14, respectively; the average markers number in each linkage group were 4.92, the average markers space was 11.29 cM, the genome coverage of maternal linkage map was 64.04%. In maternal linkage map, we have acquired a sex-specific linkage group 8. The cluster markers on linkage groups 5, 6, 8, 10 and 11 were tended to distribute by the end of linkage groups. For the paternal linkage map, 191 linked markers, including 4 co-dominant, distributed on 40 linkage groups (LOD≥4.0). The linkage groups that owned at least 4 markers number was 21, the average markers number of each linkage group was 4.77 in paternal linkage map, and average markers space is 10.58cM. The maximum length and markers number of linkage groups in paternal linkage map were 345.3cM and 19, respectively. The genome coverage of paternal linkage map was 66.26%. The linkage groups 11, 31 and 32 were found had cluster markers at the end of linkage groups. Bluegill sunfish karyotype showed that this kind of fish had diploid numbers of 48, consisting of telocentrics and acrocentrics with very short arms, in the linkage map of bluegill sunfish, many of the cluster AFLP markers distributed by the end of linkage groups, which were consistent with the chromosomes structure, but only the chromosomes become defined in bluegill sunfish, the locations of the cluster markers could be better defined. The maternal and paternal linkage group numbers were more than the haploid chromosomes number (2n=48) of bluegill, which suggested some linkage groups shared the same one chromosome, with supplement more AFLP markers and some other types marker, such as SSR, the number of linkage groups should match haploid chromosome number. The length of maternal linkage map was 129cM more than the paternal map, which was similar as in the other vertebrate organisms and other fishes, such as in rainbow trout and zebrafish. The maximum length of linkage groups in paternal map was 345.3 cM, we could not find any other matchable groups in maternal linkage map, which suggested that the difference between male and female chromosome of bluegill sunfish was obviously. Of all the 7 sex-specific markers, 6 were female specific and one was male, the chance that female owned heteromorphic sex chromosome or the female had one more chromosome than the male were good, namely the sex chromosome system of bluegill sunfish were ZW/WW or XX/XO type. The evidence that the paternal owned a much bigger linkage groups than the maternal, combined with the evidence that the green sunfish, its sex chromosome system was XX/XO, could hybridize with bluegill sunfish very easily, more suggested that the bluegill sunfish had XX/XO sex chromosome system. This work was the first study of linkage mapping of bluegill sunfish using AFLP markers, and it was the first time we tried to clear the sex determination mechanism from DNA markers and linkage mapping.更多还原