用AFLP方法分析中国对虾抗病选育群体的遗传变异

AFLP analysis of four selected generations on disease-resistance trait of Fenneropenaeus chinensis

利用AFLP技术对连续4年选育的中国对虾抗白斑病毒群体进行了遗传分析,并比较了不同的数据处理方法对遗传学参数的影响。7个EcoRⅠ/MseⅠ引物组合共产生350个位点,其中202个为多态位点。4代群体的多态位点比例分别为:39.4286%,41.4286%,33.4286%,39.1429%,Nei基因多样性指数分别为:0.1197,0.1259,0.1133,0.1249;4代群体的Shannon多样性指数分别为0.1831,0.1917,0.1702,0.1896。遗传多样性水平除了第3代群体标本明显较低外,其它3代甚为接近,维持在一个恒定的水平。比较了Nei分析、Shannon信息指数分析、AMOVA分析得出的遗传学参数,建议AMOVA分析作为应用AFLP进行群体遗传学分析时首选的统计方法。在AFLP指纹图谱中发现共显性基因座,对其中1个基因座的两个AFLP片段进行了回收、克隆及测序。序列分析表明,多态性是由引物扩增区域内4个核苷酸的插入/缺失导致的,证明了AFLP标记并不完全是显性标记。实验表明AFLP技术灵敏度高,信息量大,适用于分析亲缘关系较近的个体或品系,在中国对虾分子标记辅助育种方面有应用潜力。

Amplified fragment length polymorphism (AFLP) was used to detect the genetic variation of four successively selected specific-pathogen-resistance (SPR) generations. Seven EcoRⅠand MseⅠprimer combinations produced 202 polymorphic markers out of the total of 350 bands amplified. The proportions of polymorphic loci of the first,second,third and fourth generations were (39.4286%),41.4286%,33.4286% and (39.1429%),respectively. The Nei genetic diversity was 0.1197,0.1259,0.1133 and (0.1249),respectively. Shannon genetic diversity index was 0.1831,0.1917,0.1702 and 0.1896,respectively. The genetic diversity index of the third generation was low, while the index of the other three generations remains at a relatively constant level. The analysis showed that the SPR population has great potential in genetic breeding program. The genetic distance of four generations based on the Nei analysis ranged from 0.0282 to 0.0458. Partitioning of the genetic variation revealed that 80.54% is distributed within populations, which is similar to the value (86.72%)derived from AMOVA. Mantel tests showed good correlation of different genetic distance matrices. By comparing the genetic parameters obtained from different statistical methods, we recommended AMOVA the first choice in population genetics analysis with AFLP markers. Co-dominant loci were found in AFLP fingerprinting. Two fragments adjacent in gel positions were recovered, cloned and sequenced. The sequence analysis showed high similarity between them, which indicated that they belong to the same locus. Our results proved that AFLP markers were not complete dominant markers. Our study suggests that AFLP is sensitive to detect genetic variability and effective to find markers and it is useful in marker assisted selection.