Hunt G J, Page R E
Department of Entomology, University of California, Davis 95616.
Mol Gen Genet. 1994 Sep 1;244(5):512-8. doi: 10.1007/BF00583902.
A colony-level phenotype was used to map the major sex determination locus (designated X) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing back-cross progeny were evaluated for the trait 'low brood-viability' resulting from the production of diploid drones that were homozygous at X. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage between X and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance between X and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.
利用群体水平的表型来定位蜜蜂（西方蜜蜂）中的主要性别决定位点（命名为X）。通过器械授精将单个蜂王（有生殖能力的雌性）与单个雄蜂（可育雄性）交配。F1蜂王的单倍体雄蜂后代分别与来自一个亲本品系的蜂王女儿进行回交。对98个包含回交后代的蜂群进行评估，以检测因X位点纯合的二倍体雄蜂产生而导致的“低育雏活力”性状。这些蜂群中单倍体雄蜂父亲的DNA样本分别用于10碱基引物的聚合酶链反应（PCR）。这些反应产生随机扩增多态性DNA（RAPD）标记，分析其与群体水平表型的共分离情况。在产生低育雏活力蜂群的25只雄蜂中，有22只共享一个RAPD标记等位基因。该RAPD标记片段被克隆并进行了部分测序。设计了两个引物来定义该位点的序列标签位点（STS）。这些引物扩增出与原始RAPD标记共分离的DNA标记片段。为了更精确地估计X与STS位点之间的连锁关系，另一组由一个低育雏活力蜂群的后代组成的蜜蜂用于分离分析。检测了4只二倍体雄蜂及其181只二倍体姐妹（工蜂，无生殖能力的雌性）的RAPD和STS标记的分离情况。由于片段长度等位基因的出现，共分离的RAPD和STS标记是共显性的。这4只二倍体雄蜂对这些标记是纯合的，但181只工蜂中只有3只是纯合子（重组体）。因此，估计X与STS位点之间的距离为1.6厘摩。还发现了一个额外的连锁标记，它与STS位点相距6.6厘摩。
