摘要
我国地方畜禽品种素以肉质细嫩、肉味香浓等特性而著称于世。在过去的几十年中，家禽育种工作取得了巨大成就，尤其是肉鸡的生长速度得到了前所未有的提高。同时随着人民生活水平的提高，对肌肉品质的要求也越来越高。目前世界上已有不少国家以肌苷酸(inosine monophosphate，IMP)含量作为肉质风味及新鲜程度的衡量指标。传统的育种手段难以使产肉量和肉质均得以提高，因此育种工作者努力寻找影响肉质性状的主效基因及QTLs。候选基因法是研究动物数量性状遗传机制的方法之一。本研究选用五个地方品种鸡泰和乌骨鸡、北京油鸡、崇仁麻鸡、鹿苑鸡、霞烟鸡和一个引进肉用型品种AA鸡共448个个体作为供试群体，采用PCR-RF-SSCP技术对肌苷酸含量的候选基因AMPD1(adenosine monophosphate deaminase 1，腺苷一磷酸脱氨酶1)基因进行研究，同时对其等位基因进行克隆测序，并分析了AMPD1的不同基因型与北京油鸡IMP含量的关系，目的是为确定影响肌肉肌苷酸含量的主效基因或与其紧密连锁的遗传标记奠定基础。
利用PCR-RF-SSCP技术研究肌肉风味物质IMP的潜在候选基因AMPD1。在供试的六个鸡品种中发现，AMPD1基因有3个等位基因A、B、C，6个基因型AA、AB、AC、BB、BC、CC。各基因型在品种间分布差异显著(P＜0.01)。等位基因A、B、C除泰和乌骨鸡无A基因外，在其它5个品种中都存在，其中B基因在北京油鸡、AA鸡、鹿苑鸡、霞烟鸡为优势基因(频率分别为0.6597，0.5750，0.7051，0.7250)，而C基因在泰和乌骨鸡中为优势基因(0.6625)。通过Hardy-Weinberg平衡检验表明泰和乌骨鸡、AA鸡严重偏离遗传平衡(P＜0.01)，其余4个品种符合处于遗传平衡(P＞0.05)。崇仁麻鸡的多态信息含量(PIC)为0.5916，即高度多态，其它品种均属于中度多态。
对AMPD1等位基因A、B、C进行克隆测序发现(因无注册的鸡的AMPD1基因序列，以A为标准)，等位基因B和等位基因A只有一个碱基之差，即C→T(402)，
而等位基因C与等位基因A不同之处多达11个，它们有的缺失，有的替换.这
11处突变位点分别为G、T(361)、A缺失(373)、T缺失(404)、G*T(420)、
C一T(434)、G*A(456)、A一G(469)、G一A(473)、G，A(486)、G一A(491)、
G一A(493).
方差分析表明，A栩叨1不同基因型对IMP含量影响不显著(P)0.05)，
不同基因型与肌普酸含量关系的最小二乘均值分析表明，基因型AC所对应的
肌等酸含量最高(3.7470)，BB型次之(3.1700)，AC、BB对应的肌普酸含量
显著高于基因型AA(2 .4145)(P(0.05).肌普酸含量在月树叨了其余基因型
之间没有显著差异(P>0.05).
在后续工作中，需要更大的群体规模以及泰和乌骨鸡、AA鸡、崇仁麻鸡、
鹿苑鸡、霞烟鸡的肌普酸含量记录，结合更多相关的候选基因或DNA标记进
行分析，寻求与IMP含量紧密连锁的遗传标记，以便应用于标记辅助选择
(MAS)中.
Chinese native chicken breeds are famous for their tender and flavor. During the last years, the improvement has been made by traditional breeding methods. As the living conditions improved, the needs not only for meat production but also for meat quality are also increased. At present, more and more countries use IMP (inosine monophosphate) content as the criterion of meat flavor and freshness. But it is difficult to improve both the quantity and quality of meat by the traditional breeding methods. Therefore, the major genes and QTLs affecting meat quality are studied. Candidate gene method is one of the approaches used to study quantitative trait in animals. In this study, DNA samples from 448 chickens including five Chinese native chicken breeds: Taihe silky fowl, Beijing oil chicken, Chongren Ma chicken, Luyuan chicken, Xiayan chicken and one imported meat breed: Arbor Acres were analyzed. AMPD1 gene was studied by PCR-RF-SSCP. Meanwhile the alleles of AMPD1 gene were cloned and sequenced. The effect of different genotypes on IMP content was analyzed. The aim of this study was to determine the major gene affecting IMP content or the genetic markers tightly linked with it.
Three alleles and six genotypes for AMPD1 locus in the six chicken breeds by PCR-RF-SSCP were found. The genotype frequency was significantly different (P < 0.01) among breeds. Allele A was not found in Taihe silky fow, alleles A, B and C were all found in other five chicken breeds. Allele B was the superior gene for Beijing oil chicken, Arbor Acres, Luyuan chicken, Xiayan chicken (allele frequencies were 0.6597, 0.5750, 0.7051, 0.7250 respectively). The test of Hardy-Weinberg equilibrium showed that Taihe silky fowl and Arbor Acres much diverged from the genetic equilibrium (P < 0.01). Study on polymorphic information content for AMPDl showed that Chongren Ma had highly polymorphic (0.5916) and the other five chicken breeds had moderately polymorphic.
Cloning and sequencing of AMPD1 alleles A, B, C showed that allele B had only one base difference with allele A with C-->T(402), while allele C had eleven mutations with some base lost and others substituted: G-->T (361), the lost of A (373), the lost of T (404), G -->T (420), C-->T (434), G-->A (456), A-->G (469), G-->A (473), G-->A (486), G-->A (491), G-->A (493) (Because no sequence of AMPDl gene for chicken was enrolled in GeneBank, allele A was used as the criterion)
Analysis of variance indicated that AMPD1 genotypes didn't significantly influence the IMP content (P>0.05). Least squares means of IMP content for AC and BB (3.7470, 3.1700) were higher than that for AA (2.4145)(P<0.05), and that for other genotypes not significant (P>0.05).
It is necessary to have records of other five chicken breeds for further exploring genetic marker linked with major gene affecting IMP content by using more animals and combining
more related candidate genes. It is certain that genetic marker will play more important role in marker assisted selection of animal breeding practice.
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