Alves E, Fernández A I, Fernández-Rodríguez A, Pérez-Montarelo D, Benitez R, Ovilo C, Rodríguez C, Silió L
Departmento de Mejora Genética Animal, INIA, Carretera de la Coruña km 7.5, 28040 Madrid, Spain.
Animal. 2009 Sep;3(9):1216-23. doi: 10.1017/S1751731109004819.
Iberian pigs and wild boars are the source of highly priced meat and dry-cured products. Iberian maternal origin is mandatory for labeled Iberian products, making necessary the authentication of their maternal breed origin. Discrimination between wild and domestic pig maternal origin may be useful to distinguish labeled wild boar meat obtained from hunting or farming. In order to detect useful polymorphisms to trace Iberian, Duroc and wild boar maternal lineages, we herein investigated the complete porcine mitochondrial DNA (mtDNA) using three complementary approaches. Near-complete mtDNA sequences (16989 bp), excluding the minisatellite present in the displacement loop region (D-loop), were successfully determined in six Iberian pigs, two Duroc and six European wild boars. To complete the mtDNA analysis, the D-loop minisatellite region was also analyzed in the same set of samples by amplification and capillary electrophoresis detection. Finally, the frequencies of Asian and European Cytochrome B (Cyt B) haplotypes were estimated in Iberian (n = 96) and Duroc (n = 125) breeds. Comparison of near-complete mtDNA sequences revealed a total of 57 substitutions and two Indels. Out of them, 32 polymorphisms were potential Iberian markers, 10 potential Duroc markers and 16 potential wild boar markers. Fourteen potential markers (five Iberian and nine Duroc), were selected to be genotyped in 96 Iberian and 91 Duroc samples. Five wild boar potential markers were selected and tested in samples of wild boars (73) and domestic pigs including: 96 Iberian, 16 Duroc, 16 Large White and 16 Landrace. Genotyping results showed three linked markers (m.7998C>T, m.9111T>C, m.14719A>G) absent in Duroc and present in Iberian pigs with a frequency 0.72. Six markers (m.8158C>T, m.8297T>C, m.9230G>A, m.11859A>G, m.13955T>C, m.16933T>C), three of them linked, were absent in Iberian pigs and present in Duroc with a joint frequency of almost 0.50. Finally three linked markers (m.7188G>A, m.9224T>C, m.15823A>G) were solely detected in wild boars with a frequency 0.22. The D-loop minisatellite results showed overlapping ranges of fragment sizes and suggested heteroplasmy, a result that nullify the use of this region for the development of breed diagnostic markers. The Cyt B haplotype results showed the presence of European haplotypes in Iberian while one of the Asian haplotypes was detected in Duroc with a frequency 0.22, linked to the Duroc marker m.9230G>A. Our results are valuable to resolve the problems of Iberian and wild boar maternal origin determination but additional markers are required to achieve totally useful genetic tests.
伊比利亚猪和野猪是高价肉类和干腌制品的来源。对于贴有标签的伊比利亚产品，其母系来源必须是伊比利亚猪，因此有必要对其母系品种来源进行鉴定。区分野生猪和家养猪的母系来源，可能有助于鉴别通过狩猎或养殖获得的贴有标签的野猪肉。为了检测用于追踪伊比利亚猪、杜洛克猪和野猪母系谱系的有用多态性，我们在此使用三种互补方法对猪的完整线粒体DNA（mtDNA）进行了研究。在6头伊比利亚猪、2头杜洛克猪和6头欧洲野猪中成功测定了近乎完整的mtDNA序列（16989 bp），排除了位移环区域（D-loop）中存在的小卫星序列。为了完成mtDNA分析，还通过扩增和毛细管电泳检测对同一组样本中的D-loop小卫星区域进行了分析。最后，在伊比利亚猪（n = 96）和杜洛克猪（n = 125）品种中估计了亚洲和欧洲细胞色素B（Cyt B）单倍型的频率。近乎完整的mtDNA序列比较共发现57个替换和2个插入/缺失。其中，32个多态性是潜在的伊比利亚猪标记，10个是潜在的杜洛克猪标记，16个是潜在的野猪标记。选择了14个潜在标记（5个伊比利亚猪标记和9个杜洛克猪标记）在96头伊比利亚猪和91头杜洛克猪样本中进行基因分型。选择了5个野猪潜在标记并在野猪（73头）和家猪样本中进行测试，家猪样本包括：96头伊比利亚猪、16头杜洛克猪、16头大白猪和16头长白猪。基因分型结果显示，杜洛克猪中不存在而伊比利亚猪中存在频率为0.72的三个连锁标记（m.7998C>T、m.9111T>C、m.14719A>G）。伊比利亚猪中不存在而杜洛克猪中存在且联合频率接近0.50的六个标记（m.8158C>T、m.8297T>C、m.9230G>A、m.11859A>G、m.13955T>C、m.16933T>C），其中三个是连锁的。最后，仅在野猪中检测到频率为0.22的三个连锁标记（m.7188G>A、m.9224T>C、m.15823A>G）。D-loop小卫星结果显示片段大小范围重叠，并提示存在异质性，这一结果使该区域无法用于开发品种诊断标记。Cyt B单倍型结果显示伊比利亚猪中存在欧洲单倍型，而在杜洛克猪中检测到频率为0.22的一种亚洲单倍型，与杜洛克猪标记m.9230G>A相关。我们的结果对于解决伊比利亚猪和野猪母系来源鉴定问题很有价值，但需要更多标记才能实现完全有用的基因检测。
