乌骨鸡和珍珠鸡BF2与β微球蛋白(β2m)基因克隆及品种多态性分析

克隆了乌骨鸡（BF2＊SI）和珍珠鸡（BF2＊NM）群主要组织相容性复合体I（MHC class I）和β微球蛋白（β2m）基因，分析了其分子特征与等位基因多态性。BF2＊SI相互问氨基酸同源率为75．5％～93．9％，保留了人HLA-A2与抗原多肽结合的7个关键性氨基酸。BF2＊NM相互问氨基酸同源率为81．1％～98．5％，保留了人HLA-A2与抗原多肽结合的6个关键性氨基酸。BF2＊SI在α1和α2区（抗原多肽结合区，PBD）有24个高置换率位点，置换率最高的为69位和9位。BF2＊NM在PBD区共有6个高置换率位点。BF2＊SI PBD发生氨基酸置换的位点数大于来航鸡BF2，BF2＊NM PBD区氨基酸置换的位点数和置换率则远远小于来航鸡BF2。根据同源率，将BF2＊SI α1区分为4类，α2区分为3类，α3区分为3类，并由此聚类为5个系谱（A-E）。将BF2＊NM α1区分为3类，α2区分为3类，α3区分为1类，并由此聚类为3个系谱（A～C）。比较发现BF2＊03SI、BF2＊04SI和BF2＊05NM的α1区与抗马立克氏病的BF2＊2I同源率最高（分别为86．4％、86．4％和87．5％）；BF2＊05SI和BF2＊05NM的α2区与BF2＊2I同源率最高（均为93．4％）。另外，根据成熟肽区的氨基酸序列可将乌骨、珍珠鸡β2m分为两类，第一类和来航鸡β2m（M84767）氨基酸序列完全相同，第二类与第一类相比，氨基酸序列相互间同源率为85．7％。结果揭示了各类鸡的MHCI和β2m基因具有品种分子特征。     

Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California

Monilinia laxa is a pathogen of brown rot of stone fruit and almond in California, causing blossom blights and fruit rots. In this study, low-level resistance to the benzimidazole fungicides benomyl and thiophanate-methyl was detected in field isolates of M laxa collected from stone fruits and almonds in California. Low-resistant (LR) isolates grew in potato dextrose agar (PDA) plates amended with benomyl and thiophanate-methyl at 1 and 5 microg ml(-1), respectively, but not in plates amended with benomyl at 5 microg ml(-1) or thiophanate-methyl at 50 microg ml(-1). The benzimidazole LR isolates were characterized by temperature sensitivity and the DNA sequence of the beta-tubulin gene. The LR isolates showed high-temperature sensitivity, being sensitive to 1 microg ml(-1) of benomyl at 28 degrees C but resistant at 8-24 degrees C. Analysis of the DNA sequence of the beta-tubulin gene showed that the LR isolates had a point mutation at the amino-acid position 240, causing substitution of leucine by phenylalanine. Based on the point mutation, a pair of allele-specific PCR primers was developed for rapid detection of LR isolates of M laxa. In addition, a pair of PCR primers specific to M laxa was developed on the basis of the differences in the DNA sequence of the intron 6 of beta-tubulin gene from M laxa, M fructicola and other fungal species. The primer pair amplified the expected 376-bp DNA fragment from all M laxa isolates tested, but not from 14 other fungal species isolated from stone fruit and almond crops. The restriction endonuclease BsmA I recognized the sequence GTCTCC in the PCR products from sensitive (S) isolates only, but not the GTTTCC sequence in the PCR products from LR isolates. The endonuclease digested the 376-bp PCR products from S isolates to produce two bands (111 and 265 bp) on agarose gels. Thus, both allele-specific PCR and the PCR-restriction fragment length polymorphism (PCR-RFLP) methods could be useful for rapidly detecting benzimidazole-resistant isolates of M laxa from stone fruit and almond crops in California.     

Identification of ovalbumin phenotypes of the Asian indigenous chicken populations using polymerase chain reaction-restriction fragment length polymorphism

Three electrophoretic variations (AA, BB and AB) of ovalbumin controlled by codominant alleles Ov^A and Ov^B have been observed in various chicken populations. We compared nucleotide sequences of the open reading frame between two alleles of ovalbumin gene. The difference between the two alleles was found as a non‐synonymous substitution of asparagine to aspartic acid as a result of AAT to GAT point mutation at position 8032–8034 in exon 8. We developed polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) protocol in combination with Mbo I restriction endnuclease mapping for the detection of this substitution. By the PCR‐RFLP the allelic frequency of the Ov^B was estimated to be within the range of 0.000–0.150 in 11 Asian indigenous chicken populations and 0.000 in four improved breeds used in the present study. Gene frequency, estimated by PCR‐RFLP in the present study, paralleled that obtained by protein polymorphisms of egg white. Thus, this study provides, for the first time, information of the occurrence of ovalbumin allele Ov^A and Ov^B in Asian indigenous chicken populations.     

Characterization of a Streptococcus equi ssp. equi Isolate From a Strangles Outbreak in Thailand

Strangles, which is caused by Streptococcus equi ssp. equi, is one of the major infectious respiratory diseases in horses. Knowledge of isolates from different areas of the world is important for investigating the different strains of the disease. In contrast to many other countries, currently little is known about S. equi ssp. equi isolates in Thailand. In 2014, a farm in Thailand imported 20 horses from Europe. Approximately 1 month after arrival, 50% of the horses had developed pyrexia, mucopurulent nasal discharge, and abscesses of the mandibular lymph nodes. Nasal swabs of mucopurulent discharge were sent to a diagnostic laboratory, and two isolates of S. equi ssp. equi were identified. One of the isolates was further characterized using seM gene polymerase chain reaction and sequence analysis. The seM sequence was then compared to the database of PubMLST-seM. It was found to contain SeM allele 48, an allele isolated from horses in the United Kingdom in 2006 and 2010. This result demonstrates the usefulness of SeM allele identification as a tool for investigating the source of related strains and for the epidemiologic study of strangles. To the best of the authors' knowledge, this is the first report of the identification of an SeM allele of the S. equi ssp. equi isolate in Thailand.     

部分地方鸡种微卫星DNA指纹分析

利用20个微卫星标记对我国19个地方鸡种保种群进行了遗传检测,计算各群体的等位基因频率、平均基因杂合度、平均多态信息含量及各群体间的遗传距离,并用类平均法进行聚类分析。研究结果表明:20个微卫星标记在19个地方鸡种保种群共检测到184个等位基因,平均为9.2个,基因频率分布在0.013-0.838之间。19个地方鸡种平均杂合度在0.5824-0.7432之间。其中藏鸡最高,白耳鸡最低。平均多态信息含量在0.5238-0.7023之间,均大于0.5,表现为高度多态性;19个鸡种聚为6类。各鸡种的遗传距离及聚类结果与所保存的地方鸡种的地理分布、现实状况是相吻合的,从而表明用该方法分析品种间的亲缘关系是可行的。     

Monitoring of Venturia inaequalis harbouring the QoI resistance G143A mutation in French orchards as revealed by PCR assays

BACKGROUND: Genetic resistance to QoI fungicides may account for recent failures to control Venturia inaequalis (Cooke) Winter in French orchards. Two PCR-based assays were developed to detect the G143A point mutation in the fungal mitochondrial cytochrome b gene. The mutation is known to confer a high level of resistance to QoI fungicides. Occurrence of the G143A mutation in French field isolates collected from 2004 to 2007 was monitored. RESULTS: The QoI-resistant cytochrome b allele was specifically detected either following the cleavage of the amplified marker by a restriction endonuclease (CAPS assay) or its amplification using an allele-specific PCR primer. Using either method, the G143A mutation was found in 42% of the 291 field samples originating from French orchards in which apple scab proved difficult to be controlled. Monitoring of the G143A mutation in orchards located in 15 French administrative regions indicated that the mutation was detected at least once in nine of the regions, and its presence ranged from 33% to 64% of the orchards analysed in 2004 and in 2007 respectively. CONCLUSION: The PCR-based methods developed in this study efficiently reveal the presence of the G143A mutation in French V. inaequalis field populations.     

Identification and characterization of new S‐alleles associated with self‐incompatibility in almond

Almond is a highly heterozygous species with a high number of S‐alleles controlling its gametophytic self‐incompatibility system (GSI). In this work, we have analysed 14 Spanish local almond cultivars for S‐RNase allele diversity. Five new S‐RNase alleles were identified by cloning and sequencing, S₃₁ (804 bp) in ‘Pou de Felanitx’ and ‘Totsol’, S₃₂ (855 bp) in ‘Taiatona’, S₃₃ (1165 bp) in ‘Pou d’Establiments’ and ‘Muel’, S₃₄ (1663 bp) in ‘Pané‐Barquets’ and S₃₅ (1658 bp) in ‘Planeta de les Garrigues’. Additionally, seven already known almond alleles could be recognized in the local cultivars studied. The high number of new alleles identified reveals the wide diversity of almond germplasm still existing and requiring characterization, and points to the possibility of new findings by a wider study focusing on other provenances. The almond S‐RNases have been compared to those of other Prunus species, showing a high identity and confirming that the S‐RNase gene in this genus presents a probable common ancestor.     

四川地方小麦品种产量与品质相关性状SSR标记位点的优异等位变异遗传解析

为发掘控制小麦产量及品质相关性状的优异等位变异和携带优异等位变异的基因资源,本研究采用与小麦产量及品质相关性状显著关联的13个SSR标记,利用Breseghello提出的无效等位变异(null allele)方法对64份四川地方小麦品种构成的自然群体的等位变异进行遗传解析。共鉴定出38个控制产量相关性状、18个控制品质相关性状的等位变异。其中,7份四川地方小麦品种携带有较多的等位变异(50个)。优异等位变异分析显示,等位变异产生的表型效应值在方向和大小上均有所不同,在与产量性状关联的5个优异等位变异中,2个具有较大增效效应值(效应值3.00),其余3个则具有较大的减效效应值(效应值3.00);与品质性状关联的12个优异等位变异中,9个具有较大的增效效应值,3个具有较大的减效效应值。SSR标记Xgwm372的4个等位变异与产量和品质相关性状均显著关联。基于上述研究结果,这些与小麦产量与品质相关性状显著关联的SSR等位变异可为小麦育种杂交亲本的选择和分子辅助选择育种提供依据。     

Development of a CAPS marker for the badh2.7 allele in Sri Lankan fragrant rice (Oryza sativa)

Fragrance in rice is caused by mutations in the badh2 (betaine aldehyde dehydrogenase) gene. It was previously reported that exons 1, 2, 7, 10, 13 and 14 of badh2 are hot spots for various mutations leading to fragrance in most aromatic rice. This study was carried out to sequence the 14th exon of badh2 gene of Sri Lankan aromatic rice varieties that lack the badh2.1 allele. The aims of the study were to predict the aberrant protein structure and to develop a functional DNA marker. In view of this, we sequenced the 14th exon of four traditional aromatic accessions and compared with a published sequence. Four accessions contained a nucleotide ‘G’ insertion in the 14th exon. This novel mutation can be classified as the badh2.7 allele. The predicted three‐dimensional protein structure of the mutant shows loss of part of the oligomerization and coenzyme binding domains, a change that is predicted to result in fragrance. A CAPS‐based novel marker, Bad2.7CAPS, was developed to identify varieties possessing this badh2.7 allele, and it can be utilized in rice breeding programmes.