A genetic polymorphism and its genetic effects on goat myogenin gene in intron 1

Single nucleotide polymorphisms (SNPs) of the myogenin (MyoG) gene were tested using primer induced restriction fragment length polymorphism assay-polymerase chain reaction (PIRA-PCR) from Bore goat and its upgrading offspring to Tangshan diary goat (including F1, F2 and F3). The effects of the myogenin gene on the birth weight, 1-month body weight and the weaning weight were also analyzed. On the basis of the DNA sequence of the goat myogenin gene (FJ607135), primers were designed to amplify myogenin gene. The result showed that one polymorphism (transition of g.558C>T) was found in intron 1 of goat myogenin gene, in which two alleles (A and B) and three genotypes (AA, AB and BB) were examined. The distributions of three genotypes were basically identical in four goat populations, and allele A was the dominant gene. The effect of the myogenin genotypes on the birth weight, 1-month body weight and the weaning weight were all not significant (P > 0.05) due to the small number of BB gaots; however, the values of AA genotype goats and AB genotype goats were obviously higher than those of BB genotype goats for three growth traits, in the order of AA > AB > BB. These results suggest that the myogenin genotype has some effects on partial growth traits of goat, and selecting the individuals with A allele could be favorable to the birth weight, 1-month body weight and the weaning weight.Key words: goats, myogenin gene, primer induced restriction fragment length polymorphism assay-polymerase chain reaction (PIRA-PCR), genetic polymorphisms, genetic effects

SNP-RFLPing 2: an updated and integrated PCR-RFLP tool for SNP genotyping

<p>Abstract</p> <p>Background</p> <p>PCR-restriction fragment length polymorphism (RFLP) assay is a cost-effective method for SNP genotyping and mutation detection, but the manual mining for restriction enzyme sites is challenging and cumbersome. Three years after we constructed SNP-RFLPing, a freely accessible database and analysis tool for restriction enzyme mining of SNPs, significant improvements over the 2006 version have been made and incorporated into the latest version, SNP-RFLPing 2.</p> <p>Results</p> <p>The primary aim of SNP-RFLPing 2 is to provide comprehensive PCR-RFLP information with multiple functionality about SNPs, such as SNP retrieval to multiple species, different polymorphism types (bi-allelic, tri-allelic, tetra-allelic or indels), gene-centric searching, HapMap tagSNPs, gene ontology-based searching, miRNAs, and SNP500Cancer. The RFLP restriction enzymes and the corresponding PCR primers for the natural and mutagenic types of each SNP are simultaneously analyzed. All the RFLP restriction enzyme prices are also provided to aid selection. Furthermore, the previously encountered updating problems for most SNP related databases are resolved by an on-line retrieval system.</p> <p>Conclusions</p> <p>The user interfaces for functional SNP analyses have been substantially improved and integrated. SNP-RFLPing 2 offers a new and user-friendly interface for RFLP genotyping that can be used in association studies and is freely available at <url>http://bio.kuas.edu.tw/snp-rflping2</url>.</p

Fine mapping and DNA fiber FISH analysis locates the tobamovirus resistance gene L3 of Capsicum chinense in a 400-kb region of R-like genes cluster embedded in highly repetitive sequences

The tobamovirus resistance gene L3 of Capsicum chinense was mapped using an intra-specific F2 population (2,016 individuals) of Capsicum annuum cultivars, into one of which had been introduced the C. chinenseL3 gene, and an inter-specific F2 population (3,391 individuals) between C. chinense and Capsicum frutescence. Analysis of a BAC library with an AFLP marker closely linked to L3-resistance revealed the presence of homologs of the tomato disease resistance gene I2. Partial or full-length coding sequences were cloned by degenerate PCR from 35 different pepper I2 homologs and 17 genetic markers were generated in the inter-specific combination. The L3 gene was mapped between I2 homolog marker IH1-04 and BAC-end marker 189D23M, and located within a region encompassing two different BAC contigs consisting of four and one clones, respectively. DNA fiber FISH analysis revealed that these two contigs are separated from each other by about 30 kb. DNA fiber FISH results and Southern blotting of the BAC clones suggested that the L3 locus-containing region is rich in highly repetitive sequences. Southern blot analysis indicated that the two BAC contigs contain more than ten copies of the I2 homologs. In contrast to the inter-specific F2 population, no recombinant progeny were identified to have a crossover point within two BAC contigs consisting of seven and two clones in the intra-specific F2 population. Moreover, distribution of the crossover points differed between the two populations, suggesting linkage disequilibrium in the region containing the L locus

Evaluation of novel molecular markers for monitoring drug resistence in Plasmodium falciparum malaria

Tese de mestrado, Microbiologia Clínica, Faculdade de Medicina, Universidade de Lisboa, 2010The human malaria parasite Plasmodium falciparum is acquiring resistance to most drugs it has encountered, including the recently deployed Artemisinin Combination Therapy, on which much hope has been laid. Molecular markers for monitoring the evolution of resistance are, therefore, urgently required. Our group has recently made use of a rodent malaria model to identify a number of novel genetic markers of antimalarial drug resistance, namely a clathrin mu adaptor gene (pfcmu) involved in artemisinin resistance and an amino acid transporter gene (pfaat1) underlying chloroquine resistance. The main aim of this thesis was to characterize and evaluate the contribution of the above genes to drug resistance in natural parasite populations of P. falciparum isolates from three endemic areas: Rwanda, Democratic Republic of Sao Tomé & Principe (DRSTP) and Brazil. The global diversity of pfcmu and pfaat1 was determined, resulting in the identification of several polymorphisms. The pfaat1 gene appears to be highly conserved and no correlations were found between this gene and the in vitro resistance to 4-aminoquinolines. In contrast to pfaat1, the pfcmu gene was genetically diverse, with nine Single Nucleotide Polymorphisms and three different insertions identified across all isolates inspected. Samples could be grouped in to fourteen different pfcmu haplotypes, whose diversity was higher in both African sites than in Brazil (Hd = 0.964 ± 0.077, 0.750 ± 0.139 and 0.250 ± 0.180 in Rwanda, DRSTP and Brazil, respectively). Some of the identified polymorphisms showed geographical specificity. We found a significant association between a pfcmu G479A genotype in Rwanda samples and the in vitro sensitivity to dyhidroartemisinin (p = 0.0207). These constitute new findings to suggest that polymorphisms in pfcmu can be involved in P. falciparum defence mechanisms against artemisinin derivatives. Thus, further assessment of the gene in artemisinin responses is a top priority, in the context of effective surveillance of artemisinin resistance.A resistência do parasita Plasmodium falciparum aos fármacos é um dos principais obstáculos a uma contenção eficiente da malária. Os compostos utilizados para o combate a esta doença têm perdido sua eficácia ao longo dos anos, incluindo a artemisinina e os seus derivados, recentemente indicados como promissores no tratamento da malária. De facto, foram descritos recentemente os primeiros casos de resistência in vivo a estes compostos em parasitas de malária humana. Consequentemente, a identificação de marcadores moleculares de resistência a estes fármacos, previamente a um alastramento da resistência, apresenta-se como uma estratégia essencial. Recentemente, fazendo uso de um modelo de malária de roedores (Plasmodium chabaudi), o nosso grupo identificou um número de determinantes genéticos de resistência a diferentes antimaláricos, nomeadamente, o gene pfcmu, que codifica uma subunidade mu do complexo adaptador de clatrina e se relaciona com resistência aos derivados da artemisinina e o gene pfaat1, que codifica uma proteína transportadora de aminoácidos e está envolvido na resistência à cloroquina. O objectivo primordial deste trabalho centrou-se na caracterização dos genes acima descritos e na avaliação do seu possível papel em mecanismos de quimio-resistência em populações parasitárias provenientes de três regiões endémicas: Ruanda, República Democrática de São Tomé e Príncipe (RDSTP) e Brasil. Identificaram-se e caracterizaram-se os polimorfismos existentes em ambos os genes. O gene pfaat1 mostrou ser um gene altamente conservado e não revelou ter qualquer relação com a resistência às 4-aminoquinolinas. Pelo contrário, o gene pfcmu revelou possuir uma maior diversidade genética, tendo-se identificado nove mutações pontuais e três inserções, no conjunto de todos os isolados estudados. As amostras analisadas permitiram constituir catorze haplótipos, cuja diversidade demonstrou ser mais elevada nos países Africanos em comparação com o Brasil (Hd = 0,964 ± 0,077, 0,750 ± 0,139 e 0,250 ± 0,180 no Ruanda, RDSTP e Brasil, respectivamente). Alguns dos polimorfismos identificados revelaram especificidade geográfica. Identificou-se uma associação significativa entre uma mutação no gene pfcmu (G479A) e a susceptibilidade in vitro à dihidroartemisinina, em isolados provenientes do Ruanda (p = 0.0207). Estes resultados sugerem que polimorfismos no gene pfcmu podem estar envolvidos nos mecanismos de resistência do parasita P. falciparum aos derivados da artemisinina. Por conseguinte, estudos futuros envolvendo este gene e as respostas aos derivados da artemisinina, revestem-se de especial importância no contexto actual de uma vigilância eficaz da resistência a estes compostos

PIRA PCR designer for restriction analysis of single nucleotide polymorphisms

Primer-introduced restriction analysis (PIRA-PCR) is widely used to detect Single Nucleotide Polymorphisms (SNPs). To create artificial Restriction Fragment Length Polymorphism (RFLP), a mismatch is usually introduced near the end of the primer that is close to the mutation of interest. We describe in this report a www-based computer program that screens for the suitable mismatches, designs the primers, lists the appropriate restriction enzymes and other related information.Availability: The computer program, with related descriptions, is available at http://cedar.genetics.soton.ac.uk/public_html/primer2.htm