Background: Single nucleotide polymorphisms (SNPs) are widely used genetic markers with applications in human disease diagnostics, animal breeding, and evolutionary studies, but existing genotyping methods can be labor-intensive and costly. The aim of this study is to develop a simple and rapid method for identification of a single nucleotide change. Methods: A modified Polymerase Chain Reaction Amplification of Multiple Specific Alleles (PAMSA) and high resolution melt (HRM) analysis was performed to discriminate a bovine polymorphism in the NCAPG gene (rs109570900, 1326T > G). Results: The inclusion of tails in the primers enabled allele discrimination based on PCR product lengths, detected through agarose gel electrophoresis, successfully determining various genotypes, albeit with some time and labor intensity due to the use of relatively costly high-resolution agarose gels. Additionally, high-resolution melt (HRM) analysis with tailed primers effectively distinguished the GG genotype from the TT genotype in bovine muscle cell lines, offering a reliable way to distinguish SNP polymorphisms without the need for time-consuming AS-PCR. Conclusions: Our experiments demonstrated the importance of incorporating unique mismatched bases in the allele-specific primers to prevent cross-amplification by fragmented primers. This efficient and cost-effective method, as presented here, enables genotyping laboratories to analyze SNPs using standard real-time PCR.

Insulin-like growth factor 2 (Igf-2) and H19 genes are closely linked imprinted genes which have a pivotal role in embryogenesis and fetal development. Igf-2 and H19 are coexpressed in tissues of mesodermal, endodermal and neuroectodermal origin. Rat Igf-2 has a complex structure with three promoters and a complicate imprinting mechanism having an exception of biallelic expression in the choroid plexus, leptomeninges, and fetal tissues of neuroectodermal origin. To detect the expression of maternal and paternal alleles of Igf-2 and H19 during orofacial development, fetal and neonatal hybrid rats, obtained from Wistar and Fisher interstrain rat crosses were used. We also detected the promoter-specificity of Igf-2 transcripts by primers selected from P1, P2, and P3 of Igf-2 gene. RT-PCR analysis of Igf-2 and H19 showed the monoallelic expression of Igf-2 from the paternal allele and H19 from the maternal allele in E15.5 to E19.5 orofaciall structures including the maxilla, tongue, and salivary gland. P3 promoters were active in all tested samples, whereas transcripts derived from P2 promoter arised with approximately half of the tested cases and showed variable alternation. P1 promoter was not transcribed in all tested samples. These results suggest that Igf-2 and H19 may be involved in orofacial development and exhibit parent-of-origin monoallelic expression. On the other hand, in orofaciall development, P2 and P3 promoters except for P1 promoter are transcribed with variable alternative transcripts

사과는 배우체형 자가불화합성을 나타내는데 이는 S-locus의 복대립유전자에 의해 조절된다. 본 연구는 S-allele specific PCR분석을 통해 신품종을 포함한 24종의 사과 주요 재배품종과 7종의 꽃사과 품종의 자가불화합성 유전자형(S-genotype)을 결정하고자 수행하였다. 31종의 재배품종과 꽃사과 품종 을 23종의 S-allele specific primer을 이용하여 분석한 결과 12개의 S-allele (S1, S2, S3, S5, S7, S9, S10, S16, S21, S23, S26, S29)이 동정되었다. 그 중에서 24종의 재배품종에는 S1(41.7%), S3(58.3%), S7(29.2%), S9(54.2%)의 S-allele이 흔히 존재하는 것으로 확인되었다. 국내육성 신품종인 ‘아리수’와 ‘황옥’의 S-genotype은 각각 S3S7과 S3S9으로 동정되었다. 본 실험에서 얻은 S-genotype 정보는 안정적인 사과 과실생산에 적합한 수분수 선발과 육종프로그램에서 교배조합 작성에 유용 하게 활용될 것이다.

The use of functional markers, it is expected to make direct identification about genetic diversity at DNA level and overcome the problem of recombination /linkage. These markers can be used to identify interesting alleles in a breeding program and indirectly select for the trait, saving money, time and labor. Bacterial blight of rice caused by Xanthomonas oryzaepv. Oryzae is a destructive disease in rice production worldwide. No bactericide is effective to control the bacterial blight disease yet. Xa3, which is a gene conferring resistance to BB of the rice plant has been previously characterized by map-based cloning. We have cloned and sequenced the Xa3/xa3 gene in Korean cultivar, Hwayoung, Ilmi and Goun with gene specific primers. Our work detected polymorphisms and PCR-based allele specific SNP markers were developed. Susceptible or resistant individuals from an F2 population developed from across between Milyang244 and Ilmi, Korean germplasms and near isogenic lines carrying BB resistance genes were screened with allele specific markers. We found that the genotype completely matched their phenotype to BB using ASP-primers. These markers could be effective to marker-assisted selection for the Xa3 gene in rice breeding programs.