• BMB Reports
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• v.38 no.4
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• pp.491-499
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• 2005

DNA-based molecular markers for differentiation of five penaeid shrimps (Penaeus monodon, P. semisulcatus, Feneropenaeus merguiensis, Litopenaeus vannamei and Marsupenaeus japonicus) were developed based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-stranded conformation polymorphism (SSCP) of 16S ribosomal (r) DNA. Differentiation of P. monodon, P. semisulcatus and L. vannamei can be unambiguously carried out by PCR-RFLP of 16S $rDNA_{560}$ whereas P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16S $rDNA_{560}$. Nevertheless, the amplification success for L. vannamei and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16S $rDNA_{560}$ of an individual representing the most common mitotype of each species was cloned and sequenced. The new primer pair was designed and tested against the large sample sizes (312 bp product, N = 185). The amplification success was consistent across all species. PCR-RFLP of 16S $rDNA_{312}$ was as effective as that of 16S $rDNA_{560}$. Differentiation of all shrimp species were successfully carried out by SSCP analysis.

• Horticultural Science & Technology
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• v.29 no.1
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• pp.53-60
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• 2011

Cytoplasmic male sterility (CMS) induced by mutant mitochondria genome, has been used for commercial seed production of $F_1$ hybrid cultivars in diverse crops. In pepper (Capsicum annuum L.), two sterile cytoplasm specific gene organization, atp6-2 and coxII were identified. An open reading frame, orf456 nearby coxII gene has been speculated to induce male sterility (MS) by mutagenic analysis. Moreover, molecular markers for atp6-2 and coxII of mitochondrial genotype (mitotype) were developed. However, the Cytoplasmic MS specific markers, atp6SCAR and coxIISCAR markers appeared in both N and S cytoplasms when polymerase chain reaction (PCR) cycles prolonged more than 40 cycles. Since the reported molecular markers were dominant markers, the presence of the faint sterile-specific band in normal cytoplasm may lead to the mis-classification of pepper breeding lines. To solve this problem, one common forward primer and two different reverse primers specific to normal coxII and sterile orf456 genes were designed after analyzing their gene organizations. By using these three primers, N and S coxII specific bands were co-amplified in male-sterile lines, but only normal coxII specific band was amplified in maintainer lines. Since the reverse primer for sterile coxII was specifically designed 275 bp downstream of orf456, relatively stable PCR amplification patterns were observed regardless of the number of PCR cycles. These primer sets easily identified different mitotypes among the divergent breeding lines, commercial cultivars and diverse germplasms.