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Soil microbes are important integral components of soil ecosystem which have significant and diverse role in organic matter decomposition, nitrogen cycling, and nitrogen fixation. In this study an effective denaturing gradient gel electrophoresis (DGGE) method was employed for paddy soil microbial diversity survey. For optimum paddy soil microbial DNA extraction, different methods such as Lysis buffer, skim milk bead, sodium phosphate buffer, Epicentre Soil Master DNA extraction kit (Epicentre, USA) and Mo Bio Power Soil DNA kit (MO BIO, USA) methods were utilized. Among all the method, using Mo Bio Power Soil kit was most effective. DGGE analysis of Bacteria was carried out at 6% polyacylamide gel and 45-60% denaturing gradient in the optimal conditions. Whereas DGGE analysis of fungi was done at 6% polyacrylamide gel and 45-80% denaturing gradient in the optimal conditions. By applying the above assay, it was found that variation within the microbial community of paddy soil occurs by a factor of time. DGGE assay used in this study through for a variety of soil microbial analysis suggests the potential use of this method.

BACKGROUND: In order to develop effective assessment method for Korean paddy soil microbial community structure, reliable genomic DNA extraction method from paddy soil and quantitative real-time PCR (qRT-PCR) method are needed to establish METHODS AND RESULTS: Out of six conventional soil genomic DNA extraction methods, anion exchange resin purification method was turn to be the most reliable. Various PCR primers for distinguishing five bacterial phylum (α-Proteobacteria, β-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), all bacteria, and all fungi were tested. Various qRT-PCR temperature conditions were also tested by repeating experiment. Finally, both genomic DNA extraction and qRT-PCR methods for paddy soil were well established. CONCLUSION: Quantitative real-time PCR (qRT-PCR) method to assess paddy soil microbial community was established.

Waxy-grain sorghum is used in most of the commercial cereal products in Korea. Worldwide, three waxy mutant alleles have been identified in the sorghum germplasm, and DNA markers for these alleles have been developed to identify the waxy genotype. However, that detection method cannot be used to determine the proportion of waxy content in samples containing both waxy and non-waxy sorghum. This study developed an assay that can be used to detect and quantify the waxy content of mixed cereal samples. All Korean waxy-grain sorghum used in this study contained the wx a allele, and one wx a allele-containing individual was also heterozygous for the wx c allele. No individuals possessed the wx b allele. The genotyping results were confirmed by iodine staining and amylose content analysis. Based on the sequence of the wx a allele, three different types of primers (wx a allele-specific, non-waxy allele-specific, and nonspecific) were designed for a quantitative real-time PCR (qPCR) assay; the primers were evaluated for qPCR using the following criteria: analytical specificity, sensitivity and repeatability. Use of this qPCR assay to analyze mixed cereal products demonstrated that it could accurately detect the waxy content of samples containing both waxy and non-waxy sorghum. We developed a qPCR assay to identify and quantify the waxy content of mixed waxy and non-waxy sorghum samples as well as mixtures of cereals including sorghum, rice and barley. The qPCR assay was highly specific; the allele-specific primers did not amplify PCR products from non-target templates. It was also highly sensitive, detecting a tiny amount (>0.5%) of waxy sorghum in the mixed samples; and it was simple and repeatable, implying the robust use of the assay.