Rice Genomic DNA Research Articles

A Simplified, Efficient and Rapid DNA Extraction Protocol from Rice Grains and Leaf

DNA extraction is very complicated with different plant species because the presence of secondary metabolites that hinder with DNA isolation processes and application like DNA restriction, Gene amplification, as well as gene cloning. A simple method for preparation of rice genomic DNA was developed. The current study expressed comparatively rapid, cost-effective, less time consuming, methods for DNA extraction from seed, young leaves and old leaves of rice genotypes without using liquid nitrogen, phenol, and β–mercaptoethanol in extraction buffer. Using this method, high quality and quantity of genomic DNA was get from 0.5 g of rice seed, young leaves and 75 days old leavesand extracted total nucleic acids (genomic DNA) were amplified by employing single sequence repeats (SSR) or microsatellite markers, which produced reproducible results.This protocol resolves the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides.

A paired-end whole-genome sequencing approach enables comprehensive characterization of transgene integration in rice

Efficient, accurate molecular characterization of genetically modified (GM) organisms is challenging, especially for those transgenic events transferred with genes/elements of recipient species. Herein, we decipher the comprehensive molecular characterization of one novel GM rice event G281 which was transferred with native promoters and an RNA interference (RNAi) expression cassette using paired-end whole genome sequencing (PE-WGS) and modified TranSeq approach. Our results show that transgenes integrate at rice chromosome 3 locus 16,439,674 included a 36 bp deletion of rice genomic DNA, and the whole integration contains two copies of the complete transfer DNA (T-DNA) in a head-to-head arrangement. No unintended insertion or backbone sequence of the transformed plasmid is observed at the whole genome level. Molecular characterization of the G281 event will assist risk assessment and application for a commercial license. In addition, we speculate that our approach could be further used for identifying the transgene integration of cisgenesis/intragenesis crops since both ends of T-DNA in G281 rice were from native gene or elements which is similar with that of cisgenesis/intrasgenesis. Our results from the in silico mimicking cisgenesis event confirm that the mimic rice Gt1 gene insertion and its flanking sequences are successfully identified, demonstrating the applicability of PE-WGS for molecular characterization of cisgenesis/intragenesis crops.

Characterization of Selected Zambian Rice (Oryza sativa) Accessions using Simple Sequence Repeat (SSR) Markers

Background: Rice (Oryza sativa L.) is grown in various parts of Zambia. Response of available varieties towards biotic (bacterial and fungal pathogens) and abiotic factors such as water stress remains unavailable. A systematic rice seed system or breeding program appears to be non-existent. This study was undertaken to assess the genetic diversity among some selected Zambian rice accessions using simple sequence repeat (SSR) molecular markers. Methods: Thirty accessions were selected for genetic diversity analysis using ten SSR markers (RM5, RM168, RM7, RM13, RM225, RM452, RM211, RM205, RM413 and RM46) selected from various chromosomal loci of the rice genome. Rice genomic DNA was amplified by the polymerase chain reaction and products were analyzed by 1% agarose gel electrophoresis. Most products appeared as single alleles in form of single bands averaging between 79-200 base pairs (bp). Significant relatedness within and among accessions was observed while most of the primers showed distinct polymorphism. Cluster analysis via a dendrogram revealed that most of the accessions were closely related and clustered into two major groups designated A and B. Result: Only one accession (ZM8321) appeared to be distinct and distantly related to all the other 29 accessions Cluster B contained the rest of the 29 accessions which were subdivided into 8 sub-clusters. Of the 29, accessions ZM8295 from Samfya District and ZM8313 from Kaputa district showed 100% similarity meaning that they are the same accession.

Development and assessment of a duplex droplet digital PCR method for quantification of GM rice Kemingdao.

The implementation of genetically modified organism (GMO) labeling policies requires accurate quantitative methods to measure the GMO content in test samples. A Kemingdao/phospholipase D (KMD/PLD) duplex ddPCR method was established with rice genomic DNA (gDNA) of homozygous KMD as template by optimizing the annealing temperature and cycle number. Duplex ddPCR showed a linear response over the dynamic range from 68 to 175,000 copies, covering four orders of magnitude. The limit of detection (LOD) and limit of quantification (LOQ) for duplex ddPCR were determined to be 9 copies and 34 copies of the rice haploid genome, respectively. A very high dilution factor would result in unacceptable bias and coefficients of variation for determining copy number of the gDNA solution, and more than 1000 copies of the DNA template in one reaction is preferred to obtain accurate quantitative results by duplex PCR. Five blinded DNA samples with copy number ratio of 10%, 5%, 1%, 0.1%, and 0.05%, and three blinded real-life matrix samples with mass fraction of 5%, 1%, and 0.5% were quantified by duplex ddPCR, simplex ddPCR, and qPCR. These three methods all gave comparable GMO content and copy numbers within the required precision, but the duplex ddPCR showed the narrowest uncertainty interval and provided the highest precision in comparison to simplex ddPCR and qPCR. The ddPCR is a more appealing and reliable technology for the accurate quantification of GMO content than simplex ddPCR and qPCR considering the uncertainty and precision of quantitative results, the time consumption of generating droplets, and the cost of ddPCR reagents.

Association between microsatellite markers and rice response to the rice blast pathogen Magnaporthe oryzae in selected Zambian rice genotypes

Magnaporthe oryzae, the rice blast disease pathogen is estimated to cause losses of up to 30% in rice production worldwide, yet its impact on rice production in Zambia remains unknown. Symptomatic rice leaf tissue collected from rice fields in Mongu District of Zambia was cultured on oatmeal agar medium and fungal colonies were purified and identified through leaf lesions and conidia using published keys. An M. oryzae isolate from this study was used to challenge twenty-eight selected Zambian rice genotypes to determine their responses to infection with the pathogen estimated by lesion numbers and sizes. Twelve of the genotypes (42.8%) were highly resistant (HR) with 1 to 2 lesions per cm2, 6 (21.4%) were moderately resistant with mean lesion numbers of 3 to 4 per cm2 while 10 (35.7%) were susceptible with lesion numbers above 5 per cm2 of leaf tissue. Polymerase chain reaction (PCR)-amplification of rice genomic DNA extracted from the 28 genotypes using selected microsatellite marker primers revealed a moderate genetic diversity with three of them showing significant genetic diversity. Allelic diversity among the selected genotypes was also confirmed from a calculated mean polymorphic information content of 0.8. A correlation between 8 of the microsatellite markers in some of the rice genotypes and their resistance response to M. oryzae was also established. Results show that some of the analysed rice genotypes have a significant potential for use as a genetic resource in breeding for varieties that are resistant to the rice blast pathogen for Zambian farmers.

A simple and accurate PCR method for detection of genetically modified rice

BackgroundLegislation regulating for labeling and use of genetically modified (GM) crops are increased considerably worldwide in order to health and safety assurance of consumers. For this purpose, a polymerase chain reaction (PCR) method has been developed for detection of GM rice in people’s food diet.MethodsIn this study, eighty-one non-labeled rice samples were collected randomly from different market sites of Tehran, Iran. In order to analysis, rice genomic DNA was extracted using MBST DNA extraction kit and subsequently, sucrose phosphate synthase (SPS) gene was used to confirm the quality of extracted DNA. Then, cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium nopaline synthase (NOS) terminator were selected as screening targets for detection of GM rice sequences by PCR.ResultsAccording to our results, 2 out of 81 (2.4%) samples tested were positive for CaMV 35S promoter while no positive result was detected for NOS terminator.ConclusionThe obtained data indicated that this method is capable to identify the GM rice varieties. Furthermore, it can demonstrate the possibility of the presence of GM rice in Tehran’s market, thus putting emphasis on the requirement for developing a precise approach to evaluate this product.

Detection of honey adulteration by conventional and real-time PCR

This work applies both conventional and real-time PCR DNA amplification techniques for detecting and quantifying rice molasses in honey. Different levels of adulteration were simulated (1, 2, 5, 10, 20, 50%) using commercial rice molasses. Among the different specific genes of rice tested by PCR, the PLD1 primer was the most effective. This allowed the visualization in agarose gel of this type of adulterant up to 5–20%. Moreover, by means of real-time PCR it was possible to distinguish the different levels of rice DNA, and therefore the percentage of adulteration (1–50%). A standard curve built with the DNA serial dilutions of rice genomic DNA concentrations showed that the quantification level was between 2 and 5%. These results offer compelling evidence that DNA techniques could be useful not only for the detection of adulterations of honey with rice molasses but also for the quantification of levels lower than those of conventional techniques.

Use of a novel metal indicator to judge loop-mediated isothermal amplification for detecting the 35S promoter.

Loop-mediated isothermal amplification (LAMP) is a widely used isothermal nucleic acid amplification method. Here we developed a new closed-tube colorimetric method for judging LAMP with a novel metal indicator. First, the metal indicator, acid chrome blue K (ACBK), was evaluated in the LAMP reaction with various combinations of reaction reagents, such as reaction buffer, dNTP mixtures, primer mixtures, or Mg2+. We found that the solution color of the LAMP reaction with ACBK changed from red to blue based on a decrease in the Mg2+ concentration in the reaction solution. We then optimized the LAMP with ACBK method for detecting the Cauliflower Mosaic Virus 35S promoter. Further, the specificity of the new colorimetric assay using ACBK in the LAMP reaction for detecting the 35S promoter was tested with diverse transgenic events in different crops, and the sensitivity threshold of the assay was ∼50 copies for transgenic rice genomic DNA and 100ng of 0.1% DNA from rice, soybean, rapeseed, and maize. Finally, the applicability of the LAMP assay was successfully validated using practical maize samples. All the detection results could be easily discerned either by UV-vis spectroscopy or the naked eye. Graphical Abstract The visual detect LAMP amplification by the addition of ACBK as a signal indicator. The color of the LAMP-ACBK solution turned from red to blue as the concentration of free Mg2+ decreases. The detection results could be easily discerned either by UV-vis spectroscopy or the naked eye.

Ensifer-mediated transformation: an efficient non-Agrobacterium protocol for the genetic modification of rice.

While Agrobacterium-mediated transformation (AMT) remains the most widely used technique for gene transfer in plants, interest exists for the use of non-Agrobacterium gene delivery systems due to freedom-to-operate issues that remain with AMT across several jurisdictions. In addition, the plant pathogenic mode of action of Agrobacterium tumefaciens significantly increases the costs to passage engineered cultivars through the regulatory process. Ensifer adhaerens (OV14) is a soil-related bacterium with the proven ability to genetically modify the model plant A. thaliana and the staple crop S. tuberosum (Wendt et al., Trans Res 21:567–578, 2012). While previous work was relevant for dicotyledonous species, in this study, the efficacy of Ensifer adhaerens (OV14)-mediated transformation (EMT) was determined on two japonica rice varieties, Curinga and Nipponbare, and the recalcitrant indica variety, IR64. The results indicated that E. adhaerens (OV14) exhibits infection efficiencies ranging between 50–70 %, 90–100 % and 90–95 % for Curinga, Nipponbare and IR64 respectively. Curinga and Nipponbare plants transformed with E. adhaerens (OV14) and A. tumefaciens (LBA4404 and EHA105) were regenerated achieving transformation efficiencies of 16 % and 26–32 % for Curinga and 7 and 4 % for Nipponbare respectively. Separately, the transformation of IR64 was only recorded via EMT (transformation efficiency ~1 %). Integration analyses conducted on 24 transgenic rice lines illustrated that T-DNA insertion occurred randomly throughout the rice genome for EMT (and AMT), with similar integration patterns in the rice genomic DNA observed for both bacterial species.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1369-9) contains supplementary material, which is available to authorized users.

A simplified genomic DNA extraction protocol for pre-germination genotyping in rice.

Genotyping is a critical step for molecular marker-assisted selection in rice. Rice genomic DNA samples for genotyping are typically isolated from living tissues such as seedlings. This requires the germination of all candidate seeds and extraction of DNA from the seedlings. Currently, an ideal individual is selected from a very large number of plants, which is time- and labor-consuming, requiring several transplantations of materials and sampling processes. In this study, we developed a simplified genomic DNA extraction protocol in rice by using amylase to treat half-seeds. The yields of genomic DNA from a half-seed of Indica and Japonica rice were greater than 203.8 ± 32.5 and 143.2 ± 25.5 ng, respectively, and the 260/280 nm absorbance ratio was 1.75-2.10. The DNA was confirmed to be sufficient for polymerase chain reaction amplification and can be used in a marker-assisted selection program.

Development of disease-resistant rice by optimized expression of WRKY45.

The rice transcription factor WRKY45 plays a central role in the salicylic acid signalling pathway and mediates chemical-induced resistance to multiple pathogens, including Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. Previously, we reported that rice transformants overexpressing WRKY45 driven by the maize ubiquitin promoter were strongly resistant to both pathogens; however, their growth and yield were negatively affected because of the trade-off between the two conflicting traits. Also, some unknown environmental factor(s) exacerbated this problem. Here, we report the development of transgenic rice lines resistant to both pathogens and with agronomic traits almost comparable to those of wild-type rice. This was achieved by optimizing the promoter driving WRKY45 expression. We isolated 16 constitutive promoters from rice genomic DNA and tested their ability to drive WRKY45 expression. Comparisons among different transformant lines showed that, overall, the strength of WRKY45 expression was positively correlated with disease resistance and negatively correlated with agronomic traits. We conducted field trials to evaluate the growth of transgenic and control lines. The agronomic traits of two lines expressing WRKY45 driven by the OsUbi7 promoter (PO sUbi7 lines) were nearly comparable to those of untransformed rice, and both lines were pathogen resistant. Interestingly, excessive WRKY45 expression rendered rice plants sensitive to low temperature and salinity, and stress sensitivity was correlated with the induction of defence genes by these stresses. These negative effects were barely observed in the PO sUbi7 lines. Moreover, their patterns of defence gene expression were similar to those in plants primed by chemical defence inducers.

Flexible and scalable genotyping-by-sequencing strategies for population studies.

BackgroundMany areas critical to agricultural production and research, such as the breeding and trait mapping in plants and livestock, require robust and scalable genotyping platforms. Genotyping-by-sequencing (GBS) is a one such method highly suited to non-human organisms. In the GBS protocol, genomic DNA is fractionated via restriction digest, then reduced representation is achieved through size selection. Since many restriction sites are conserved across a species, the sequenced portion of the genome is highly consistent within a population. This makes the GBS protocol highly suited for experiments that require surveying large numbers of markers within a population, such as those involving genetic mapping, breeding, and population genomics. We have modified the GBS technology in a number of ways. Custom, enzyme specific adaptors have been replaced with standard Illumina adaptors compatible with blunt-end restriction enzymes. Multiplexing is achieved through a dual barcoding system, and bead-based library preparation protocols allows for in-solution size selection and eliminates the need for columns and gels.ResultsA panel of eight restriction enzymes was selected for testing on B73 maize and Nipponbare rice genomic DNA. Quality of the data was demonstrated by identifying that the vast majority of reads from each enzyme aligned to restriction sites predicted in silico. The link between enzyme parameters and experimental outcome was demonstrated by showing that the sequenced portion of the genome was adaptable by selecting enzymes based on motif length, complexity, and methylation sensitivity. The utility of the new GBS protocol was demonstrated by correctly mapping several in a maize F2 population resulting from a B73 × Country Gentleman test cross.ConclusionsThis technology is readily adaptable to different genomes, highly amenable to multiplexing and compatible with over forty commercially available restriction enzymes. These advancements represent a major improvement in genotyping technology by providing a highly flexible and scalable GBS that is readily implemented for studies on genome-wide variation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-979) contains supplementary material, which is available to authorized users.

Genomic regions and underlying candidate genes associated with coleoptile length under deep sowing conditions in a wheat RIL population

Longer coleoptile is a desirable trait in wheat for sowing under drought stress environments with moisture availability in the deeper layers of the soil. In the present study, WL711/C306 wheat RIL population and the parents were phenotyped for coleoptile length under deep sowing conditions in the field and plant growth chamber. C306, a tall traditional cultivar had longer coleoptile compared to the semi dwarf parent WL711 carrying Rht-B1b gene. The RIL population showed considerable variation, normal distribution and transgressive segregation for coleoptile length under field and controlled environment conditions. The genetic linkage map of WL711/C306 RIL population was constructed comprising of 346 markers. The total map distance was 4,526.8 cM with an averaged interval of 12.9 cM between the adjacent markers. Major novel consistent QTLs for coleoptile length were identified on 4BS, co-located with the Rht-B1b gene and on 3BS. Also major QTL was identified on chromosome 3BL for coleoptile length explaining up to 12.4 % of phenotypic variation. On comparison with the rice (Oryza sativa L.) genomic DNA sequences syntenic to wheat chromosome 4BS and 3BS, two candidate genes were identified namely gibberellin C-20 oxidase 1 and α-Expansin. These genes play a role in cell wall expansion in the young expanding tissue in the coleoptile. QTLs qCL.4B.1 and qCL.3B.1 emerge to be important for coleoptile length in the WL711/C306 RIL population suggesting their potential value for use in marker assisted selection after validation for longer coleoptile length and improved establishment.

The detection of T-Nos, a genetic element present in GMOs, by cross-priming isothermal amplification with real-time fluorescence.

An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06 × 10(3) copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO.

Genetic Dissection of Yield and Its Component Traits Using High-Density Composite Map of Wheat Chromosome 3A: Bridging Gaps between QTLs and Underlying Genes

Earlier we identified wheat (Triticum aestivum L.) chromosome 3A as a major determinant of grain yield and its component traits. In the present study, a high-density genetic linkage map of 81 chromosome 3A-specific markers was developed to increase the precision of previously identified yield component QTLs, and to map QTLs for biomass-related traits. Many of the previously identified QTLs for yield and its component traits were confirmed and were localized to narrower intervals. Four novel QTLs one each for shoot biomass (Xcfa2262-Xbcd366), total biomass (wPt2740-Xcfa2076), kernels/spike (KPS) (Xwmc664-Xbarc67), and Pseudocercosporella induced lodging (PsIL) were also detected. The major QTLs identified for grain yield (GY), KPS, grain volume weight (GVWT) and spikes per square meter (SPSM) respectively explained 23.2%, 24.2%, 20.5% and 20.2% of the phenotypic variation. Comparison of the genetic map with the integrated physical map allowed estimation of recombination frequency in the regions of interest and suggested that QTLs for grain yield detected in the marker intervals Xcdo549-Xbarc310 and Xpsp3047-Xbarc356 reside in the high-recombination regions, thus should be amenable to map-based cloning. On the other hand, QTLs for KPS and SPSM flanked by markers Xwmc664 and Xwmc489 mapped in the low-recombination region thus are not suitable for map-based cloning. Comparisons with the rice (Oryza sativa L.) genomic DNA sequence identified 11 candidate genes (CGs) for yield and yield related QTLs of which chromosomal location of two (CKX2 and GID2-like) was confirmed using wheat aneuploids. This study provides necessary information to perform high-resolution mapping for map-based cloning and for CG-based cloning of yield QTLs.

Development of a SCAR Marker for Discrimination of a Thai Jasmine Rice (<i>Oryza sativa</i> L. cv. KDML105) Mutant, BKOS6, and Associated with Purple Color Trait in Thai Jasmine Rice-Related Varieties

The potential of SCAR marker for discrimination of a Thai jasmine rice (Oryza sativa L. cv. KDML 105) mutant, BKOS6, obtained from ion-beam-induced mutation, was evaluated. The improved rice variety, BKOS6, exhibited many remarkable characteristics which fitted the multiple cropping system characteristics of progressive agriculture including photoperiod insensitivity, early flowering, short in stature, and purple pigment accumulation in pericarp. The BKOS6 rice grain extract has already been proved that it exhibited higher antioxidant properties than the KDML 105 and other tested rice grain extracts. In this study, the BKOS6 specific SCAR marker was developed by HAT-RAPD analysis of rice genomic DNA. The marker was successfully used to identify BKOS6 variety and its hybrid varieties containing purple pigment accumulation in plant tissues. Moreover, it was found that this marker could be used to detect other purple pigmented rice varieties that genetically related to Thai jasmine rice. Recently, a wide variety of anthocyanin-based foods are believed to provide significant potential health benefits, and become more attractive. KDML 105 is also a Thai premier fragrant rice variety which is one of the main varieties of country’s rice export. Thus this molecular marker could be useful for commercial and breeding purposes of BKOS6 mutant and other developed varieties from KDML 105 which contain anthocyanin accumulation.

Quantitative detection of the rice false smut pathogen Ustilaginoidea virens by real-time PCR

Rice false smut (RFS) is an important rice disease that is caused by the pathogen Ustilaginoidea virens. In this study, we developed a real-time polymerase chain reaction (PCR) assay to detect U. virens and to estimate the level of disease. The genomic DNAs of U. virens and rice were extracted together from the rice samples. Real-time PCR assays were performed and compared to conventional nested-PCR assays. The real-time PCR assay presented a consistent linearity of the standard curve (R(2) = 0.9999). The detection limit could be as low as 40 fg U. virens DNA with a rice genomic DNA background on using the real-time PCR assay, which showed significantly higher sensitivity than the conventional nested-PCR assay. We conclude that the real-time PCR quantitative assay is a useful tool for detecting U. virens and for early defense and control of RFS.

A transgenic system for generation of transposon Ac/Ds-induced chromosome rearrangements in rice

The maize Activator (Ac)/Dissociation (Ds) transposable element system has been used in a variety of plants for insertional mutagenesis. Ac/Ds elements can also generate genome rearrangements via alternative transposition reactions which involve the termini of closely linked transposons. Here, we introduced a transgene containing reverse-oriented Ac/Ds termini together with an Ac transposase gene into rice (Oryza sativa ssp. japonica cv. Nipponbare). Among the transgenic progeny, we identified and characterized 25 independent genome rearrangements at three different chromosomal loci. The rearrangements include chromosomal deletions and inversions and one translocation. Most of the deletions occurred within the T-DNA region, but two cases showed the loss of 72 kilobase pairs (kb) and 79 kb of rice genomic DNA flanking the transgene. In addition to deletions, we obtained chromosomal inversions ranging in size from less than 10 kb (within the transgene DNA) to over 1 million base pairs (Mb). For 11 inversions, we cloned and sequenced both inversion breakpoints; in all 11 cases, the inversion junctions contained the typical 8 base pairs (bp) Ac/Ds target site duplications, confirming their origin as transposition products. Together, our results indicate that alternative Ac/Ds transposition can be an efficient tool for functional genomics and chromosomal manipulation in rice.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-012-1925-4) contains supplementary material, which is available to authorized users.

Comparative genomic in situ hybridization analysis on the chromosomes of five grass species with rice genomic DNA probe

Comparative genomic in situ hybridization (cGISH) with biotin-labeled rice genomic DNA to the chromosomes of Zea mays, Hordeum vulgare, Sorghum bicolor, Setaria italic and Secale cereale were conducted to analyze genomic homology between rice and other grass (Gramineae) speices. At 75% stringency, the rice DNA probe generated large numbers of signals dispersedly distributed over all chromosomes in all target species. The nucleolar organizing regions (NORs), a few telomeres, most centromeric regions and numerous interstitial sites were detected. The signals in small genomes were relatively sparse and unevenly distributed along chromosomes, whereas those in large genomes were dense and basically evenly distributed. Chromosomal in situ suppression (CISS) experiments indicated that the signals represented the hybridization of repetitive DNAs is common between rice and the target genome. cGISH conducted at low and high stringency revealed that repetitive DNAs with ～65% homology are rich and those with ～90% homology are rare between rice and maize or barley. cGISH using maize genomic DNA probe to the rice chromosomes generated relatively sparse and unevenly distributed signals. Our results reveal that there exist besides rDNA and telomeric repeats, some other conserved repetitive DNAs among grass species of different subfamilies. Our results also suggest that ancient conserved repetitive DNAs had undergone considerable amplification in large grass genomes during evolution. Key words: Comparative genomic in situ hybridization (cGISH), genomic homology, chromosome, grass genome, repetitive DNA.

A Simple Method for Preparation of Rice Genomic DNA

The extraction of DNA is often the most time consuming and laborious step in high-throughput molecular genetic analysis and marker assisted selection (MAS) programs. A simple method for preparation of rice genomic DNA was developed. A small amount (1-50 mg) of leaf tissue of rice seedling, 500 μL of extraction buffer, and one steel bead were put into a 2-mL microcentrifuge tube. After vigorously mashing for 2 min, 5 μL of supernatant was directly applied to PCR amplification. Otherwise, the supernatant was precipitated with two times volume of ethanol to obtain high quality genomic DNA. This method is simple, rapid, low cost, and reliable for PCR analysis. One person can manipulate as many as 96 samples for PCR in 10 min. It is especially suitable for genotyping of large number of samples.