Successful amplification of rice chloroplast microsatellites from century-old grass samples from the park grass experiment

We report the successful amplification of microsatellite markers for the chloroplast genome from century-old samples of 2 grasses growing in the Park Grass Experiment (PGE):Anthoxanthum ordoratum andFestuca rubra. This opens the possibility of establishing a long-term genetic time series for the PGE, which began in 1856 and is believed to be the oldest ecological experiment in existence. Although the plant samples used were not originally prepared or stored with molecular analysis in mind, the hexadecyltrimethylammonium bromide (CTAB) method of DNA extraction was successfully used. Obtained DNA was degraded but could be amplified by means of PCR. It produced bands around the expected size for chloroplast microsatellite primers derived from rice. When sequenced, bands showed good homology with sequences from rice chloroplast genomes listed in GenBank (accession No X15901).     

Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations(termed as the 1st,2nd,3rd and 4th DNA sample, respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.     

Conversion of an RAPD marker to an STS marker for barley variety identification

Barley (Hordeum vulgare L.) variety identification is important to the malting and brewing industries. Because many new malting cultivars (varieties) are closely related, new and more effective identification techniques are needed. We report on a series of techniques used to convert an RAPD marker to a more stable STS marker that can identify barley Stander from Robust, an important distinction for the American malting and brewing industries. The techniques included DNA extraction, RAPD amplification, random cloning of all amplified fragments, selection of clones by insert size, DNA sequencing of select inserts, design of a barley-based primer pair, and detection of a single nucleotide polymorphism using restriction endonucleaseAlu I. The barley-based primer pair was used to further sequence the RAPD fragment. Five single nucleotide polymorphisms between Robust and Stander exist, one of which was detected by electrophoresing DNA fragments differentially restricted byAlu I. The conversion technique was different from ones previously reported in that it did not require manual extraction of DNA fragments from a gel. This could be applied to other situations in which RAPD marker conversion would be desirable.     

A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.     

Construction of a BAC library of pearl millet, Pennisetum glaucum

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from pearl millet (Pennisetum glaucum), and used as a resource for the isolation of microsatellite sequences. The library contains a total of 159,100 clones with an average insert size of 90 kb, and corresponds to 5.8 haploid genome equivalents. The BAC library was pooled for screening by the polymerase chain reaction (PCR) as well as robotically gridded on high-density filters. PCR-based screening of a subset of the library (4.7 haploid genome equivalents) using five sequence-tagged site (STS) and six microsatellite markers identified between 2 and 11 positives superpools (5.4 on average). The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be less than 1% by hybridisation with a rice chloroplast probe. Received: 30 January 2000 / Accepted: 16 October 2000     

PSIIP680 ChlaAP基因在水稻叶绿体基因组中的定位

本实验总结出一套水稻叶绿体DNA的提取方法,并获得清晰的叶绿体DNA限制性内切酶图谱。Southern杂交结果表明,菠菜PSIIP680ChlaAP基因探针与水稻叶绿体DNA的Pst-1,Pst-14,Pvu-2和Sal-1片段的部分顺序有较高的同源性。根据Hirai和赵衍的水稻叶绿体基因组物理图,可以确定该基因位于紧靠RuBPCaseLS基因,距反向重复区约26kb处。高等植物叶绿体基因组中这种基因排列方式还未见报道。     

Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice

Summary The entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare) was identified using clone banks that cover the chloroplast and mitochondrial genomes. The mitochondrial fragments that were homologous to chloroplast DNA were mapped and sequenced. The nucleotide sequences around the termini of integrated chloroplast sequences in the rice mtDNA revealed no common sequences or structures that might enhance the transfer of DNA. Sixteen chloroplast sequences, ranging from 32 bases to 6.8 kb in length, were found to be dispersed throughout the rice mitochondrial genome. The total length of these sequences is equal to approximately 6% (22 kb) of the rice mitochondrial genome and to 19% of the chloroplast genome. The transfer of segments of chloroplast DNA seems to have occurred at different times, both before and after the divergence of rice and maize. The mitochondrial genome appears to have been rearranged after the transfer of chloroplast sequences as a result of recombination at these sequences. The rice mitochondrial DNA contains nine intact tRNA genes and three tRNA pseudogenes derived from the chloroplast genome.     

One-step isolation of plant DNA suitable for PCR amplification

We report a one-step extraction technique for the isolation of plant DNA, DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm² in less than 20 min, with no tube changes. The method was tested on several plant specA00AK020ies. The described method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated by the novel method to PCR products generated using standard DNA isolation techniques.     

杂草稻叶绿体籼粳分化的多重PCR分析

通过分析籼稻93-11和粳稻培矮64S的叶绿体全基因组,优化和构建了籼粳分化的叶绿体分子标记ORF100和ORF29-TrnC^GCA的多重PCR。应用这个多重PCR对200余份世界各地杂草稻和其它水稻材料进行分析。结果表明:杂草稻中有明显的叶绿体籼粳分化,表现出明显的地域性,且与传统的中国栽培稻的南籼北粳能较好的对应。推测粳型杂草稻可能是栽培稻突变或粳型水稻(作母本)与其它类型水稻材料杂交而形成的。     

The Mutant Form of Acetolactate Synthase Genomic DNA from Rice is an Efficient Selectable Marker for Genetic Transformation

The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.     

Marker assisted selection of bacterial blight resistance genes in rice

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases affecting rice production in Asia. We were interested in surveying rice genotypes that are popularly used in the Indian breeding program for conferring resistance to bacterial blight, using 11 STMS and 6 STS markers. The basis of selection of these DNA markers was their close linkage to xa5, xa13, and Xa21 genes and their positions on the rice genetic map relative to bacterial blight resistance genes. Eight lines were found to contain the xa5 gene while two lines contained Xa21 gene and none of the lines contained the xa13 gene with the exception of its near-isogenic line. Using the polymorphic markers obtained in the initial survey, marker-assisted selection was performed in the F3 population of a cross between IR-64 and IET-14444 to detect lines containing multiple resistance genes. Of the 59 progeny lines analyzed, eight lines contained both the resistance genes, xa5 and Xa4.     

Grass consensus STS markers: an efficient approach for detecting polymorphism in Lolium

For ryegrass and many forage crops, characterization of varieties is often difficult for two reasons: few of discriminant morphological traits and a great within-varieties variation. Futhermore, few molecular markers are publicly accessible. In this paper we describe two approaches for the development of 42 sequence-tagged-site (STS) markers. Firstly, 14 STS markers were developed from Lolium sequences found in data bases. Secondly, 28 STS markers were developed from sequences found in related species of Gramineae. Out of 42 STS markers developed, 85.8% yielded successfull amplification and 62% revealed a high level of polymorphism with an average of five alleles per locus. The analysis of amplicons reveals a high STS marker specificity, a high conservation in gene structure and a strong intron sequence homology between allelic forms. Moreover, the majority of the STS markers can be considered as "universal markers" because 81% of these STS markers amplified successfully across 20 related grass species. These results permit us to consider the use of these markers in synteny studies.Communicated by G. Wenzel     

Single-seeded InDel fingerprints in rice:An effective tool for indica-japonica rice classification and evolutionary studies

Asian cultivated rice(Oryza sativa L.),an important cereal crop worldwide,was domesticated from its wild ancestor 8000 years ago.During its long-term cultivation and evolution under diverse agroecological conditions, Asian cultivated rice has differentiated into indica and japonica subspecies.An effective method is required to identify rice germplasm for its indica and japonica features,which is essential in rice genetic improvements.We developed a protocol that combined DNA extraction from a single rice seed and the insertion/deletion(InDel) molecular fingerprint to determine the indica and japonica features of rice germplasm.We analyzed a set of rice germplasm,including 166 Asian rice varieties,two African rice varieties,30 accessions of wild rice species,and 42 weedy rice accessions,using the single-seeded InDel fingerprints(SSIF).The results show that the SSIF method can efficiently determine the indica and japonica features of the rice germplasm.Further analyses revealed significant indica and japonica differentiation in most Asian rice varieties and weedy rice accessions.In contrast,African rice varieties and nearly all the wild rice accessions did not exhibit such differentiation.The pattern of cultivated and wild rice samples illustrated by the SSIF supports our previous hypothesis that indica and japonica differentiation occurred after rice domestication under different agroecological conditions.In addition,the divergent pattern of rice cultivars and weedy rice accessions suggests the possibility of an endoferal origin(from crop)of the weedy rice included in the present study.     

Chloroplast DNA insertions into the nuclear genome of rice: the genes,sites and ages of insertion involved

Rice (Oryza sativa) is one of three predominant grain crops, and its nuclear and organelle genomes have been sequenced. Following genome analysis revealed many exchanges of DNA sequences between the nuclear and organelle genomes. In this study, a total of 45 chloroplast DNA insertions more than 2 kb in length were detected in rice nuclear genome. A homologous recombination mechanism is expected for those chloroplast insertions with high similarity between their flanking sequences. Only five chloroplast insertions with high sequence similarity between two flanking sequences from an insertion were found in the 45 insertions, suggesting that rice might follow the non-homologous end-joining (NHEJ) repair of double-stranded breaks mechanism, which is suggested to be common to all eukaryotes. Our studies indicate that the most chloroplast insertions occurred at a nuclear region characterized by a sharp change of repetitive sequence density. One potential explanation is that regions such as this might be susceptible target sites or “hotspots” of DNA damage. Our results also suggest that the insertion of retrotransposon elements or non-chloroplast DNA into chloroplast DNA insertions may contribute significantly to their fragmentation process. Moreover, based on chloroplast insertions in nuclear genomes of two subspecies (indica and japonica) of cultivated rice, our results strongly suggest that they diverged during 0.06–0.22 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chloroplast genome sequences from total DNA for plant identification

Chloroplast DNA sequence data are a versatile tool for plant identification or barcoding and establishing genetic relationships among plant species. Different chloroplast loci have been utilized for use at close and distant evolutionary distances in plants, and no single locus has been identified that can distinguish between all plant species. Advances in DNA sequencing technology are providing new cost‐effective options for genome comparisons on a much larger scale. Universal PCR amplification of chloroplast sequences or isolation of pure chloroplast fractions, however, are non‐trivial. We now propose the analysis of chloroplast genome sequences from massively parallel sequencing (MPS) of total DNA as a simple and cost‐effective option for plant barcoding, and analysis of plant relationships to guide gene discovery for biotechnology. We present chloroplast genome sequences of five grass species derived from MPS of total DNA. These data accurately established the phylogenetic relationships between the species, correcting an apparent error in the published rice sequence. The chloroplast genome may be the elusive single‐locus DNA barcode for plants.     

Shotgun法测序制备高纯度水稻基因组DNA的方法探讨(英)

在用散弹 (shotgun)法测定水稻 (OryzasativaL .ssp .indica)基因组全序列的过程中 ,叶绿体和线粒体DNA的污染问题非常严峻 .应用脉冲场电泳 (PFGE)技术对水稻基因组DNA进行纯化 ,结果表明它能够有效去除叶绿体和线粒体DNA ,使其污染率从 3%降低到 0 2 % .同时 ,比较了水稻绿苗和黄化苗的DNA得率 ,以及HB法和NIB法制备大分子质量(HMW)DNA的异同 .最后提出一套制备水稻基因组DNA的方法 ,包括黄化苗培养 ;细胞核的分离、包埋和裂解 ;脉冲场电泳纯化、回收聚集在低熔点 (LMP)胶中的水稻HMWDNA .用该方法所得的水稻基因组DNA ,纯度高 (无叶绿体和线粒体DNA污染 )、基因组完整 (机械剪切和降解少 )、回收率高 (操作过程中DNA损失少 ) .另外 ,首次报道在融化的低熔点(LMP)胶中对水稻HMWDNA于 38℃进行超声波处理 ,能够获得用于shotgun文库和梯度文库构建所需要的各种DNA片段(1 5～ 3kb ,3～ 12kb) ,并且效果优于在TE中进行操作     

Rapid Isolation of DNA from Fresh and Preserved Fish Scales for Polymerase Chain Reaction

We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales. The procedure is based on boiling the scales in 5% Chelex 100, followed by digestion with proteinase K and subsequent absorption of genomic DNA using silica. A single fresh scale from larger species (e.g., tilapia) or a few scales from smaller species (e.g., 4 scales from zebrafish) provide over 200 ng of DNA, enough for at least 40 polymerase chain reaction amplifications. The procedure is applicable for DNA isolation not only from fresh and ethanol-preserved scales, but also from dried and formaldehyde-treated samples, and thus might be useful for investigating specimens stored in museums and other collections. Since the removal of a few scales is a gentle means of sample collection, this technique will allow analysis of genetic diversity, mating systems, and parentage in populations of endangered or ornamental fish with minimal experimental influence. Received November 6, 2000; accepted February 26, 2001.     

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa.

A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by mapping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chloroplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-1,5-bisphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA.     

Direct Chloroplast Sequencing: Comparison of Sequencing Platforms and Analysis Tools for Whole Chloroplast Barcoding

Direct sequencing of total plant DNA using next generation sequencing technologies generates a whole chloroplast genome sequence that has the potential to provide a barcode for use in plant and food identification. Advances in DNA sequencing platforms may make this an attractive approach for routine plant identification. The HiSeq (Illumina) and Ion Torrent (Life Technology) sequencing platforms were used to sequence total DNA from rice to identify polymorphisms in the whole chloroplast genome sequence of a wild rice plant relative to cultivated rice (cv. Nipponbare). Consensus chloroplast sequences were produced by mapping sequence reads to the reference rice chloroplast genome or by de novo assembly and mapping of the resulting contigs to the reference sequence. A total of 122 polymorphisms (SNPs and indels) between the wild and cultivated rice chloroplasts were predicted by these different sequencing and analysis methods. Of these, a total of 102 polymorphisms including 90 SNPs were predicted by both platforms. Indels were more variable with different sequencing methods, with almost all discrepancies found in homopolymers. The Ion Torrent platform gave no apparent false SNP but was less reliable for indels. The methods should be suitable for routine barcoding using appropriate combinations of sequencing platform and data analysis.