RESUMEN
A linkage map of expressed sequence tag (EST)-based markers in radish (Raphanus sativus L.) was constructed using a low-cost and high-efficiency single-nucleotide polymorphism (SNP) genotyping method named multiplex polymerase chain reaction-mixed probe dot-blot analysis developed in this study. Seven hundred and forty-six SNP markers derived from EST sequences of R. sativus were assigned to nine linkage groups with a total length of 806.7 cM. By BLASTN, 726 markers were found to have homologous genes in Arabidopsis thaliana, and 72 syntenic regions, which have great potential for utilizing genomic information of the model species A. thaliana in basic and applied genetics of R. sativus, were identified. By construction and analysis of the genome structures of R. sativus based on the 24 genomic blocks within the Brassicaceae ancestral karyotype, 23 of the 24 genomic blocks were detected in the genome of R. sativus, and half of them were found to be triplicated. Comparison of the genome structure of R. sativus with those of the A, B, and C genomes of Brassica species and that of Sinapis alba L. revealed extensive chromosome homoeology among Brassiceae species, which would facilitate transfer of the genomic information from one Brassiceae species to another.
Asunto(s)
Etiquetas de Secuencia Expresada/química , Marcadores Genéticos/genética , Polimorfismo de Nucleótido Simple/genética , Arabidopsis/genética , Brassicaceae/clasificación , Brassicaceae/genética , Mapeo Cromosómico/métodos , Ligamiento Genético , Genoma de Planta , Genómica , Immunoblotting , Reacción en Cadena de la Polimerasa Multiplex , Raphanus/genética , Sinapis/genética , Especificidad de la Especie , SinteníaRESUMEN
DNA methylation is an important epigenetic modification regulating gene expression and transposon silencing. Although epigenetic regulation is involved in some agricultural traits, there has been relatively little research on epigenetic modifications of genes in Brassica rapa, which includes many important vegetables. In B. rapa, orthologs of DDM1, a chromatin remodeling factor required for maintenance of DNA methylation, have been characterized and DNA hypomethylated knock-down plants by RNAi (ddm1-RNAi plants) have been generated. In this study, we investigated differences of DNA methylation status at the genome-wide level between a wild-type (WT) plant and a ddm1-RNAi plant by methylation-sensitive amplification polymorphism (MSAP) analysis. MSAP analysis detected changes of DNA methylation of many repetitive sequences in the ddm1-RNAi plant. Search for body methylated regions in the WT plant revealed no difference in gene body methylation levels between the WT plant and the ddm1-RNAi plant. These results indicate that repetitive sequences are preferentially methylated by DDM1 genes in B. rapa.
Asunto(s)
Brassica rapa/genética , Metilación de ADN/genética , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Southern Blotting , Sistemas de Lectura Abierta/genética , Interferencia de ARNRESUMEN
An OsWRKY11 gene, which encodes a transcription factor with the WRKY domain, was identified as one of the genes that was induced by both heat shock and drought stresses in seedlings of rice (Oryza sativa L.). To determine if overexpression of OsWRKY11 confers heat and drought tolerance, OsWRKY11 cDNA was fused to the promoter of HSP101 of rice and introduced into a rice cultivar Sasanishiki. Overexpression of OsWRKY11 was induced by heat treatment. After heat pretreatment, the transgenic lines showed significant heat and drought tolerance, as indicated by the slower leaf-wilting and less-impaired survival rate of green parts of plants. They also showed significant desiccation tolerance, as indicated by the slower water loss in detached leaves. Our results indicate that the OsWRKY11 gene plays a role in heat and drought stress response and tolerance, and might be useful for improvement of stress tolerance.
Asunto(s)
Oryza/fisiología , Proteínas de Plantas/metabolismo , Plantones/fisiología , Estrés Fisiológico , Factores de Transcripción/metabolismo , ADN Complementario/genética , Sequías , Regulación de la Expresión Génica de las Plantas , Respuesta al Choque Térmico , Calor , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , ARN de Planta/genética , Plantones/genética , Plantones/metabolismo , Factores de Transcripción/genética , Agua/fisiologíaRESUMEN
Identification of single nucleotide polymorphisms (SNPs) in a large number of genes will enable estimation of the number of genes having different alleles in a population. In the present study, SNPs between 21 rice cultivars including 17 Japanese cultivars, one upland rice, and three indica cultivars were analyzed by PCR-RF-SSCP. PCR-RF-SSCP analysis was found to be a more efficient method for detecting SNPs than mismatch-cleavage analysis, though both PCR-RF-SSCP and mismatch-cleavage are useful for screening SNPs. The number of DNA fragments showing polymorphism between Japanese cultivars was 134 in the 1,036 genes analyzed. In 137 genes, 638 DNA polymorphisms were identified. Out of 52 genes having polymorphisms in the exons, one had a frame-shift mutation, three had polymorphism causing amino acid insertions or deletions, and 16 genes had missense polymorphisms. The number of genes having frame-shift mutations and missense polymorphisms between the 17 Japanese cultivars was estimated to be 41 and 677 on average, respectively, and those between japonica and indica to be 425 and 6,977, respectively. Chromosomal regions of cultivars selected in rice breeding processes were identified by SNP analysis of genes.
Asunto(s)
Alelos , Genes de Plantas , Oryza/genética , Polimorfismo de Nucleótido Simple , Disparidad de Par Base/genética , Mapeo Cromosómico , Cromosomas de las Plantas/genética , Genoma de Planta , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
BACKGROUND AND AIMS: Glycinebetaine (GB), a quaternary ammonium compound, is a very effective compatible solute. In higher plants, GB is synthesized from choline (Cho) via betaine aldehyde (BA). The first and second steps in the biosynthesis of GB are catalysed by choline monooxygenase (CMO) and by betaine aldehyde dehydrogenase (BADH), respectively. Rice (Oryza sativa), which has two genes for BADH, does not accumulate GB because it lacks a functional gene for CMO. Rice plants accumulate GB in the presence of exogenously applied BA, which leads to the development of a significant tolerance to salt, cold and heat stress. The goal in this study was to evaluate and to discuss the effects of endogenously accumulated GB in rice. METHODS: Transgenic rice plants that overexpressed a gene for CMO from spinach (Spinacia oleracea) were produced by Agrobacterium-mediated transformation. After Southern and western blotting analysis, GB in rice leaves was quantified by (1)H-NMR spectroscopy and the tolerance of GB-accumulating plants to abiotic stress was investigated. KEY RESULTS: Transgenic plants that had a single copy of the transgene and expressed spinach CMO accumulated GB at the level of 0.29-0.43 micromol g(-1) d. wt and had enhanced tolerance to salt stress and temperature stress in the seedling stage. CONCLUSIONS: In the CMO-expressing rice plants, the localization of spinach CMO and of endogenous BADHs might be different and/or the catalytic activity of spinach CMO in rice plants might be lower than it is in spinach. These possibilities might explain the low levels of GB in the transgenic rice plants. It was concluded that CMO-expressing rice plants were not effective for accumulation of GB and improvement of productivity.
Asunto(s)
Betaína/metabolismo , Oryza/metabolismo , Oxigenasas/metabolismo , Cloruro de Sodio/metabolismo , Betaína/química , Southern Blotting , Western Blotting , Genes de Plantas , Espectroscopía de Resonancia Magnética , Oryza/genética , Oxigenasas/deficiencia , Oxigenasas/genética , Hojas de la Planta/genética , Plantas Modificadas Genéticamente , Rhizobium/genética , Spinacia oleracea/enzimología , Spinacia oleracea/genética , Temperatura , Transformación GenéticaRESUMEN
Nuclear so-called fertility-restorer genes reverse the pollen sterility of cytoplasmic male-sterile (CMS) plants caused by disturbed mitochondrial-nuclear interactions. We identified a CMS-associated chimeric mitochondrial gene in an alloplasmic CMS line of Brassica oleracea in the 'mur' system. This novel chimeric gene, orf72, was found in the mitochondrial genome of donor cytoplasm. It was located downstream of normal rps7 and contained part of atp9 (atp9-b). It was expressed specifically on the nuclear background of CMS B. oleracea, partially suppressed in the fertility-restored line and entirely suppressed in the cytoplasmic donor.
Asunto(s)
Brassica/genética , ADN Mitocondrial/genética , ADN de Plantas/genética , Genes de Plantas , Sistemas de Lectura Abierta , Infertilidad Vegetal/genética , Secuencia de Aminoácidos , Secuencia de Bases , Brassica/clasificación , Brassicaceae/clasificación , Brassicaceae/genética , Genes Mitocondriales , Genoma de Planta/genética , Datos de Secuencia Molecular , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión/análisisRESUMEN
DNA polymorphism of randomly selected genes in rice cultivars was analyzed by the polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) technique. Single DNA fragments were amplified from genomic DNA of the Nipponbare cultivar by 671 primer pairs among the 1000 primer pairs tested. PCR-RF-SSCP analysis using the 671 primer pairs detected polymorphism in 108 DNA fragments between 17 japonica paddy-rice cultivars. An average of 36.9 DNA fragments showed polymorphism between any pair of japonica paddy-rice cultivars. The nucleotide sequences of the polymorphic DNA fragments were determined for 50 alleles of 45 genes together with Nipponbare alleles. In these genes, 142 SNPs and 32 insertions/deletions were identified. Among these 174 sequence variations, 71 were in exons, 78 in introns, and 25 in unassigned regions. There were 28 alleles which had sequence variations in the exons. One allele had a 1-bp deletion in the exon causing a frame-shift mutation, 15 alleles had missense mutations, and the other 12 alleles had synonymous changes and/or sequence variations in 3' untranslated regions. The number of genes having sequence variations between the rice cultivars and the functional implications of the identified SNPs are herein discussed.
Asunto(s)
Genes de Plantas , Oryza/genética , Polimorfismo de Nucleótido Simple , Alelos , Secuencia de Aminoácidos , Cruzamiento , ADN de Plantas/genética , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo Conformacional Retorcido-Simple , Selección Genética , Eliminación de SecuenciaRESUMEN
In Brassica species, alloplasmic lines displaying cytoplasmic male sterility (CMS) are established by combining the nucleus from B. rapa with the cytoplasm from Diplotaxis muralis. The failure to observe restriction fragment length polymorphism (RFLP) patterns of mitochondrial genes ( coxII, coxIII, atpA, atp6, atp9, cob, nad3, nad6, and nad9) between alloplasmic lines of B. rapa and D. muralis indicates that introgression of the B. rapa nucleus into the cytoplasm of D. muralis does not cause any alterations in the structure of the mitochondrial genome. To investigate how the nucleus influences the cytoplasm, we examined the expression of mitochondrial genes in the leaves of euplasmic and alloplasmic lines of B. rapa and D. muralis. We detected higher levels of mitochondrial gene mRNAs in alloplasmic lines of B. rapa than in D. muralis. Patterns of mitochondrial gene transcription also differed among the alloplasmic lines of B. rapa. Thus, expression of mitochondrial genes in alloplasmic lines of B. rapa differed in the leaves compared to D. muralis. Overexpression of mitochondrial genes may be the result of novel interactions between the nucleus and the mitochondria in alloplasmic lines of B. rapa. Further study is necessary to clarify how these phenomena are involved in CMS.
Asunto(s)
Brassica rapa/genética , Brassicaceae/genética , Mitocondrias/genética , Núcleo Celular/genética , Citoplasma/genética , ADN Mitocondrial/genética , Proteínas de Plantas/genética , Polimorfismo de Longitud del Fragmento de Restricción , ARN Mensajero/genética , Mapeo RestrictivoRESUMEN
The level of cis-unsaturated fatty acids in phosphatidylglycerol (PG) from rice leaves was genetically altered from 19.3% in the wild-type to 29.4 and 32.0% in T1 plants segregated with cDNAs for glycerol-3-phosphate acyltransferase of chloroplasts (GPAT; EC 2.3.1.15) from Arabidopsis (+AGPAT plant) and spinach (+SGPAT plant), respectively; and to 21.4% in a non-transformant segregated from +SGPAT plants (-SGPAT plant). In all these plants, O2 evolution from leaves was similar at 25 degrees C and was impaired to a similar extent at 5 and 11 degrees C. However, in parallel with the levels of cis-unsaturated fatty acids in PG, +AGPAT and +SGPAT plants showed less impaired rates of O(2) evolution from leaves than the wild-type and -SGPAT plants at 14 and 17 degrees C. In agreement with this, the fresh weight of 14-day-old seedlings increased to 571 + or - 18, 591 + or - 23, 687 + or - 32 and 705 + or - 31 mg in the wild-type, -SGPAT, +AGPAT and +SGPAT plants, respectively, after 6 weeks at 17/14 degrees C (day/night). These results demonstrate the practical importance of the present technology with GPAT in improvement of the chilling sensitivity of crops.
