Linkage analysis of the nuclear homologues of mitochondrial plasmid-like DNAs in rice.

A genetical study on the nucleotide sequences of the nuclear DNAs which share homology with rice mitochondrial plasmid-like DNAs, B1, B2, B3 and B4 was carried out. Restriction fragments of the nuclear DNAs hybridized with these plasmid-like DNAs showed polymorphisms in their length between Indica and Japonica rice cultivars. The hybridized signals found specifically in Indica or Japonica cultivars segregated in the F2 population derived from a cross between these two subspecies. The observed ratio of the nuclear homologues in the F2 population demonstrated that they were transmitted according to the Mendelian inheritance. The co-segregation of homologues was examined and the linkage was detected between the B1-nuclear homologue of Japonica and the B4-nuclear homologue of Indica, and also between the nuclear homologues of B2 and B3 of Indica. The linkage between the B1-nuclear homologue of Japonica and the B4-nuclear homologue of Indica was conserved in the different rice cultivars.     

Distribution of mitochondrial plasmid-like DNA in cultivated rice (Oryza sativa L.) and its relationship with varietal groups

Summary Mitochondrial (mt) plasmid-like DNA was found in most of more than 100 rice cultivars (Oryza sativa L.) by the use of 0.7% agarose gel electrophoresis (AGE). The DNA varied in molecular weight and number. By electron microscopy, small circular DNAs of different sizes could be detected in addition to the DNAs of high molecular weight, even in cultivars in which mt plasmid-like DNA was not detected by AGE. The detection of the mt plasmid-like DNAs by AGE did not depend on their presence or absence, but on their high stoichiometry. The relationship between cytoplasms with mt plasmid-like DNAs and varietal (for example, Indica rice) groups was close. The geographical distribution of cytoplasms is discussed.     

Classification of rice germplasm. I. Analysis using ALP and PCR-based RFLP

The potential of using a PCR-based approach to detect DNA polymorphism for rice germplasm classification was compared with that of Southern-based RFLP analysis. Thirty-five Iranian rice varieties were studied along with 2 typical Indica and 3 typical Japonica varieties. Thirteen mapped RFLP markers were used as hybridization probes against Southern blots containing digests of one restriction endonuclease; 12 of the 13 probes detected polymorphism in the varieties. Fifteen sets of oligonucleotides derived from sequences near the ends of the same probes and of two other mapped probes were used as primers for PCR amplification of total genomic DNA of the varieties. Amplicon length polymorphisms (ALPs) were detected with 6 of the 15 sets of primers. To identify additional polymorphism, the PCR products were digested with nine different restriction endonucleases recognizing 4- or 5-bp DNA sequences and analyzed by gel electrophoresis in agarose and polyacrylamide. RFLPs were detected for 11 sets of primers, due to point mutations and to addition/deletion events that were too small to be detected as ALPs. Because PCR products are easily generated and may be analyzed in detail through the use of restriction endonucleases that cut rice DNA frequently, PCR-based RFLP analysis is a useful tool for the classification of rice germplasm.     

Differentiation of Indica-Japonica rice revealed by insertion/deletion (InDel) fragments obtained from the comparative genomic study of DNA sequences between 93-11 (Indica) and Nipponbare (Japonica)

DNA polymorphisms from nucleotide insertion/deletions (InDels) in genomic sequences are the basis for developing InDel molecular markers.To validate the InDel primer pairs on the basis of the comparative genomic study on DNA sequences between an Indica rice 93-11 and a Japonica rice Nipponbare for identifying Indica and Japonica rice varieties and studying wild Oryza species,we studied 49 Indica,43 Japonica,and 24 wild rice accessions collected from ten Asian countries using 45 InDel primer pairs.Results indicated that of the 45 InDel primer pairs,41 can accurately identify Indica and Japonica rice varieties with a reliability of over 80%.The scatter plotting data of the principal component analysis (PCA) indicated that:(i) the InDel primer pairs can easily distinguish Indica from Japonica rice varieties,in addition to revealing their genetic differentiation;(ii) the AA-genome wild rice species showed a relatively close genetic relationship with the Indica rice varieties;and (iii)the non-AA genome wild rice species did not show evident differentiation into the Indica and Japonica types.It is concluded from the study that most of the InDel primer pairs obtained from DNA sequences of 93-11 and Nipponbare can be used for identifying lndica and Japonica rice varieties,and for studying genetic relationships of wild rice species,particularly in terms of the Indica-Japonica differentiation.     

Genetic differentiation for nuclear,mitochondrial and chloroplast genomes in common wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.) and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The genetic differentiation of nuclear, mitochondrial (mt) and chloroplast (cp) genomes was investigated by Southern and PCR analysis using 75 varieties of cultivated rice ( Oryza sativa L.) and 118 strains of common wild rice (CWR, Oryza rufipogon Griff.) from ten countries of Asia. The distinguishing differences between the Indica and Japonica cultivars were detected both in the nuclear genome and the cytoplasmic genome, confirming that the Indica-Japonica differentiation is of major importance for the three different classes of genome in cultivated rice. This differentiation was also detected in common wild rice with some differences among the genome compartments and the various regions. For nuclear DNA variation, both Indica-like and Japonica-like types were observed in the Chinese CWR, with the latter more-frequent than the former. No Japonica-like type was found in South Asia, and only two strains of the Japonica-like type were detected in Southeast Asia, thus the Indica-like type is the major type among South and Southeast Asian CWR. For mtDNA, only a few strains of the Japonica-like type were detected in CWR. For cpDNA, the Japonica type was predominant among the CWR strains from China, Bangladesh and Burma, while the Indica type was predominant among the CWR strains from Thailand, Malaysia, Cambodia and Sri Lanka, and both types were found in similar frequencies among the Indian CWR. Altogether, however, the degree of Indica-Japonica differentiation in common wild rice was much-less important than that in cultivated rice. Cluster analyses for nuclear and mitochondrial DNA variation revealed that some CWR strains showed large genetic distances from cultivated rice and formed clusters distinct from cultivated rice. Coincidence in the genetic differentiation between the three different classes of genome was much higher in cultivated rice than in CWR. Among the 75 cultivars, about 3/4 entries were "homoeotype" showing congruent results for nuclear, mt and cpDNA regarding the Indica-Japonica differentiation. In CWR, the proportions of homoeotypes were 5.7%, 15% and 48.8% in China, South Asia and Southeast Asia, respectively. Based on the average genetic distance among all the strains of CWR and cultivated rice for nuclear and mitochondrial genomes, the variability of the nuclear genome was found to be higher than that of the mitochondrial genome. The global pattern based on all genomes shows much-more diversification in CWR than that in cultivated rice.     

Genetic basis of hybrid breakdown in a Japonica/Indica cross of rice,Oryza sativa L

Reproductive barriers often arise in hybrid progeny between two varietal groups of Asian cultivated rice ( Oryza sativa L.), Japonica and Indica. Hybrid breakdown showing poor growth habit, and complete sterility was found in the backcrossed progeny derived from a cross between a Japonica variety, Asominori, and an Indica variety, IR24. We employed RFLP analysis in the segregating population to study the genetic basis underlying hybrid breakdown. It was found that the hybrid breakdown is caused by a set of two nuclear genes, which were symbolized as hwe1 and hwe2. The parental varieties, Asominori and IR24, carry hwe1(+) hwe1(+) hwe2hwe2 and hwe1hwe1hwe2(+) hwe2(+) genotypes, respectively, whereas the progenies that showed a weakness performance carry the double recessive genotype ( hwe1hwe1hwe2hwe2). Abnormality was not observed in the progenies that carry the other genotypes, indicating that a single dominant allele at either locus is necessary for normal growth. Based on linkage analysis with RFLP markers, the hwe1 locus was located between RFLP markers R1869 and S1437 on chromosome 12 and the hwe2 locus was located between R3192 and C1211 on chromosome 1. The genetic basis was reconfirmed using near-isogenic lines carrying the genes with reciprocal genetic backgrounds. The present study provides clear evidence, viewed by previous workers, that hybrid breakdown is attributed to complementary genes from both parents.     

重复DNA顺序pRRD9在稻属中的拷贝数测定

本文应用狭缝印渍杂交方法,把水稻基因组总DNA和含水稻中度重复顺序片段的质粒(pRRD9)DNA分别转移到尼龙膜上形成狭缝印渍、然后用~(32)P标记的 pRRD9插入片段进行杂交、根据各狭缝印渍的放射性强度,测定水稻(Oryza)一些栽培种和野生种基因组中重复DNA顺序的拷贝数,并就拷贝数与水稻进化关系及基因组型的联系进行讨论.     

Polymorphism at the esterase isozyme locus Est10 associated with phylogenetic differentiation in rice

A new esterase isozyme locus, Est10, with 6 alleles including the null form, has been found in rice by using polyacrylamide gel electrophoresis. Thirty F(2) populations of all possible combinations between 5 different band morphs were studied. The segregation pattern indicated that bands 1, 2, 3, 4, and the null form (0) were allelic with each other. The alleles of Est10 were distributed at different frequencies among different varietal groups of rice and also between cultivated rice and its wild relatives (Oryza rufipogon Griff.). Alleles 1 and 2 were frequently found in Japonica and Indica types, respectively. Allele 3 showed a high frequency in Aus and Boro, both Indica types cultivated in South Asia. Allele 4 was frequent in wild rice O. rufipogon. Judging from the linkage between Est10 and RFLP marker RG220 and isozyme marker Est5, Est10 is located on chromosome 1. The importance of this locus in evolutionary studies of rice is discussed.     

Distribution of the genes causing F2 chlorosis in rice cultivars of the Indica and Japonica types

Summary Chlorotic plants were segregated in F₂ populations in varietal crosses of common rice. The genetic basis and distribution of the genes causing F₂ chlorosis in native cultivars were studied to examine the role of the F₂ chlorosis in varietal differentiation of rice. It was proven that this F₂ chlorosis was controlled by a set of duplicate genes, hca-1 and hca-2. The hca-2 gene was widely distributed in native cultivars of the Japonica type, while many Indica types carried its dominant allele hca-2 ⁺. Japanese cultivar J-147 carried hca-2. The hca-1 gene was frequently distributed in cultivars containing the Hwc-2 gene for F₁ weakness. We concluded that F₂ chlorosis does not cause or promote varietal differentiation in rice.     

Efficient detection of DNA polymorphism in cabbage and rice cultivars by PCR-RF-SSCP (PRS)

PCR (polymerase chain reaction)-RF(restriction fragment)-SSCP (single-strand conformation polymorphism) - designated here as PRS - is a combined method of SSCP and PCR-RFLP (restriction fragment length polymorphism) - designated as CAPS (cleaved amplified polymorphic sequence) - and was efficient in detecting intraspecific variation of the SLR1 gene in Brassica oleracea. One to six nucleotide changes in restriction fragments of the SLR1 gene were detected as different bands in PRS. In an analysis of randomly chosen DNA fragments in cabbage, PRS detected DNA polymorphism between different cultivars with more than 60% of the primer pairs used except for a combination of two cultivars having highly similar characteristics. In rice, no DNA polymorphism was found between two Japonica cultivars, while more than 80% of the primer pairs showed DNA polymorphism between Japonica cultivars and Indica cultivars. PRS had a 1.5- to twofold greater ability to detect DNA polymorphism in these cabbage and rice cultivars than CAPS. The present study indicated that PRS is potentially useful for the identification of crop cultivars and genetic mapping of DNA fragments including genes of interest.     

影响籼粳稻DNA限制性片段长度多态性检测的因素分析

对窄叶青(籼稻)和京系17(梗稻)的RFLP进行了系统分析。结果表明,某种酶多态性检测能力与同时检测到此多态性的其余酶的数目之间存在极显著的正相关(r=0.962**)。由此推论,籼粳稻大部分RFLP可能来自大范围DNA的结构变化而不是碱基取代。cDNA克隆虽然具有高度的保守性,但却具有比基因组克隆更高的多态性检测能力。在实验使用的探针范围内,探针长度和检测到的多态性无相关性。由探针检测到的多态性位点均匀地散布在水稻的12对染色体上,这可能是这两个品种所属的亚种之间的遗传距离较大的原因。     

Abundance of plastid DNA insertions in nuclear genomes of rice and Arabidopsis

Pairwise comparison of whole plastid and draft nuclear genomic sequences of Arabidopsis thaliana and Oryza sativa L. ssp. indica shows that rice nuclear genomic sequences contain homologs of plastid DNA covering about 94 kb (83%) of plastid genome and including one or more full-length intact (without mutations resulting in premature stop codons) homologues of 26 known protein-coding (KPC) plastid genes. By contrast, only about 20 kb (16%) of chloroplast DNA, including a single intact plastid-derived KPC gene, is presented in the nucleus of A. thaliana. Sixteen rice plastid genes have at least one nuclear copy without any mutation or with only synonymous substitutions. Nuclear copies for other ten plastid genes contain both synonymous and non-synonymous substitutions. Multiple ESTs for 25 out of 26 KPC genes were also found, as well as putative promoters for some of them. The study of substitutions pattern shows that some of nuclear homologues of plastid genes may be functional and/or are under the pressure of the positive natural selection. The similar comparative analysis performed on rice chromosome 1 revealed 27 contigs containing plastid-derived sequences, totalling about 84 kb and covering two thirds of chloroplast DNA, with the intact nuclear copies of 26 different KPC genes. One of these contigs, AP003280, includes almost 57 kb (45%) of chloroplast genome with the intact copies of 22 KPC genes. At the same time, we observed that relative locations of homologues in plastid DNA and the nuclear genome are significantly different.     

RAPD, RFLP and SSLP analyses of phylogenetic relationships between cultivated and wild species of rice.

RAPD, RFLP, nuclear SSLP and chloroplast SSLP analyses were carried out to clarify the phylogenetic relationships among A-genome species of rice. In total, 12 cultivars of Oryza sativa (4 Japonica, 3 Javanica and 5 Indica), one cultivar of O. glaberrima, and 17 wild accessions (12 O. rufipogon, 2 O. glumaepatula, 1 O. longistaminata, 1 O. meridionalis and 1 O. barthii) were used. Their banding patterns were scored and compared to evaluate the similarity between accessions. Genetic differentiation within and between taxa was examined based on the average similarity indices. Except for chloroplast SSLP analysis, the average similarities were higher within O. sativa than within O. rufipogon, and O. sativa Indica had greater intrasubspecific variation than Japonica and Javanica. Comparisons between cultivated and wild species showed that O. sativa was closely related to O. rufipogon, while O. glaberrima was closely related to O. barthii. This indicated that two cultivated species, O. sativa and O. glaberrima, originated from O. rufipogon and O. barthii, respectively. Domestication of O. sativa seemed to be diphyletic, since strong similarity was observed between O. sativa Japonica-Javanica and O. rufipogon from China and between O. sativa Indica and O. rufipogon from tropical Asia. In addition, dendrograms for RAPD, RFLP, and nuclear and chloroplast SSLP analyses were constructed to reveal the overall genetic relationships among A-genome species. In all analyses, O. sativa and O. glaberrima formed groups with O. rufipogon and O. barthii, respectively. However, their manners of clustering with other wild species were not the same. The results of RAPD and RFLP analyses indicate that O. glumaepatula was relatively close to the groups of O. sativa and O. glaberrima whereas O. longistaminata and O. meridionalis were highly differentiated from other A-genome species. On the other hand, clear interspecific relationships were not obtained by nuclear or chloroplast SSLP analyses.     

Distribution and organization of a tandemly repeated 352-bp sequence in the oryzae family

Summary A 352-bp EcoRI fragment from rice DNA was cloned and shown to be a member of a tandem repeat. Sequence determination revealed homologies with human alpha satellite DNA and maize knob heterochromatin specific repeat. This 352-bp sequence is highly specific for the AA genome of rice. However, copy number and sequence organization are variable, depending on the accession analyzed. Several examples of amplification were observed in O. rufipogon and O. longistaminata. Use of resolutive polyacrylamide gel electrophoresis and 4-bp cutter enzymes allowed one to distinguish between the Indica and Japonica subtypes of O. sativa. The same method also discriminates between two groups of O. rufipogon, the presumed ancestor of O. sativa, suggesting that the present day Indica and Japonica subtypes originated independently from two O. rufipogon distinct populations.     

水稻品种多样性遗传分析与稻瘟病控制

以2个籼型杂交稻——汕优63(A)和汕优22(B)、2个地方糯稻品种——黄壳糯(C)和紫糯(D)和3个粳稻品种——合系41(E)、楚粳12(F)和8126(G)为材料进行抗病基因同源序列(Resistance Gene Analogue,RGA)遗传分析。结果表明,杂交稻品种间以及粳稻品种间的抗性遗传较为相似,其相似系数分别为0．86和0．84。糯稻品种间以及糯稻、杂交稻和粳稻间的抗性遗传差异较大,相似系数为0．45。聚类分析表明,RGA结果与品种的系谱来源相吻合,与品种的田间抗性基本一致。根据品种的抗性遗传差异、农艺性状和经济性状的不同,在云南籼稻区的建水和石屏县以及温暖粳稻区的泸西县分别选用5种(A／C、A／D、B／C、B／D和A／B)和2种(E／C和E／F／G)不同的品种组合进行品种多样性混合间栽控制稻瘟病田间试验,结果表明,抗性遗传差异大(相似性：0．45～0．77)的5个品种混合间栽组合对稻瘟病有极为显著的控制效果,尤其是在混合间栽中高度感病的优质地方稻品种稻瘟病的发病率、病情指数均有极显著的下降,防治效果达54．47％～92．18％;遗传差异较小(相似性：0．84～0．90)的2个混栽组合混栽对稻瘟病的控制效果不明显,稻瘟病的防治效果在15．12％～25．54％。此外,品种抗性遗传和株高差异大的品种组合具有显著的增产效果,与品种净栽相比,平均增产539．0～900．0kg／ha,增幅5．57％-10．38％;品种抗性遗传和株高相似的品种组合没有增产效果。     

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.     

Rice allelopathy and the possibility for weed management

In attempts to control weeds in rice, much effort has been focused on rice allelopathy research for more than 30 years. Among screening methods that have been developed, some estimate the allelopathic potential of various rice cultivars in a limited time and space, which is less costly and can be conducted year round. Rice allelopathy activity is variety dependent and origin dependent, where Japonica rice shows greater allelopathic activity than Indica and Japonica–Indica hybrids. Allelopathic characteristics in rice are quantitatively inherited and several allelopathy‐involved traits have been identified. Numerous phytotoxins such as cytokinins, diterpenoids, fatty acids, flavones, glucopyranosides, indoles, momilactones (A and B), oryzalexins, phenols, phenolic acids, resorcinols and stigmastanols have been identified and determined as growth inhibitors in rice. However, the fate and actual modes of action of these compounds as well as other potent rice phytotoxins in nature are not well understood. The question of which compounds play a major role in rice allelopathy has remained obscure; however, rice allelopathy might be attributable to the interaction of all present allelochemicals. Despite locating genes determining or involving allelopathy in rice having attracted much effort, the introduction of these genes into target rice cultivars has not yet been achieved. Success in breeding new rice cultivars having good weed‐suppressing ability would benefit farmers in rice‐cultivating countries and play an important role in sustainable agricultural production.     

Indica-Japonica differentiation of rice cultivars viewed from variations in key characters and isozymes,with special reference to landraces from the Himalayan hilly areas

Summary To assess the extent of differentiation into the Indica and Japonica types of Asian rice in its diversity center, we investigated landraces collected from the hilly areas of Nepal, Sikkim and Assam. We examined variations in four key characters and six isozyme loci known to be diagnostic for classifying Indica and Japonica types, and compared the results with those from a control set of rice cultivars representing the whole of Asia. The hill cultivars showed a high level of genetic diversity in key characters as well as in isozymes. A marked feature found in their character variation was the occurrence of various atypical cultivars that were intermediate between the Indica and Japonica types. With respect to isozymes, however, the hill cultivars could be classified mostly as either Indicas or Japonicas, although the patterns of allelic association were more random than in the control cultivars. Indica-Japonica variation in key characters and in isozymes corresponded well with each other in the controls, but not in the hill cultivars. This means that nonrandom association in characters as well as in genes (gametic disequilibrium) is not fully developed in the diversity center. Populations of hill cultivars were highly polymorphic genetically, but did not show a trend to Indica-Japonica differentiation within their populations. The process of Indica-Japonica differentiation is discussed in view of these observations.     

Inhibitory Effects of Oxygen on the Germination of Oryza sativa L. Seeds

Some workers have reported that the breaking of seed dormancyin rice (O. sativa L.) is usually enhanced by higher oxygentension, whereas others have shown that rice seed dormancy canbe broken by incubation under anaerobic conditions. This articleaims to clarify this paradox. The results show that high oxygentensions inhibit seed germination for a certain period afterharvest in Japonica rice, whereas Indica rice cultivars arenot inhibited by oxygen at any stage. Oxygen inhibition, seed germination, aquatic plants, Japonica rice, Indica rice