Genetic diversity and differentiation of indica and japonica rice detected by RFLP analysis

Summary Genetic diversity and differentiation in indica and japonica groups of the cultivated rice (Oryza sativa L.) were studied by assaying DNA restriction fragment length polymorphisms of 12 indica and 14 japonica rice lines digested with three restriction endonucleases. A total of 49 probes were selected to represent the entire RFLP map at intervals of 20–30 cM. It was shown that 95 of the 145 possible probe/enzyme combinations, involving 43 probes and all three enzymes, detected restriction fragment length variation, and the degree of polymorphism varied greatly from one probe/enzyme combination to another. These results demonstrate that indica rice is genetically more diverse than japonica type. Significant differentiation between the two rice groups was detected by 33 probes representing 11 of the 12 rice chromosomes. It was deduced that the processes leading to differentiation involved a combination of molecular events that include base substitutions and insertion/deletions.     

Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L.

Restriction fragment length polymorphisms (RFLPs) were studied in fourteen accessions of CCDD genome allotetraploid wild rice species (Oryza latifolia, O. alta and O. grandiglumis). Fourteen nuclear RFLP markers previously mapped in AA genome-cultivated rice were used as probes. A phylogenetic tree, constructed by parsimony analysis based on RFLPs, grouped the accessions according to their geographic origin from Central or South America. Oryza alta, O. grandiglumis and one accession of O. latifolia grouped together as a subgroup, and our results suggested that the three taxa should be considered as populations of a single complex species. Duplicate loci, representing the two constituent genomes of the allotetraploid, were observed for most RFLP markers. By comparing RFLPs from the allotetraploids with those from a CC genome diploid wild species (O. officinalis), it was possible to detect RFLPs specific for both the CC and DD genomes of the allotetraploid. In inter-accession F2 populations, independent segregation of RFLP markers for CC and DD genomes was observed.     

RFLP analysis of rice (Oryza sativa L.) introgression lines

Summary Fifty-two introgression lines (BC₂F₈) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.     

Overexpression of the rFCA RNA Recognition Motif Affects Morphologies Modifications in Rice (Oryza sativa L.)

RNA recognition motifs as important regulators of gene expression are highly conserved in animals and plants. The FCA floral promotion gene in Arabidopsis encodes a protein, containing two RNA recognition motifs (RRM) and a WW protein interaction domain. Here we isolated FCA cDNA from rice. FCA in rice (rFCA) was homologous to FCA-gamma of Arabidopsis and contained conserved domains. To investigate the function of RRM domain, fragment RRM1 and RRM2 of rFCA were introduced into rice subspecies Oryza sativa L. subsp. Indica var. 9311 and another rice subspecies Oryza sativa L. subsp. Japonica var. zhonghua11 transformation. Two transgenic lines exhibited similar phenotypes, flowering time delay, seed size and cell volume of transgenic plants was increased. These results showed that constitutive expression of RRMs could regulate cellular size. The patterns of overexpression of two RRM domains and their similar morphologies indicate they may play a same role.     

Molecular mapping of rice chromosomes

Summary We report the construction of an RFLP genetic map of rice (Oryza sativa) chromosomes. The map is comprised of 135 loci corresponding to clones selected from a PstI genomic library. This molecular map covers 1,389 cM of the rice genome and exceeds the current classical maps by more than 20%. The map was generated from F₂ segregation data (50 individuals) from a cross between an indica and javanica rice cultivar. Primary trisomics were used to assign linkage groups to each of the 12 rice chromosomes. Seventy-eight percent of the clones assayed revealed RFLPs between the two parental cultivars, indicating that rice contains a significant amount of RFLP variation. Strong correlations between size of hybridizing restriction fragments and level of polymorphism indicate that a significant proportion of the RFLPs in rice are generated by insertions/delections. This conclusion is supported by the occurrence of null alleles for some clones (presumably created by insertion or deletion events). One clone, RG229, hybridized to sequences in both the indica and javanica genomes, which have apparently transposed since the divergence of the two cultivars from their last common ancestor, providing evidence for sequence movement in rice. As a by product of this mapping project, we have discovered that rice DNA is less C-methylated than tomato or maize DNA. Our results also suggest the notion that a large fraction of the rice genome (approximately 50%) is single copy.     

Biased distribution of microsatellite motifs in the rice genome

Microsatellites are useful tools to study the extent of divergence between two taxonomic groups that show high sequence similarity. We have compared microsatellite distribution to illustrate genetic variation between the two rice genomes, Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica. Microsatellite distribution proved to be non random as certain regions of very high microsatellite density have been identified. Microsatellite density in the subspecies japonica was computed marginally higher than in the subspecies indica in the genomic regions compared between the two subspecies. Unexpectedly high microsatellite densities were observed in 5′-untranslated regions of genes. These regions also displayed a clear motif bias. Some of the longest microsatellite repeats were found in intron sequences. Frequency, as well as motif bias was also noted with respect to the association of microsatellites with transposable elements. Microsatellite mutability values were exemplarily estimated for 90 loci by aligning the microsatellite containing regions between the two genomes. Poor rates of finding an orthologue corresponded with high microsatellite mutability in rice. These insights are likely to play a significant role in selecting microsatellite loci to be used in molecular breeding and studying evolutionary dynamics of the two subspecies.     

Chromosomal rearrangements differentiating the ryegrass genome from the Triticeae, oat, and rice genomes using common heterologous RFLP probes

An restriction fragment length polymorphism (RFLP)-based genetic map of ryegrass (Lolium) was constructed for comparative mapping with other Poaceae species using heterologous anchor probes. The genetic map contained 120 RFLP markers from cDNA clones of barley (Hordeum vulgare L.), oat (Avena sativa L.), and rice (Oryza sativa L.), covering 664 cM on seven linkage groups (LGs). The genome comparisons of ryegrass relative to the Triticeae, oat, and rice extended the syntenic relationships among the species. Seven ryegrass linkage groups were represented by 10 syntenic segments of Triticeae chromosomes, 12 syntenic segments of oat chromosomes, or 16 syntenic segments of rice chromosomes, suggesting that the ryegrass genome has a high degree of genome conservation relative to the Triticeae, oat, and rice. Furthermore, we found ten large-scale chromosomal rearrangements that characterize the ryegrass genome. In detail, a chromosomal rearrangement was observed on ryegrass LG4 relative to the Triticeae, four rearrangements on ryegrass LGs2, 4, 5, and 6 relative to oat, and five rearrangements on ryegrass LGs1, 2, 4, 5, and 7 relative to rice. Of these, seven chromosomal rearrangements are reported for the first time in this study. The extended comparative relationships reported in this study facilitate the transfer of genetic knowledge from well-studied major cereal crops to ryegrass.     

RFLP analysis of the wild potato species,Solanum acaule Bitter (Solanum sect. Petota)

Summary Intraspecific variation of a wild potato species, Solanum acaule Bitt., was analyzed by RFLPs of genomic DNA. One hundred and five accessions were selected throughout the distribution area, including all subspecies, i.e., ssp. albicans (hexaploid), ssp. punae (tetraploid), ssp. acaule (tetraploid) and ssp. aemulans (tetraploid). Twenty-seven low-copy DNA clones (probes) were Southern hybridized with EcoRI, EcoRV, HindIII, and XbaI digests of total DNA of all accessions. In total, 238 RFLPs were detected from 94 enzyme x probe combinations. Among them, 49 RFLPs were specific to ssp. albicans, suggesting that the additional third genome is distinct from its two other genomes. RFLPs between and within subspecies were analyzed by principal component analysis. DNA similarities between subspecies coincided with a former taxonomic treatment in the sense that ssp. albicans is the most distantly related to ssp. acaule and ssp. aemulans is distantly related. Subspecies acaule and ssp. punae were indistinguishable. In addition, RFLPs could be used to distinguish groups within subspecies. Subspecies aemulans, confined to Argentina, was divided into two populations, one from the province of La Rioja and the other from the province of Jujuy. In ssp. acaule, some accessions from the southernmost distribution area were clearly distinguishable, while the others varied continuously, showing a geographical cline from Peru to Argentina.Reference to a specific brand or firm name does not constitute endorsement by the US Department of Agriculture over others of similar nature not mentioned     

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa)

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F₂ progeny, which were derived from a single F₁ plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.     

Linkage Arrangement of RFLP loci in progenies from crosses between doubled haploid Asparagus officinalis L. clones

A preliminary genetic map of the dioecious species Asparagus officinalis L. (2n = 20) has been constructed on the basis of restriction fragment length polymorphism (RFLP) and isozyme marker data. With DNA samples digested with either EcoRI or HindIII 61 out of 148 probes (41%) identified RFLPs in six families of doubled haploid lines obtained through anther culture. A higher level of polymorphism (65%) was observed when a single family was screened for RFLPs using six distinct restriction enzymes. Segregation analysis of the BC progenies (40–80 individuals) resulted in a 418-cM extended map comprising 43 markers: 39 RFLPs, three isozymes and one morphological (sex). These markers are clustered in 12 linkage groups and four of them exhibited significant deviations from the expected 11 ratio. One isozyme and three RFLP markers were assigned to the sex chromosome.     

Genetic structure and differentiation of <Emphasis Type="Italic">Oryza sativa</Emphasis> L. in China revealed by microsatellites

China is one of the largest centers of genetic diversity of Oryza sativa L. in the world. Using a genetically representative primary core collection of 3,024 rice landraces in China, we analyzed the genetic structure and intraspecific differentiation of O. sativa, and the directional evolution of SSR. The genetic structure was investigated by model-based structure analysis and construction of neighbor-joining phylogenetic tree. Comparison between genetic structure and predefined populations according to Ting’s taxonomic system revealed a hierarchical genetic structure: two distinct subspecies, each with three ecotypes and different numbers of geo-ecogroups within each ecotype. Two subspecies evidently resulted from adaptation to different environments. The different cropping systems imposed on the subspecies led to further differentiation, but the variation within each subspecies resulted from different causes. Indica, under tropical-like or lowland-like environments, exhibited clear differentiation among seasonal ecotypes, but not among soil-watery ecotypes; and japonica showed clear differences between soil water regime ecotypes, but not among seasonal ecotypes. Chinese cultivated rice took on evident directional evolution in microsatellite allele size at several aspects, such as subspecies and geographical populations. Japonica has smaller allele sizes than indica, and this may partly be the result of their different domestication times. Allele size was also negatively correlated with latitude and altitude, and this may be interpreted by different mutation rates, selection pressures, and population size effects under different environments and cropping systems.     

Construction and characterization of a rice YAC library for physical mapping

Genomic libraries of rice,Oryza sativa L. cv. Nipponbare, in yeast artificial chromosomes were prepared for construction of a rice physical map. High-molecular-weight genomic DNA was extracted from cultured suspension cells embedded in agarose plugs. After size fractionation of theEco RI- andNot I-digested DNA fragments, they were ligated with pYAC4 and pYAC55, respectively, and used to transformSaccharomyces cerevisiae AB1380. A total of 6932 clones were obtained containing on average ca. 350 kb DNA. The YAC library was estimated to contain six haploid genome equivalents. The YACs were examined for their chimerism by mapping both ends on an RFLP linkage map. Most YACs withEco RI fragments below 400 kb were intact colinear clones. About 40% of clones were chimeric. Genetic mapping of end clones from large size YACs revealed that the physical distance corresponding to 1 cM genetic distance varies from 120 to 1000 kb, depending on the chromosome region. To select and order YAC clones for making contig maps, high-density colony hybridization using ECL was applied. With several probes, at least one and at most ten YAC clones could be selected in this library. The library size and clone insert size indicate that this YAC library is suitable for physical map construction and map-based cloning.     

Identifying different types of de-differentiated microspores from indica-japonica F1 hybrids with subspecies-differentiating RFLP probes in rice

The indica, japonica and intermediary types of de-differentiated microspores from indica-japonica hybrids were identified with 11 subspecies-differentiating RELP probes in rice (Oryza sativa L.). The results showed that the distribution of indica, japonica and intermediary types of de-differentiated microspores could be easily detected in a simple and quick way using the RFLP method. Moreover, the microspores from the same hybrid but inoculated onto different media, or microspores from different hybrids when inoculated onto the same medium, often displayed distinctive distribution curves of de-differentiated microspores types, indicating that the media employed in this experiment had high selectivity for the de-differentiation of certain types of microspores. The application of the RELP method to de-differentiated microspore identification is of great theoretical and practical significance in rice doubled-haploid breeding. Received: 27 February 1996 / Accepted: 14 June 1996     

Comparative proteomic analysis of indica and japonica rice varieties

Indica and japonica are two main subspecies of Asian cultivated rice (Oryza sativa L.) that differ clearly in morphological and agronomic traits, in physiological and biochemical characteristics and in their genomic structure. However, the proteins and genes responsible for these differences remain poorly characterized. In this study, proteomic tools, including two-dimensional electrophoresis and mass spectrometry, were used to globally identify proteins that differed between two sequenced rice varieties (93–11 and Nipponbare). In all, 47 proteins that differed significantly between 93–11 and Nipponbare were identified using mass spectrometry and database searches. Interestingly, seven proteins were expressed only in Nipponbare and one protein was expressed specifically in 93–11; these differences were confirmed by quantitative real-time PCR and proteomic analysis of other indica and japonica rice varieties. This is the first report to successfully demonstrate differences in the protein composition of indica and japonica rice varieties and to identify candidate proteins and genes for future investigation of their roles in the differentiation of indica and japonica rice.     

RICD: A rice <Emphasis Type="Italic">indica</Emphasis>cDNA database resource for rice functional genomics

Background  

The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones.     

Enigmatic double-stranded RNA in Japonica rice

We have found a linear, 16 kb, double-stranded RNA (dsRNA) in symptomless Japonica rice (Oryza sativa L.) that is not found in Indica rice (Oryza sativa L.). The dsRNA was detected in every tissue and at every developmental stage, and its copy number was approximately constant (about 20 copies/cell). Double-stranded RNA was also detected in two strains of Oryza rufipogon (an ancestor of O. sativa). Hybridization experiments indicated that the dsRNA of O. rufipogon was homologous but not identical to that of O. sativa. The sequence of about 13.2 kb of the dsRNA was determined and two open reading frames (ORFs) were found. The larger ORF (ORF B) was more than 12 351 nucleotides long and encoded more than 4 117 amino acid residues.     

Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Summary Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.     

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.     

Differentiation of a Miniature Inverted Transposable Element （MITE） System in Asian Rice Cultivars and Its Inference for a Diphyletic Origin of Two Subspecies of Asian Cultivated Rice

In the present study, we report a survey on a Miniature Inverted Transposable Element （MITE） system known as mPing in 102 varieties of Asian cultivated rice （Oryza sativa L.）. We found that mPing populations could be generalized Into two families, mPing-1 and mPing-2, according to their sequence structures. Further analysis showed that these two families of mPing had significant bias in their distribution pattern in two subspecies of rice, namely O. sativa ssp. japonica and indica. 0. sativa japonica has a higher proportion of mPing-1 as a general trait, whereas 0. sativa indica has a higher proportion of roPing-2. We also examined the mPing system In a doubled haploid （DH） cross-breeding population of jingxi 17 （japonica） and zhaiyeqing 8 （indica） varieties and observed that the mPing system was not tightly linked to major subspecies-determining genes. Furthermore, we checked the mPing system in 28 accessions of Asian common wild rice O. rufipogon and found the roPing system in 0. rufipogon. The distribution pattern of the roPing system in O. rufipogon indicated a diphyletlc origin of the Asian cultivated rice O. sativa species. We did not find the mPing system in another 20 Oryza species. These results substantiated a previous hypothesis that O. ruflpogon and O. nivara species were the closest relatives of O. sativa and that the two extant subspecies of O. sativa were evolved independently from corresponding ecotypes of O. ruflpogon.     

RFLP variation in diploid and tetraploid alfalfa

Summary Alfalfa (Medicago sativa L.) is a major forage crop throughout the world. Although alfalfa has many desirable traits, continued breeding is required to incorporate pest resistances and other traits. We conducted this study to determine the amount of restriction fragment length polymorphism (RFLP) variability present within and between diploid and tetraploid alfalfa populations, and whether or not this variability is sufficient for construction of an RFLP map. Diploid plants from M. sativa ssp. falcata, ssp. coerulea, and ssp. sativa and tetraploid spp. sativa cultivars Apollo, Florida 77, and Spredor 2 were included. A total of 19 cDNA clones was probed onto genomic Southern blots containing DNA digested by EcoRI, HindIII, or BamHI. Phylogenetic trees were produced, based on parsimony analysis of shared restriction fragments. Evidence for extensive gene duplication was found; most probes detected complex patterns of restriction fragments. Large amounts of variation are present within all diploid subspecies. M. sativa ssp. falcata plants formed clusters distinct from ssp. sativa or ssp. coerulea plants, which were not distinctly clustered. Some M. sativa ssp. falcata plants were more similar to the other groups than to other plants within ssp. falcata. Variation among tetraploid cultivars showed that Florida 77 and Apollo had more similarities than either showed with Spredor 2. All three cultivars showed large within-population variation, with Apollo being the most diverse and Spredor 2 the least. Based on these results, development of an RFLP map at the diploid level appears possible. Also, differentiation of cultivars, particularly ones of divergent origin, seems possible based on RFLP patterns.