Genetic characterization of weedy rice (Oryza sativa L.) based on morpho-physiology, isozymes and RAPD markers

 Weedy rice (Oryza sativa L.) is an important resource for breeding and for studying the evolution of rice. The present study was carried out to identify the genetic basis of the weedy rices distributed in various countries of the world. One hundred and fifty two strains of weedy rice collected from Bangladesh, Brazil, Bhutan, China, India, Japan, Korea, Nepal, Thailand and the USA were tested for variations in six morpho-physiological characteristics and in 14 isozyme loci. Twenty six weedy strains selected from the above materials were assayed for the Est-10 locus, six RAPD loci of the nuclear genome, and one chloroplast locus. From the results of multivariate analysis based on the morpho-physiological characteristics and the isozymes, weedy rice strains were classified into indica and japonica types, and each type was further divided into forms resembling cultivated and wild rice. Thus, four groups designated as I, II, III and IV were identified. Weedy strains of group I (indica-type similar to cultivars) were distributed mostly in temperate countries, group II (indica-type similar to wild rice) in tropical countries, group III (japonica-type similar to cultivars) in Bhutan and Korea, group IV ( japonica-type similar to wild rice) in China and Korea. In group I, classified as indica, several strains showed japonica-specific RAPD markers, while some others had japonica cytoplasm with indica-specific RAPD markers in a heterozygous state at several loci. One weedy strain belonging to group II showed a wild rice-specific allele at the Est-10 locus. However, in groups III and IV, no variation was ound either for the markers on Est-10 or for the RAPD loci tested. Judging from this study, weedy rice of group I might have originated at least partly from gene flow between indica and japonica, whereas that of group II most probably originated from gene flow between wild and cultivated indica rice. Weedy rice of group III is thought to have originated from old rice cultivars which had reverted to a weedy form, and that of group IV from gene flow between japonica cultivars and wild rice having japonica backgrounds. Received: 2 May 1996 / Accepted: 30 August 1996     

Molecular breeding of a novel herbicide-tolerant rice by gene targeting

We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant rice plants generated by conventional breeding approaches that depend on spontaneous mutations. Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are superior to those produced by conventional mutation breeding protocols or transgenic technology.     

Identification of quantitative trait loci controlling rice mature seed culturability using chromosomal segment substitution lines

The genetic transformation efficiency of a rice variety is largely determined by its tissue culturability. Establishment of a highly efficient tissue-culture system has greatly accelerated the wide spread application of transgenic japonica varieties. However, such process for indica rice was hampered because this type of variety is recalcitrant to in vitro culture. This study aimed to map the quantitative trait loci (QTLs) for mature seed culturability using a chromosomal segment substitution lines (CSSL) population derived from a cross between an indica variety “Zhenshan 97B” and a japonica variety “Nipponbare”. The CSSLs consist of 139 lines each containing a single or a few introgression segments, and together covering the whole “Nipponbare” genome. Every CSSL was tested by culturing on the two medium systems developed for the respective indica and japonica parental varieties. The performance of culturability was evaluated by four indices: frequency of callus induction (CIF), callus subculture capability (CSC), frequency of plant regeneration (PRF) and the mean plantlet number per regenerated callus (MNR). All four traits displayed continuous variation among the CSSLs. With the culture system for japonica rice, three CIF QTLs, three CSC QTLs, three PRF QTLs and three MNR QTLs were detected. With the culture system for indica variety, six CIF QTLs, two CSC QTLs, three PRF QTLs and six MNR QTLs were identified, and these QTLs distributed on nine rice chromosomes. Two QTLs of CIF and two QTLs of MNR were detected in both the japonica and indica rice culture system. The correlation coefficients of all the four traits varied depending on the culture systems. These results provide the possibilities of enhancing the culturability of indica rice by marker-assisted breeding with those desirable alleles from the japonica. Lina Zhao and Hongju Zhou have contributed equally to this work.     

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.     

Quantification of total genomic DNA and selected repetitive sequences reveals concurrent changes in different DNA families in indica and japonica rice

This paper describes a fluorescence in situ hybridization (FISH) analysis of three different repetitive sequence families, which were mapped to mitotic metaphase chromosomes and extended DNA fibers (EDFs) of the two subspecies of rice (Oryza sativa), indica and japonica (2n=2x=24). The repeat families studied were (1) the tandem repeat sequence A (TrsA), a functionally non-significant repeat; (2) the [TTTAGGG]_n telomere sequence, a non-transcribed, tandemly repeated but functionally significant repeat; and (3) the 5S ribosomal RNA (5S rDNA). FISH of the TrsA repeat to metaphase chromosomes of indica and japonica cultivars revealed clear signals at the distal ends of twelve and four chromosomes, respectively. As shown in a previous report, the 17S ribosomal RNA genes (17S rDNA) are located at the nucleolus organizers (NORs) on chromosomes 9 and 10 of the indica cultivar. However, the japonica rice lacked the rDNA signals on chromosome 10. The size of the 5S rDNA repeat block, which was mapped on the chromosome 11 of both cultivars, was 1.22 times larger in the indica than in the japonica genome. The telomeric repeat arrays at the distal ends of all chromosome arms were on average three times longer in the indica genome than in the japonica genome. Flow cytometric measurements revealed that the nuclear DNA content of indica rice is 9.7% higher than that of japonica rice. Our data suggest that different repetitive sequence families contribute significantly to the variation in genome size between indica and japonica rice, though to different extents. The increase or decrease in the copy number of several repetitive sequences examined here may indicate the existence of a directed change in genome size in rice. Possible reasons for this phenomenon of concurrent evolution of various repeat families are discussed. Received: 9 August 1999 / Accepted: 29 December 1999     

Rice anther culture: callus initiation and androclonal variation in progenies of regenerated plants

Summary Anthers from rice (Oryza sativa L.) subspecies japonica initiated more callus than their indica or indica x japonica counterparts. A mild stress, either by slow desiccation or heat shock, prior to the plating of anthers enhanced the ability to initiate callus. Slow dessication of anthers enhanced the ability of the japonica anthers to initiate callus even in medium that was supplemented with NaCl. The ability to initiate callus by the anthers plated on NaCl-supplemented medium decreased as the NaCl level in the medium increased. Among the regenerated plants 2.5% were albino and another 2% were haploid. Androclonal variation for tiller numbers, shoot height, plant dry matter and flowering were noticed in the progenies of the regenerated plants.Abbreviations NAA -napthaleneacetic acid - KT Kinetin - 2,4-D 2, 4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - BAP N⁶ benzylaminopurine - NFM NaCl-free medium     

Identification of specific proteins from seed embryos by two-dimensional gel electrophoresis for the discrimination between indica and japonica rice

Summary Proteins extracted from seed embryos of 29 different cultivated rice (Oryza sativa L.) and one wild rice (O. rufipogon Griff.) were compared by two-dimensional gel electrophoresis analysis. Among more than 300 protein spots on the gel we found some interesting variations in ten spots which were individually designated as proteins A-J. Protein E was observed in all indica cultivars but was not found in those of the subspecies japonica. In contrast, protein F was only detected in japonica cultivars. Protein A existed in all japonica cultivars but, with the exception of IR-36, could not be found in other indica cultivars. Therefore, proteins A, E and F can be used as markers for the identification of indica and japonica. Some so-called Javanica cultivars showed the characteristics of japonica subspecies with regard to proteins A and F, while one other cultivar of Javanica expressed a type intermediate between indica and japonica interms of proteins A and E. One feature discriminating between Javanica and japonica cultivars was found in the D, G, and J proteins which were expressed strongly in Javanica cultivars but were scarcely expressed in those of japonica. Expression of subspecies-specific proteins E and F in f₁ hybrids was also investigated.     

Analysis of indica- and japonica-specific markers of Oryza sativa and their applications

Asian rice, Oryza sativa L., is one of the most important crop species. Genetic analysis has established that rice consists of several genetically differentiated variety groups, with two main groups, namely, O. sativa ssp. japonica kata and ssp. indica kata. To determine the genetic diversity of indica and japonica rice, 45 rice varieties, including domesticated rice and Asia common wild rice (O. rufipogon Griff.), were analyzed using sequence-related amplified polymorphism, target region amplified polymorphism, simple sequence repeat, and intersimple sequence repeat marker systems. A total of 90 indica- and japonica-specific bands between typical indica and japonica subspecies were identified, which greatly helped in determining whether domesticated rice is of the indica or japonica type, and in analyzing the consanguinity of hybrid rice with japonica, which were bred from indica and japonica crossed offspring. These specific bands were both located in the coding and non-encoding region, and usually connected with quantitative trait loci. Utilizing the indica-japonica-specific markers, japonica consanguinity was detected in sterile hybrid rice lines. Many indica-japonica-specific bands were found in O. rufipogon. This result supports the multiple-origin model for domesticated rice. Javanica exhibited a greater number of indica-japonica-specific bands, which indicates that it is a subspecies of O. sativa L.     

Mature seed-derived callus of the model indica rice variety Kasalath is highly competent in Agrobacterium-mediated transformation

We previously established an efficient Agrobacterium-mediated transformation system using primary calli derived from mature seeds of the model japonica rice variety Nipponbare. We expected that the shortened tissue culture period would reduce callus browning—a common problem with the indica transformation system during prolonged tissue culture in the undifferentiated state. In this study, we successfully applied our efficient transformation system to Kasalath—a model variety of indica rice. The Luc reporter system is sensitive enough to allow quantitative analysis of the competency of rice callus for Agrobacterium-mediated transformation. We unexpectedly discovered that primary callus of Kasalath exhibits a remarkably high competency for Agrobacterium-mediated transformation compared to Nipponbare. Southern blot analysis and Luc luminescence showed that independent transformation events in primary callus of Kasalath occurred successfully at ca. tenfold higher frequency than in Nipponbare, and single copy T-DNA integration was observed in ~40% of these events. We also compared the competency of secondary callus of Nipponbare and Kasalath and again found superior competency in Kasalath, although the identification and subsequent observation of independent transformation events in secondary callus is difficult due to the vigorous growth of both transformed and non-transformed cells. An efficient transformation system in Kasalath could facilitate the identification of QTL genes, since many QTL genes are analyzed in a Nipponbare × Kasalath genetic background. The higher transformation competency of Kasalath could be a useful trait in the establishment of highly efficient systems involving new transformation technologies such as gene targeting.     

Genetic Transformation of Rice

Rice was the first major monocot crop species to be transformed and regenerated. Initially, rice transformation was limited to japonica cultivars. Subsequently, a number of indica and javanica cultivars have also been transformed and regenerated into fertile transgenic plants. Most transformation studies in rice have used direct DNA uptake into protoplasts, induced by polyethylene glycol treatment or electroporation. Recently, other transformation methods have been developed that are less genotype dependent, such as microprojectile bombardment of cell suspensions and immature embryos. This review summarizes progress in both protoplast-based and other transformation methods.     

TOND1 confers tolerance to nitrogen deficiency in rice

Nitrogen (N), the most important mineral nutrient for plants, is critical to agricultural production systems. N deficiency severely affects rice growth and decreases rice yields. However, excessive use of N fertilizer has caused severe pollution to agricultural and ecological environments. The necessity of breeding of crops that require lower input of N fertilizer has been recognized. Here we identified a major quantitative trait locus on chromosome 12, Tolerance Of Nitrogen Deficiency 1 (TOND1), that confers tolerance to N deficiency in the indica cultivar Teqing. Sequence verification of 75 indica and 75 japonica cultivars from 18 countries and regions demonstrated that only 27.3% of cultivars (41 indica cultivars) contain TOND1, whereas 72.7% of cultivars, including the remaining 34 indica cultivars and all 75 japonica cultivars, do not harbor the TOND1 allele. Over‐expression of TOND1 increased the tolerance to N deficiency in the TOND1‐deficient rice cultivars. The identification of TOND1 provides a molecular basis for breeding rice varieties with improved grain yield despite decreased input of N fertilizers.     

Genetic diversity and classification of Oryza sativa with emphasis on Chinese rice germplasm

Despite extensive studies on cultivated rice, the genetic structure and subdivision of this crop remain unclear at both global and local scales. Using 84 nuclear simple sequence repeat markers, we genotyped a panel of 153 global rice cultivars covering all previously recognized groups and 826 cultivars representing the diversity of Chinese rice germplasm. On the basis of model-based grouping, neighbour-joining tree and principal coordinate analysis, we confirmed the widely accepted five major groups of rice cultivars (indica, aus, aromatic, temperate japonica and tropical japonica), and demonstrated that rayada rice was unique in genealogy and should be treated as a new (the sixth) major group of rice germplasm. With reference to the global classification of rice cultivars, we identified three major groups (indica, temperate japonica and tropical japonica) in Chinese rice germplasm and showed that Chinese temperate japonica contained higher diversity than that of global samples, whereas Chinese indica and tropical japonica maintained slightly lower diversity than that present in the global samples. Particularly, we observed that all seasonal, drought-tolerant and endosperm types occurred within each of three major groups of Chinese cultivars, which does not support previous claims that seasonal differentiation exists in Indica and drought-tolerant differentiation is present in Japonica. It is most likely that differentiation of cultivar types arose multiple times stemming from artificial selection for adaptation to local environments.     

A tissue culture system for different germplasms of indica rice

Agrobacterium-mediated transformation of indica rice has been manipulated in only a limited number of cultivars because the majority of indica varieties are recalcitrant to in vitro response. Establishment of a highly efficient and widely used tissue culture system for indica rice will accelerate the application of transformation technology in breeding programs and the study of the functions of indica-specific genes. By manipulating plant growth regulators, organic components and salts within the culture media, we established two media for callus induction and subculture, respectively, in tissue culture of indica rice. The modified media could guarantee the production and proliferation of a great number of embryogenic calli with high regeneration capacity from mature seeds representing different indica rice germplasms. The calli obtained from this system should be ideal material for Agrobacterium-mediated transformation. The results suggest that this optimized tissue culture system will be widely applicable for the tissue culture of indica varieties. Electronic Supplementary Material Supplementary material is available for this article at The first two authors contributed equally to this work.     

Integration of genomic tools to assist breeding in the <Emphasis Type="Italic">japonica</Emphasis> subspecies of rice

The cultivated rice (Oryza sativa L.) has two subspecies, indica and japonica. The japonica rice germplasm has a narrower genetic diversity compared to the indica subspecies. Rice breeders aim to develop new varieties with a higher yield potential, with enhanced resistances to biotic and abiotic stresses, and improved adaptation to environmental changes. In order to face some of these challenges, japonica rice germplasm will have to be diversified and new breeding strategies developed. Indica rice improvement could also profit from more “genepool mingling” for which japonica rice could play an important role. Interesting traits such as low-temperature tolerance, and wider climate adaptation could be introgressed into the indica subspecies. In the past decade, huge developments in rice genomics have expanded our available knowledge on this crop and it is now time to use these technologies for improving and accelerating rice breeding research. With the full sequence of the rice genome, breeders may take advantage of new genes. Also new genes may be discovered from the genepool of wild relatives, or landraces of the genus Oryza, and incorporated into elite japonica cultivars in a kind of “gene revolution” program. Expectedly, new technologies that are currently being optimized, aiming for novel gene discovery or for tracking the regions under selection, will be suggested as new breeding approaches. This paper revisits breeding strategies successfully employed in indica rice, and discusses their application in japonica rice improvement (e.g. ideotype breeding, wide hybridization and hybrid performance).     

Better performance of germination in hyperosmotic solutions in conspecific weedy rice than cultivated rice

Weeds and crops that grow together often confront similar types of environmental stress, especially drought stress. Weedy rice (Oryza sativa f. spontanea) and cultivated rice (O. sativa L.) provide a unique pair consisting of a weed and a conspecific model crop that can be used to study the drought tolerance of plants across a large distributional range. The investigation on weedy rice's damage to paddy fields showed that it was more serious in dry direct seeding than water direct seeding. Compared with water direct seeding, the seeds of cultivated rice and weedy rice in dry direct seeding will absorb water and germinate under the condition of insufficient soil moisture. Our hypothesis is that weedy rice seeds have evolved stronger germination ability than coexisting cultivated rice under water stress, so that they can obtain more growth space in the early stage in dry direct seeding and thus obtain higher fitness. Seeds of weedy rice populations and coexisting rice cultivars were collected from 61 sites across China and were germinated with 20% polyethylene glycol‐6000 to simulate drought stress. Two drought response indices, which assessed germination rate and germination index, plus one germination stress tolerance index, indicated significantly greater drought tolerance in weedy rice populations than in coexisting rice cultivars (P < 0.01). Drought tolerance for the three indexes were indica weedy rice > indica rice cultivars, japonica weedy rice > japonica rice cultivars, and indica weedy rice > japonica rice cultivars. These results indicate that weedy rice populations show stronger drought stress tolerance than coexisting rice cultivars at various sites, specifically during the seed germination period. Furthermore, Pearson's correlation found that drought response of weedy rice populations and coexisting rice cultivars were significantly different with these environmental factors: latitude, altitude, annual mean precipitation, mean annual temperature, mean precipitation in the sowing month, mean temperature in the sowing month, and sowing methods. Weedy rice shows different patterns of drought tolerance variation across geographical (latitude and altitude) and environmental (precipitation) gradients compared to coexisting rice cultivars. This study suggests that weedy rice might have evolved new drought tolerance and could provide a useful source of genetic resources for improving drought tolerance of crop cultivars and breeding direct seeded cultivars to reduce the usage of seeds in direct seeding.     

Genetic variation detected with RAPD markers among upland and lowland rice cultivars (Oryza sativa L.)

Genetic variation of nine upland and four lowland rice cultivars (Oryza sativa L.) was investigated at the DNA level using the randomly amplified polymorphic DNA (RAPD) method via the polymerase chain reaction (PCR). Forty-two random primers were used to amplify DNA segments and 260 PCR products were obtained. The results of agarosegel electrophoretic analysis of these PCR products indicated that 208 (80%) were polymorphic. All 42 primers used in this experiment were amplified and typically generated one-to-four major bands. Only two primers showed no polymorphisms. In general, a higher level of polymorphism was found between japonica and indica subspecies while fewer polymorphisms were found between upland and lowland cultivars within the indica subspecies. A dendrogram that shows the genetic distances of 13 rice cultivars was constructed based on their DNA polymorphisms. Classification of rice cultivars based on the results from the RAPD analysis was identical to the previous classification based on isozyme analysis. This study demonstrated that RAPD analysis is a useful tool in determining the genetic relationships among rice cultivars.     

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.     

Identification of SNPs in the<Emphasis Type="Italic"> waxy</Emphasis> gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice

Common non-waxy (Wx) rice cultivars contain two different alleles at the waxy locus, designated Wx ^a and Wx ^b, which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wx ^a allele was predominant in non-waxy indica cultivars, whereas the Wx ^b allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wx ^a from Wx ^b have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AGGT to AGTT) at a donor site of the first intron within the Wx gene. In the case of waxy (wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wx ^a or Wx ^b alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wx ^a allele (AGGT) but not in the Wx ^b allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wx ^a- and Wx ^b-derived alleles, but that the Wx ^b-derived allele was predominant, and its distribution was independent of indica-japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples.Communicated by J. Heslop-Harrison     

Origin of cytoplasm substituted rice cultivars found in Japan

Genetic variation of Japanese rice cultivars were examined. Five of 450 lowland cultivars and another five of 200 upland cultivars were determined as the indica type by using isozyme genotypes and the remainder were of the japonica type. The major characteristics of these indica cultivars, revealed a slender shape of grains, a short apiculus hair length, a positive allele for Ph reaction, and allele-3 for the Pgd1 locus. Three of these indica cultivars showed a non-deletion ORF100, which is essential to the japonica-type plastid. The plastid subtype identity (PS-ID) sequences of these plastids is 6C7A, which is also a japonica-specific repeat unit. Thus, these cultivars were concluded to be naturally generated cytoplasm substituted lines. These plastids were introduced into a indica genetic background from japonica cultivars grown elsewhere. The rest of the indica cultivars revealed a deletion-type ORF100 and plastid subtype 8C8A, both of which are indica-specific. These cultivars carried indica-type allelic constitutions for diagnostic isozyme loci. However, other characters were identical to the cytoplasm-substituted cultivars in Japan. In East and Southeast Asia, cultivars carrying a indica-type nuclear genotype with a japonica-type plastid are restricted to Aus cultivars in the Bengal region. Genetic and historical records suggest that Japanese indica cultivars and the Aus cultivars are closely related. The Aus cultivars acquire necessary genetic constitutions from both indica and japonica cultivars through naturally occurring out-crossing to adapt to a particular cultivation condition in the region. The wide adaptability enabled them to be introduced into a northern region like Japan.