Research Progress on Genetic Diversity and Its Ecological Conservation of Endangered Dongxiang Wild Rice (Oryza rufipogon Griff.)

It is of great importance for the analysis on genetic evolution mechanism, and rice breeding utilization by estimation and protection of genetic diversity of pre- sent DXWR （Oryza rufipogon Griff.）. This study has summarized genetic diversity and protection achievements of DXWR from many aspects like varied conservations, years, generations, other species of wild rice and cultivated rice. In this study, a u- nified and scientific evaluation system of genetic diversity is encouraged to be built and suggestions on research prospect and ecological conservation of DXWR genetic diversity have been proposed.

Research on Flowering Habits and Fertility of Gaozhou Common Wild Rice (Oryza rufipogon) in Lingshui of Hainan Province

[Objective] The paper was to explore the flowering habits and fertility of Gaozhou common wild rice （Oryza rufipogon Griff.） in Lingshui of Hainan Province. [Method] The flowering habits, pollen fertility and seed setting rate of Gaozhou common wild rice in Lingshui of Hainan Province were studied, and compared with the flowering habits of common wild rice in Hainan Province. [Result] Gaozhou common wild rice was able to flower twice a year. The first flowering stage was from mid-April to mid-June for about 60 d, and the daily peak flowering dates were from May 13 to May 22. The daily flowering time was mainly between 11：20 and 13：00, and the peak flowering was at 11：30. The second flowering stage was from late August to late October for about 45 d, and the peak flowering dates lasted from September 8 to September 18. The daily flowering time was mainly from 11：15 to 12：50, and the daily peak flowering was at 11：30. In both flowering stages, the panicles had the same flowering characteristics. The stigma exsertion reached a high value of 95.37% with single （46.23%） and dual （49.14%） stigmas exsertion respectively. A spikelet remained open for 1.5-2.5 h, the angle of spikelet opening varied from 29.9° to 32.4°. At the two flowering stage the average pollen fertility rates were 89.67% and 85.62%, the average seed setting rates were 58.81% and 55.79%, respectively. Hainan population flowered only once （from mid-October to mid-December）, this had significant difference with the performance of Gaozhou common wild rice which flowered twice a year. [Conclusion] The results provided basis for the research on gene flow frequency from transgenic rice to wild rice and its relationship with flowering habits of common wild rice.

Irrigation regimes modulate non-structural carbohydrate remobilization and improve grain filling in rice(Oryza sativa L.)by regulating starch metabolism

Recently developed ‘super’ rice cultivars with greater yield potentials often suffer from the problem of poor grain filling, especially in inferior spikelets. Here, we studied the activities of enzymes related to starch metabolism in rice stems and grains, and the microstructures related to carbohydrate accumulation and transportation to investigate the effects of different water regimes on grain filling. Two ‘super’ rice cultivars were grown under two irrigation regimes of well-watered(WW) and alternate wetting and moderate soil drying(AWMD). Compared with the WW treatment,the activities of ADP glucose pyrophosphorylase(AGPase), starch synthase(StSase) and starch branching enzyme(SBE), and the accumulation of non-structural carbohydrates(NSCs) in the stems before heading were significantly improved, and more starch granules were stored in the stems in the AWMD treatment. After heading, the activities of α-amylase, β-amylase, sucrose phosphate synthase(SPS) and sucrose synthase in the synthetic direction(SSs)were increased in the stems to promote the remobilization of NSCs for grain filling under AWMD. During grain filling, the enzymatic activities of sucrose synthase in the cleavage direction(SSc), AGPase, StSase and SBE in the inferior spikelets were increased, which promoted grain filling, especially for the inferior spikelets under AWMD.However, there were no significant differences in vascular microstructures. The grain yield and grain weight could be improved by 13.1 and 7.5%, respectively, by optimizing of the irrigation regime. We concluded that the low activities of key enzymes in carbon metabolism is the key limitation for the poor grain filling, as opposed to the vascular microstructures, and AWMD can increase the amount of NSC accumulation in the stems before heading, improve the utilization rate of NSCs after heading, and increase the grain filling, especially in the inferior spikelets, by altering the activities of key enzymes in carbon metabolism.

Progress on Transferring Elite Genes from Non-AA Genome Wild Rice into Oryza sativa through Interspecific Hybridization

The progress of research on transferring elite genes from non-AA genome wild rice into Oryza sativa through interspecific hybridization are in three respects, that is, breeding monosomic alien addition lines （MAALs）, constructing introgression lines （ILs） and analyzing the heredity of the characters and mapping the related genes. There are serious reproductive barriers, mainly incrossability and hybrid sterility, in the interspecific hybridization of O. sativa with non-AA genome wild rice. These are the ＇bottleneck＇ for transferring elite genes from wild rice to O. sativa. Combining traditional crossing method with biotechnique is a reliable way to overcome the reproductive barriers and to improve the utilizing efficiency of non-AA genome wild rice.

Identification and Genetic Analysis of Fertility Restoration Ability in Dongxiang Wild Rice (Oryza rufipogon)

Two populations of Dongxiang wild rice （Oryza rufipogon）, Shuitaoshu and Dongtangshang, were crossed with five male sterile rice lines with different cytoplasmic backgrounds （B06S, Zhenshan 97A, Xieqingzao A, Zhong 9A, and Yuetai A）, and the seed setting rate of the F1 was used to judge the fertility restoration ability in the Dongxiang wild rice. With P1, F1, P2, and F2 populations as materials, the Akaike＇s Information Criterion （AIC） was used to identify the major genes affecting quantitative traits, and when the major genes existed, the genetic effects of the major gene and the polygene and their genetic variance were estimated through segregation analysis. The seed setting rates of the F1 generation varied from 45.98% to 76.57%, suggesting that the Dongxiang wild rice had the fertility restoration ability. One major gene plus polygene mixed inheritance model was the most fitted genetic model for this trait in all the F2 populations. The heritability values of the major genes varied from 56.63% to 88.29% and those of the polygenes from 2.74% to 30.97%, and the total heritability values were from 63.17% to 94.01%. The major gene inheritance of the combination Zhong 9A/Dongtangshang was controlled by the additive effect without dominant effect, and the other nine combinatfons were by the completely dominant inheritance.

Development of Novel Cytoplasmic Male Sterile Source from Dongxiang Wild Rice(Oryza rufipogon)

This study was conducted to develop and characterize a novel cytoplasmic male sterile （CMS） source which was identified from Dongxiang wild rice （Oryza rufipogon） by crossing Dongxiang wild rice as female with Zhongzao 35, an indica inbred variety, as male and continuous backcrossing with Zhongzao 35. Observation under optical microscope manifested that this novel CMS belonged to typical abortion type with less pollen compared with wild abortive type cytoplasm （CMS-WA）. Sequential planting showed that this novel CMS has complete and stable male sterility. Testcross experiment showed that all the 24 tested materials including maintainer and restorer lines of CMS-WA and Honglian type cytoplasm （CMS-HL） and other indica inbred varieties are the maintainers with complete maintaining ability, suggesting that this novel CMS has fertility restoration totally different from CMS-WA and CMS-HL and belongs to a novel type of CMS. So far, we only discovered a unique fertility restoration source for this novel CMS. Inheritance analysis showed that the fertility restoration of this CMS was governed by three pairs of independent dominant genes. Prospect for application of this novel CMS system in hybrid rice breeding was also discussed.

Genetic Analysis on Characteristics to Measure Drought Resistance Using Dongxiang Wild Rice(Oryza rufupogon Griff.) and Its Derived Backcross Inbred Lines Population at Seedling Stage

Drought stress is one of the major constraints to rice （Oryza sativa L.） production and yield stability especially in rainfed ecosystems and is getting worse as the climate changes worldwide. Dongxiang wild rice （DXWR） Oryza rufipogon Griff., contains drought resistant gene. Improving drought resistance of cultivars is crucial to increase and stabilize rice grain yield via transferring resistant gene from species related to rice. In this paper, four upland rice, sixty backcross inbred lines （BILs） derived from BC1F5 of R974//DXWR/R974, and their parents were employed to evaluate drought-resistance at seedling stage in the greenhouse. Nine traits were recorded for assessment of drought resistance, including maximum root length （MRL）, number of roots （NR）, shoot length （SL）, dry root weight （DRW）, fresh root weight （FRW）, root relative water content （RRWC）, leaf relative water content （LRWC）, level for rolling leaf （LRL）, and seedling survivability under repeat drought （SSRD）. Using more than 88% of accumulative contribution resulted from the principal component analysis （PCA）, the nine traits were classified into five independent principal components and the line 1949 showed the highest resistance. Analysis on the stepwise regression equation and correlation demonstrated that MRL, RN, FRW, and RRWC significantly influenced the drought resistance, thus could be used as comprehensive index for drought resistance at the seedling stage. Using the major gene plus polygene mixed inheritance model of quantitative traits, the inheritance of drought-resistance of BIL population at seedling stage was mostly controlled by two independent genes plus polygene. As a result, the DXWR could be precious resources for genetic improvement of drought resistance in cultivated rice.

Novel QTLs from Wild Rice Oryza longistaminata Confer Strong Tolerance to High Temperature at Seedling Stage

Global warming poses a threat to rice production.Breeding heat-tolerant rice is an effective and economical approach to address this challenge.African rice is a valuable genetic resource for developing heat-tolerant crops due to its intricate mechanism for adapting to high temperatures.Oryza longistaminata,a widely distributed wild rice species in Africa,may harbor an even richer gene pool for heat tolerance,which remains untapped.In this study,we identified three heat tolerance QTLs from O.longistaminata at the seedling stage,including novel heat tolerance loci qTT4 and qTT5.Our findings demonstrated that the O.longistaminata alleles for these two QTLs can enhance the heat tolerance of rice seedlings.Remarkably,qTT5 was mapped to a region spanning approximately 287.2 kb,which contains 46 expressing genes.Through the analysis of Gene Ontology and expression differences under heat induction,we identified four candidate genes.Our results lay the foundation for discovering heat tolerance genes underlying O.longistaminata and developing new genetic resources for heat-tolerant rice breeding.

Combining QTL mapping and expression profile analysis to identify candidate genes of cold tolerance from Dongxiang common wild rice(Oryza rufipogon Griff.)

Rice（Oryza sativa L.）, a tropical and subtropical crop, is susceptible to low temperature stress during seedling, booting, and flowering stages, which leads to lower grain quality levels and decreasing rice yields. Cold tolerance is affected by multiple genetic factors in rice, and the complex genetic mechanisms associated with chilling stress tolerance remain unclear. Here, we detected seven quantitative trait loci（QTLs） for cold tolerance at booting stage and identified one cold tolerant line, SIL157, in an introgression line population derived from a cross between the indica variety Guichao 2, as the recipient, and Dongxiang common wild rice, as the donor. When compared with Guichao 2, SIL157 showed a stronger cold tolerance during different growth stages. Through an integrated strategy that combined QTL-mapping with expression profile analysis, six candidate genes, which were up-regulated under chilling stress at the seedling and booting developmental stages, were studied. The results may help in understanding cold tolerance mechanisms and in using beneficial alleles from wild rice to improve the cold tolerance of rice cultivars through molecular marker-assisted selection.

The Magnaporthe oryzae effector Avr-PikD suppresses rice immunity by inhibiting an LSD1-like transcriptional activator

Avirulence effectors(Avrs),encoded by plant pathogens,can be recognized by plants harboring the corresponding resistance proteins,thereby initiating effector-triggered immunity(ETI).In susceptible plants,however,Avrs can function as effectors,facilitating infection via effector-triggered susceptibility(ETS).Mechanisms of Avr-mediated ETS remain largely unexplored.Here we report that the Magnaporthe oryzae effector Avr-PikD enters rice cells via the canonical cytoplasmic secretion pathway and suppresses rice basal defense.Avr-PikD interacts with an LSD1-like transcriptional activator AKIP30 of rice,and AKIP30 is also a positive regulator of rice immunity,whereas Avr-PikD impedes its nuclear localization and suppresses its transcriptional activity.In summary,M.oryzae delivers Avr-PikD into rice cells to facilitate ETS by inhibiting AKIP30-mediated transcriptional regulation of immune response against M.oryzae.

Molecular identification of the cytoplasmic male sterile source from Dongxiang wild rice(Oryza rufipogon Griff.)

The use of cytoplasm male sterility （CMS） is crucial for three-line hybrid seed production. Two types of CMS have been discovered from Dongxiang wild rice, namely the wild abortion type CMS （CMS-WA） and the Dongxiang wild type CMS （CMS-DW）. In this study, we show the molecular identification of the two types of CMS in Dongxiang wild rice. WA352, which conferred CMS-WA, was not detected in Dongxiang wild iice, implying Dongxiang wild rice does not carry the CMS- WA source. Further analysis of WA352 in DYIA, a CMS-DW line, by PCR amplification and sequencing, revealed two insertion-deletion polymorphisms occurred in CMS-DW compared to WA352 of CMS-WA. It was reported that WA352 was comprised of an unknown origin sequence and partial sequences of three open reading frames （ORFs）, orf284, orf224 and orf288. The 42-bp insertion was located between the two segments of orf224 and orf288, which created a new chimeric ORF, orf216. This new ORF was also detected in CMS-HL. Based on the 9-bp deletion in orf284, a specific mitochon- drial marker of DW-M1 was developed, which could be used to specifically distinguish the DW-type source. Moreover, semi-quantitative RT-PCR analysis preliminarily suggested that both orf216 and orf284 could be considered as candidates for CMS-DW. These findings present a preliminary understanding of CMS-DW at the molecular level.

The auxin transporter OsAUX1 regulates tillering in rice(Oryza sativa)

Tillering is an important agronomic trait of rice(Oryza sativa)that affects the number of effective panicles,thereby affecting yields.The phytohormone auxin plays a key role in tillering.Here we identified the high tillering and semi-dwarf 1(htsd1)mutant with auxin-deficiency root characteristics,such as shortened lateral roots,reduced lateral root density,and enlarged root angles.htsd1 showed reduced sensitivity to auxin,but the external application of indole-3-acetic acid(IAA)inhibited its tillering.We identified the mutated gene in htsd1 as AUXIN1(OsAUX1,LOC_Os01g63770),which encodes an auxin influx transporter.The promoter sequence of OsAUX1 contains many SQUAMOSA PROMOTER BINDING PROTEIN-LIKE(SPL)binding sites,and we demonstrated that SPL7 binds to the OsAUX1 promoter.TEOSINTE BRANCHED1(OsTB1),a key gene that negatively regulates tillering,was significantly downregulated in htsd1.Tillering was enhanced in the OsTB1 knockout mutant,and the external application of IAA inhibited tiller elongation in this mutant.Overexpressing OsTB1 restored the multi-tiller phenotype of htsd1.These results suggest that SPL7 directly binds to the OsAUX1 promoter and regulates tillering in rice by altering OsTB1 expression to modulate auxin signaling.

Genetic Diversity of Dongxiang Wild Rice(Oryza rufipogon Griff.)Detected by SSLP Markers

The genetic diversity of Dongxiang wild rice populations was evaluated by using 14 SSR primer pairs. Twelve of the primer pairs amplified polymorphic bands. A total of 70 polymorphic DNA bands were generated with average 5. 83 polymorphic bands per primer pair, indicating that the genetic diversity of Dongxiang wild rice was high. Cluster analysis showed that there were highly variant individuals within and among populations, while others were highly similar to each other. The within-population genetic distances were 0. 23 - 0.47, being smaller than 0.40 - 0.55 of among-population. Strict management must be taken for the preservation of Dongxiang wild rice.

Genetic Homozygosis of Resistance of Common Wild Rice (Oryza rufipogon) against Brown Planthopper

[ Objective] The stability of resistance heredity of common wild rice against brown planthopper was studied to screen wild rice germplasm with stable resistance, so as to provide resources for rice breeding with resistance against brown planthopper. [ Method] A total of 1 591 accessions of common wild rice, collected from 46 proterozoic growth locations of Guangxi, were screened to analyze resistance against brown planthopper, and genetic homozygosis in their offspring was detected using bag-selfing or anther culture methods. [ Result] The results showed that most of common wild rice germplasm were heterozygotic, and growth habit and awn of their first self-bred generation showed segregation. Among 1 591 accessions of common wild rice, only 30 accessions showed third to fifth grades of resist- ance against brown planthopper, and most of them showed fifth grade of resistance. Among selfing offspring of 11 resistant materials, the populations from Z1 to Z3 showed segregation of resistant genes, while some materials showed stable resistance in Z4. Out of 11 accessions of materials, resistance of five accessions increased from third grade to first, second and third grades; four accessions increased from fifth grade to first, second and third grades ; two accessions increased from fifth to third grade. Anther culture results showed that 125 accessions of callus from independent origins were obtained after induction by in vitro culture of 6 000 anthers; eight clusters of diploid and two clusters of haploid green seedlings were differentiated, with induction rate of callus as 2.0% and differentiation rate of green seed- lings as 6.4%. Out of eight offspring of anther culture, six showed fifth grade of resistance against brown planthopper and two showed third grade of resistance. [ Conclusion] The stable highly-resistant resources could be obtained and resistance level of common wild rice could be enhanced through bag-selfing and multigeneration identification of resistance against brown planthopper. Anther culture is beneficial to promote genetic homozygosis of resistance of common wild rice against brown planthopper and shorten the screening time.

Improvement of Wild Rice Oryza Longistaminata through Mutation Induction

Influence of a mutation to improve the undesirable traits （shattering, red caryopsis etc.） of wild rice O. longistaminata while preserving its useful genes by radiation 20 Kr gamma rays from 60Co was studied. The mutants issued this irradiation were crossed with the interspecific variety SIK385-b-42-28-28 （O. glaberrima x O. sativa）. Irradiation followed by crossing with interspecific variety generated a large genetic variability, in the subsequent generations, in plant height, maturity, non-shattering grain, kernel colour, spikelets fertility, panicle length, and grain size. This has resulted in identification of promising mutants which possess all the traits of cultivated rice O. sativa （white kernel, non-shattering grain, secondary branchies etc.）. During 2012 rainy season, nineteen selected mutants from M5 and M7 generations were evaluated for yield potential in replicated trials at Longorola station.

<i>In Situ</i>Conservation of Wild Rice Populations: A Targeted Study of Common Wild Rice <i>Oryza rufipogon</i>from China

Although great accomplishments of in situ conservation have been made during the last decade throughout the world, there is an urgent need to conduct more targeted research to explore many basic questions about crop wild relatives (CWR) in situ conservation such as how to better identify and manage the target in situ conservation populations of CWR. In this study, we performed an extensive field investigation of 201 natural populations or habitats of O. rufipogon, a seriously endangered wild progenitor of cultivated rice in China. Our updated information suggests that: 1) the majority of the natural populations have been extinct throughout China, which leads to serious fragmentation of the population system as a whole;and 2) the survived populations have become small in size and thus fragmented within the population as a result of the loss of subpopulations. To assess the relationships between the biodiversity education and conservation of wild rice, we employed participatory approaches in the field investigation. Our data indicate that the continuous rapid decline of biodiversity education seemed closely related to the extinction of wild rice germplasm. These findings imply the potential necessity and huge challenge for making in situ conservation plans in the future. By means of our understanding of ecogeography, sociology and culture, and population genetics of the species, we propose a strategy for selecting in situ conservation locations as well as priority sites for establishing in situ conservation practices in China.

Identification of quantitative trait loci and candidate genes associated with ABA sensitivity in common wild rice (Oryza rufipogon Griff,)

Abscisic acid （ABA）, as one of the foremost signaling molecules in plants, is an important hormone which plays versatile functions in regulating developmental process and adaptive stress process. A set of introgression lines were previously generated via a backcrossing program using an elite indica cultivar rice Teqing （O. sativa L.） as recipient and an accession of Yuanjiang common wild rice （O. rufipogon Griff.） as donor. In this study, the previously developed introgression lines were evaluated for ABA sensitivity. Here we reported that a total of 14 quantitative trait loci （QTLs） associated with ABA sensitivity were identified. An ABA sensitive introgression line, YIL53, was identified and characterized. Physiological characterization, including chlorophyll content, malondialdehyde content, soluble sugar content, and stomata movement, demonstrated that YIL53 exhibited the characteristics associated with ABA sensitivity. Genotypic analysis revealed that YIL53 harbored one QTL related to ABA sensitivity, qASS1-2, which was located on chromosome 1 within one introgressed segment derived from the Yuanjiang common wild rice. Furthermore, the qASS1-2 was finally narrowed down to a 441-kb region between simple sequence repeats （SSR） marker RM212 and single nucleotide polymorphism （SNP） marker M3 using the segregation population derived from the cross between Teqing and YIL53, and three candidate genes associated with ABA sensitivity were identified using a strategy combined gene expression analysis with QTL mapping. Identification of the QTLs related to ABA sensitivity and characterization of the ABA sensitive line YIL53 would provide a helpful basis for isolating novel genes related to ABA sensitivity.

Identification of Quantitative Trait Loci Controlling Floral Morphology of Rice Using a Backcross Population between Common Cultivated Rice, <i>Oryza sativa</i>and Asian Wild Rice, <i>O. rufipogon</i>

Differences in floral morphologies affect pollination behaviour in many flowering plants. In the genus Oryza, several differences in the size of floral organs are known. In this study, we focused on the differences in the size of floral organs between common cultivated rice, Oryza sativa L. and its wild ancestor, O. rufipogon. We compared floral morphologies between cultivated rice O. sativa cv. Nipponbare and O. rufipogon W630. We first evaluated temporal changes in filament and anther lengths. W630 had longer filaments with rapid elongation within 15 min after spikelet opening. W630 also had longer anthers than Nipponbare, and size of anther was consistent throughout all time examined. We also analysed other six floral traits, and found that W630 had higher stigma and style length, as well as lemma and palea length, but lower lemma and palea width. Quantitative trait locus (QTL) analysis was performed to identify the loci controlling these floral traits, using backcross recombinant inbred lines derived from a cross between Nipponbare and W630. A total of 11 significant QTLs were identified. Of these, two pairs of QTLs for lemma and palea length and one pair for lemma and palea width overlapped, suggesting that common genetic factors may be the reason for the differences in these traits. In addition, we performed QTL analysis for grain size, and found that QTLs for grain size coincided with those for lemma and palea size, indicating that grain size is partly controlled by glume capacity. The QTLs identified in this study will be informative for understanding genetic changes associated with rice domestication.

Developmental Change of Vegetative Plant Architecture of Annual-Form-Wild Rice (<i>Oryza rufipogon</i>Griff.) Elevates Competitive Ability during the Late Development under a Dense Condition

Annual-form-wild (AFW) rice developmentally changes the vegetative plant architecture from flat to elect shape. The main objective of the present study is to suggest that the change might be related to competitive ability (CA). Recombinant inbred lines (RILs) derived from a cross between an AFW rice and a cultivar were grown under high-density (H) and low-density (L) conditions in a weedy paddy field. At two and three months after transplanting (MATs), we evaluated the two traits associated with utilization of light resource, tiller angle (TA) and actual plant height (APH), and amount of the growing weeds using an index, weed-denseness score (WDS). Then, DC-TA, DC-APH and SC-WDS was defined as the amount of change in TA, APH, and WDS from two to three MATs, respectively. Multiple regression analysis revealed that only DC-TA significantly affected SC-WDS specific under H condition that is relatively similar to habitats of AFW rice. Quantitative trait locus (QTL) analyses identified that one of the two QTLs associated with DC-TA shared a linked molecular marker with a QTL associated with SC-WDS only under H condition. Further, RILs with AFW-rice-derived allele on the QTL associated with DC-TA tended to show drastic changes of TA toward narrow angle as well as large decreases of weed amount. All the QTLs associated with DC-APH were not linked to the QTL associated with SC-WDS under H condition. Therefore, it is suggested that under the crowded conditions during the late plant development, AFW-rice plants might not survive through canopy domination but elevate CA through increases of efficiency of capturing light due to changes from wide to narrow tiller angle. Since flat-plant shape of AFW rice during the early development would have been evolved by trampling pressures, the present finding suggest that different selective pressures in life time might have cooperatively evolved the developmental change.

ITS1 SEQUENCES OF NUCLEAR RIBOSOMAL DNA IN WILD RICES AND CULTIVATED RICES OF CHINA AND THEIR PHYLOGENETIC IMPLICATIONS

The first internal transcribed spacer(ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced with automated fluorescent sequencing. The sequences of ITS1 ranged from 193 bp to 218 bp in size and G/C content varied from 69.3% to 72.7%. In pairwise comparisons among the five taxa, sequence site divergence ranged from 1.5% to 10.6%. Phylogenetic analysis of ITS1 sequences using Wagner parsimony generated a single well resolved tree, which revealed that Oryza rufipogon was much more closely related to cultivated rice species than to the other two wild species. Oryza granulata was less closely related to either cultivated rice species or the other two wild species, and might be a unique and isolated taxon in the genus Oryza. The phylogenetic relationships of the three wild rice species and two cultivated rice subspecies inferred from ITS1 sequences is highly concordant with those based on the molecular evidence from isozyme, chloroplast DNA (cpDNA), mitochondrial DNA(mtDNA) and nuclear DNA (nDNA) of the genus Oryza .