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.

Physiological Analysis on Mechanisms of Cold-tolerance of Dongxiang Wild Rice(Ⅰ)

[Objective] The research aimed to study the effects of soluble substance concentration in cell and the scavenging activity of active oxygen radicals on the cold-tolerance physiology of Dongxiang wild rice.[Method] Dongxiang wild rice was used as materials to detect and compare the content of soluble sugar,free proline and soluble protein,POD activity,CAT activity,MDA content in the leaves in different growth periods.Using with control of two species of common cultivated rice Xieqingzao and Nongda 288.[Resul...

Studies on the Microbial Community in the Rhizosphere Soil of Dongxiang Wild Rice

[Objective] The aim was to study the characteristics of microbial community in the rhizosphere soil of Dongxiang wild rice（Oryza rufipogon Griff.）.[Method] The microbial biomass carbon and nitrogen was estimated by the chloroform fumigation extraction method;the microbial community composition and Nitrogen cycling microbial functional groups were investigated by the Dilution plate culture method and the most probable number methods respectively.[Result] The microbial biomass carbon and nitrogen,in rhizosphere soil of Dongxiang Wild Rice was 83.02±18.23 mg/kg soil and 16.98±2.54 mg/kg soil,which was lower than that of ordinary cultivated rice;The relationship between the number of culturable microbial groups was bacteriaactinomycetesfungi,and the Nitrogen cycling microbial physiological groups was as the following：ammonifying bacteriaaerobic azotobacteriadenitrobacteriaanaerobic azotobacterianitrobacterianitrosobacteria.[Conclusion] The microbial community in the rhizosphere soil of Dongxiang Wild Rice was different from that of the ordinary cultivated rice.

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 Progress on the Protection of Wild Rice Resources in China

[Objective] The research aimed to summarize the research progresses on the protection of wild rice resources in China in recent 20 years.[Method] The protection effects of wild rice by using the methods of in situ protection,ectopic protection,the combination of in situ protection and ectopic protection were expounds,the research progresses on the protection of wild rice resources at present were summarized.[Result] Some scholars thought that in situ protection was an effective way for the protection of wil...

Mapping QTLs for Aluminum Tolerance in Introgression Lines of Wild Rice at Seedling Stage

In this study, wild rice chromosome segment substitution lines bred by Yuanjiang common wild rice and elite indica rice variety Teqing were used as ex-perimental materials to detect aluminum tolerancerelated QTLs based on the phe-notypic data of indoor and outdoor plant height inhibition rate and dry weight inhibi-tion rate at seedling stage. According to experimental results, 11, 18, 14 and 5 QTLs related with aluminum tolerance were detected respectively on different chro-mosomes. Analysis of phenotypic data of indoor and outdoor plant height inhibition rate indicated that QTLs around RM38 on chromosome 8 and RM277 chromosome 12 exhibited the highest contribution rates （12% and 11%）, belonging to major QTLs. Analysis of phenotypic data of indoor and outdoor dry weight inhibition rate indicated that the highest contribution rate was only 9% and 8%; therefore, no major QTL was detected. QTLs detected repeatedly were located on chromosomes 7, 8, 9, 11 and 12, respectively. Two QTLs were located on chromosome 8. QTLs around RM310 were detected three times and other QTLs were detected twice, which were identified as stable QTLs.

The Taxonomy and the SEM Observation on Tubercles on Lemma of Yihuang Wild Rice

To distinguish the taxonomy of Yihuang wild rice found in Yihuang County, Jiangxi Province, China in 1999, the botanical characteristics of Yihuang wild rice were retrieved by genus, and species; at the same time, the tubercle characters on lemma in Yihuang wild rice, O. rufipogon, 0. officinalis, 0. meyeriana, and O. granulata, were also observed by scanning electron microscopy. The retrieval result by genus showed that Yihuang wild rice is Leersia Soland. Ex Swartz., not Oryza L., Hygroryza Nees and Chikusichloa Koidz. The retrieval result by species showed that Yihuang wild rice is L. sayanuka Ohwi, not L. hexandra Swartz., L. japonica （Makino） Honda and L. oryzoides （L.） Swartz. The results of the SEM observation on tubercles on lemma showed that the tubercle characters on lemma of Yihuang wild rice belongs to ＂inlaid- silica-peak＂ type, which is different from the three types of wild rice existing in China, O. rufipogon, O. officinalis, O. Meyeriana, and also O. granulata, but is similar to L. tisseranti, thereby gives a proof of the above retrieval results. It could be concluded that Yihuang wild rice does not belong to Oryza L., but to Leersia sayanuka Ohwi of Leersia Soland. Ex Swartz.

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.

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.

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.

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.

Systematic Characterization of Long Non-Coding RNAs and Their Responses to Drought Stress in Dongxiang Wild Rice

Long non-coding RNAs(lncRNAs) play important roles in response to various biotic and abiotic stresses. So far, systematic identification and characterization of lncRNAs have been reported in a few model plant species and major crops, but their roles in abiotic stress response have not yet been reported in common wild rice(Oryza rufipogon). Dongxiang wild rice(DXWR) possesses a high degree of drought resistance and has been well recognized as a precious genetic resource for drought resistant rice breeding. We presented the reference catalog of 1 655 novel lncRNA transcripts in DXWR using strand-specific RNA sequencing and bioinformatics approaches. Meanwhile, a total of 1092 lncRNAs were determined as differentially expressed lncRNAs under drought stress. Quantitative real-time PCR results exhibited a high concordance with RNA sequencing data, which confirmed that the expression patterns of lncRNAs based on RNA sequencing were highly reliable. Furthermore, 8 711 transcripts were predicted as target genes of the differentially expressed lncRNAs. Functional annotation analysis based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases showed that the target genes were significantly enriched in cellular and metabolic processes, cell part, binding and plant hormone signal transduction, as well as many other terms related to abiotic stress resistance. These results expanded our understanding of lncRNA biology and provided candidate regulators for genetic improvement of drought resistance in rice cultivars.

Genetic Diversity of Wild Rice Species in Yunnan Province of China

Yunnan Province of China is one of the important centers for origin and evolution of cultivated rice worldwide. Wild rice is the ancestor of the cultivated rice. Many elite traits of wild rice have widened the genetic basis in cultivated rice. However, many populations of wild rice species have disappeared in the past few years. Therefore, the current status of wild rice resources should be updated and the genetic diversity of wild rice species should be examined for further germplasm preservation and utilization. Our investigations showed that the number of natural wild rice populations declined sharply in Yunnan Province during the past few years due to various reasons. Fortunately, one population of Oryza rufipogon, three of O officinalis and ten of O. granulata have been newly found in different ecological sites, which were confirmed by inter-simple sequence repeat （ISSR） marker analysis in this study. ISSR analysis and investigation of some important traits of nutritional values indicated that the genetic diversity of the currently existing wild rice resources in Yunnan is still rich. The demonstration of genetic diversity of wild rice by a combined use of geographical distribution, morphological traits, nutrition contents and ISSR markers would be helpful for the conservation and exploration of these important wild rice resources.

Identification and Characterization of Drought Stress-Responsive Novel microRNAs in Dongxiang Wild Rice

MicroRNAs （miRNAs） are non-coding small RNAs, which play important regulatory roles in response to biotic and abiotic stresses. Dongxiang wild rice （Oryza rufipogon, DXWR） can survive in extreme drought environment, but its molecular mechanism of drought resistance is still largely unknown. To further explore miRNA regulatory mechanisms involved in drought resistance, we identified 138 novel miRNAs in DXWR using small RNA sequencing and bioinformatics approaches, and found that the expression levels of 67 novel miRNAs were significantly affected by drought stress. In total, 200 candidate target genes were predicted and annotated for the drought stress-responsive novel miRNAs. Gene Ontology （GO） analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways suggested that most of the target genes were related to metabolism. Stem-loop quantitative real-time PCR （qRT-PCR） results exhibited high concordance with sequencing data, which confirmed that miRNA expression patterns based on small RNA sequencing in the present study were reliable. Meanwhile, qRT-PCR validated the inverse expression patterns between several miRNAs and their target genes. These results will enhance our understanding of miRNA regulatory mechanisms in response to drought stress in DXWR, and can serve as an important reference for the protection and utilization of this valuable genetic resource.

Conservation and Utilization of Genetic Resources of Wild Rice in China

The abundant genetic resources of wild rice in China represent a key gene pool for modern rice breeding,contributing to food production and agricultural development in China and worldwide.Between the 1970s and the 2010s,two national wild rice surveys were carried out in China.More than 20000accessions of three species Oryza rufipogon,O.officinalis and O.meyeriana have been conserved ex situ.An in situ conservation system has also been set up to protect notable and endangered populations.This review summarized the geographical distribution of wild rice in China,the current status of conservation,the discovery of elite genes,and the application of research into the origin and domestication of rice.

Repellent Activity of Extracts of Wild Rice Species against Panonychus citri and Aphis citricola in Associated with Esterase Isoenzyme in Insests

Six species of wild rice with different ecophenotypes including Oryza grandiglumis （E6-1, E6-3 / 6-4）, O. minuta （E13-9, E13-13）, O. officinalis（E15-8, E15-13）, O. punctata （E16-1, E16-3, E16-13）, O. granulata（E7-4）, and O. latifolia（101392, E9-1, E9-10） were extracted with methnol and the repellent activity of the extracts against the two insects Aphis citricola and Panonychus citri were studied. The extracts of O. officinalis E15-8 showed higher repellent rate to the two insects than those of the other species. The repellent rates of the extracts of E15-8 to P. citriand A. citricola were 83.26% and 87.86% at 5×10^4 μg/mL in 24 h and 87.95% and 82.43% in 48 h, respectively. The extracts of O. officinalis E15-8 had the effect of inhibition to the esterase of the two insects.

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.

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.

Cloning and Sequence Analysis of Disease Resistance Gene Analogues from Three Wild Rice Species in Yunnan

Two sets of degenerate oligonucleotide primers were designed according to amino acid conserved regions of reported plant disease resistance genes which encode proteins that contain nucleotide-binding site and leucine-rich repeats(NBS-LRR), and the plant disease resistance genes which encode serine/threonine protein kinase(STK). By polymerase chain reaction(PCR), disease resistance gene analogues have been amplified from three wild rice species in Yunnan Province, China. The DIN A fragments from amplification have been cloned into the pGEM-T vector respectively. Sequencing of the DNA fragments indicated that 7 classes, 2 classes and 6 classes NBS-LRR disease resistance gene analogues from Oryza rufipogon Griff. , Oryza officinalis Wall. , and Oryza meyeriana Baill. were obtained respectively. The two representative fragments of TO12 from Oryza officinalis Wall, and TR19 from Oryza rufipogon Griff, belong to the same class and homology of their sequences are 100%. The result shows that the sequences of the same class disease resistance gene analogues have no difference among different species of wild rice. 5 classes STK disease resistance gene analogues were also obtained among which 4 classes from Oryza rufipogon Griff. , 1 class from Oryza officinalis Wall. By comparison analysis of amino acid sequences. we found that the obtained disease resistance gene analogues have very low identity(low to 25%) with the reported disease resistance gene L6, N, Bs2, Prf, Pto, Lr10 and Xa21 etc. The finding suggests that the obtained disease resistance gene analogues are analogues of putative disease resistance genes that have not been isolated so far.