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.

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.

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.

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.

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.

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.

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.

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.

Dissecting Genetic Basis of Deep Rooting in Dongxiang Wild Rice

Deep rooting is an important trait in rice drought resistance.Genetic resources of deep-rooting varieties are valuable in breeding of water-saving and drought-resistant rice.In the present study,234BC2F7 backcross introgression lines were derived from a cross of Dongye 80(an accession of Dongxiang wild rice as the donor parent)and R974(an indica restorer line as the recurrent parent).A genetic linkage map containing 1977 bin markers was constructed by ddRADSeq for QTL analysis.Thirty-one QTLs for four root traits(the number of deep roots,the number of shallow roots,the total number of deep roots and the ratio of deep roots)were assessed on six rice chromosomes in two environments(2020 Shanghai and 2021 Hainan).Two of the QTLs,qDR5.1 and qTR5.2,were located on chromosome 5 in a 70-kb interval.They were detected in both environments.qDR5.1 explained 13.35%of the phenotypic variance in 2020 Shanghai and 12.01%of the phenotypic variance in 2021 Hainan.qTR5.2 accounted for 10.88%and 10.93%of the phenotypic variance,respectively.One QTL(qRDR2.2)for the ratio of deep roots was detected on chromosome 2 in a 210-kb interval and accounted for 6.72%of the phenotypic variance in 2020.The positive effects of these three QTLs were all from Dongxiang wild rice.Furthermore,nine and four putative candidate genes were identified in qRDR2.2 and qDR5.1/qTR5.2,respectively.These findings added to our knowledge of the genetic control of root traits in rice.In addition,this study will facilitate the future isolation of candidate genes of the deep-rooting trait and the utilization of Dongxiang wild rice in the improvement of rice drought resistance.

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.

论wild rice不一定指野生稻

野生稻,英文译为wild rice,但笔者在查阅有关野生稻(wild rice)的英文文献时发现,wild rice除指代禾本科稻属中的野生稻之外;还有文献指代的是禾本科菰属的菰米。因此,本文分两部分阐述,野生稻与菰的比较、wild rice不一定指野生稻,为后人查阅wild rice时辨别菰或野生稻提供有效的帮助。

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.

Study on Young Panicle Culture in vitro From Wild Rice of Different Genomes

Researches have been made on young panicle culturein vitro from wild rice of different genomes. Main results are as follows: 1. The induction frequencies of young panicle culturedin vitro from wild rice varied largely a relation to its genome. The optimal induction period of callus is the stamen and pistil differentiation stage of young panicle development. 2. Plantlets were regenerated through two ways: first, culture method, the induced calli were transferred onto differentiation medium; second, regenerate plantlets directly from young panicles of wild rice that were cultured on the differentiation medium. 3. The regeneration rate of green plantlets that obtained through cryopreservated calli inO. meyeriana was 10 times higher than that of control.