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.

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.

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.

Development of Chromosomal Segment Substitution Lines from a Backcross Recombinant Inbred Population of Interspecific Rice Cross

A backcross recombinant inbred line population consisting of 202 lines was developed from Xieqingzao B//Xieqingzao B / Dongxiang wild rice. The population was assayed with DNA markers and phenotyped on planthopper resistance and yield traits. A linkage map consisting of 119 DNA markers and spanned for 1188 cM over the 12 rice chromosomes was constructed. Thirty-two chromosomal segment substitution lines were selected based on the percentage of Xieqingzao B allele at marker loci. These lines are of great potential for gene mapping and alien gene introgression.

利用东乡野生稻染色体片段置换系鉴定穗部性状主效QTL

【目的】稻穗是水稻产量的主要承载者,发掘穗部性状相关的主效QTL对进一步精细定位、克隆和育种利用具有重要意义。【方法】以粳稻日本晴为受体、东乡野生稻为供体构建的一套染色体片段置换系(chromosome segment substitution lines,CSSLs)群体为材料,在4个生态环境下进行穗部性状表型鉴定与主效QTL定位分析。【结果】不同生态环境下CSSL群体穗部性状存在较大幅度变异,东乡野生稻染色体片段的导入显著改变背景亲本的穗部性状,具有明显的增产效应;早季、中季、晚季和海南环境下共检测到64个控制每穗颖花数和籽粒大小的QTL,其中qPL2、qSN1.2、qSN2、qFGN3、qTGW2、qTGW12.1和qSL2在3个环境下稳定表达,21个QTL在2个环境下被重复检测到,其余QTL仅在单一环境下被发现;主效QTLqSN1.1、qSN1.2、qSN2、qSN3和qSN12对每穗颖花数具有明显的影响,多个QTL聚合具有明显调控每穗颖花数的功能;主效QTLqSL2、qSL3.2、qSLW3.1、qSLW3.2、qTGW8.1和qSLW10对籽粒大小具有明显的影响。这些位点分别作用于粒长、粒宽和长宽比,相互作用、共同决定籽粒大小;同时,穗部性状QTL成簇分布于10个染色体区段,其中主效QTL簇qGNS1.1、qGNS1.3、qGNS2.1、qGNS3.1、qGNS9、qGNS10和qGNS12包含多个调控每穗颖花数及籽粒大小性状的QTL,可在不同环境下稳定表达。来自东乡野生稻的等位基因具有明显调控产量表型的功能。【结论】研究结果不仅为东乡野生稻优异基因发掘奠定基础,也可为水稻穗部性状的分子育种提供新基因源。

东乡野生稻苗期耐冷性的QTL定位

【目的】研究东乡野生稻苗期耐冷性的QTL和连锁标记,为水稻耐冷种质资源的利用及分子标记辅助选择育种提供理论和实践依据。【方法】以东乡野生稻作为非轮回亲本,南京11号为轮回亲本,构建144株BC2F1分离群体。通过SSR标记以根电导率作为耐冷性指标,以复合区间定位法对东乡野生稻苗期耐冷性进行QTL定位。【结果】检测到2个QTL qRC10-1和qRC10-2均位于第10染色体,对表型的贡献率分别为34.13%和37.02%,是两个主效的QTL。在与2个QTL的连锁标记RM171周围发展分子标记进一步定位,检测到3个QTL位于标记RM171附近。【结论】东乡野生稻第10染色体上的2个QTLqRC10-1,qRC10-2与苗期耐冷性有关,并位于SSR标记RM304-RM1108区间,可用于水稻耐冷性分子标记辅助选择育种。

东乡野生稻耐冷性的遗传改良初步研究

以强耐冷优异种质资源———东乡野生稻为骨干亲本 ,采用“双重”低温加压筛选法对粳稻耐冷性进行遗传改良 ,取得了良好效果 ,育成的 491 3— 1粳稻品种 (品系 )其耐冷水平 ,在苗期 (二叶一心 )冰箱 0℃处理48h,完好无损 ;在南昌自然条件下越冬再生 3年 ,通过生产示范 ,产量、品质和抗性都得到了较理想的效果。并且育成了粳型三系不育系和两用系不育系 ,可以在南昌越冬 ,可望实现杂种优势固定大面积应用于生产。

应用微卫星标记分析东乡野生稻遗传多样性初探

利用SSLP标记对东乡野生稻进行多样性分析。结果表明 ,在 14对SSLP标记引物中 ,12对引物扩增的产物呈现多态 ,多态性探针百分率为 85 .71% ,共扩增出 70条多态性带 ,平均每对引物扩增出多态性带 5 .83条 ,表明东乡野生稻具有较丰富的遗传多样性。聚类分析结果表明 ,东乡野生稻 9个居群内及居群间存在一些差异较大的材料 ,同时也存在一些相同或遗传距离很小的材料 ;居群内平均遗传距离为 0 .2 3～ 0 .4 7,居群间平均遗传距离为0 .4 0～ 0 .5 5 ,居群间的遗传距离略大于居群内的遗传距离 ,为此 ,必须对东乡野生稻现有居群进行严格的保护。

东乡野生稻耐冷性研究进展

对东乡野生稻耐冷性方面的研究进展作了综述 ,主要内容包括水稻冷害和东乡野生稻耐冷性的鉴定、遗传分析、利用等。并对今后东乡野生稻的研究利用提出了几点建议。

东乡野生稻的研究与利用

从多角度对东乡野生稻的研究和利用现状进行了综合评述,指出了研究中存在的问题,对未来的研究方向提出了建议。

东乡野生稻BILs群体苗期抗旱性综合评价及其遗传分析

本研究选用旱稻、R974//R974/东乡野生稻回交重组自交系及其亲本等66份水稻材料,进行苗期抗旱性鉴定及其综合指标筛选,测定了水分胁迫下最大根长、根基数、茎长、根干重、根鲜重、根系相对含水量、叶片相对含水量、卷叶级别及干旱反复存活率等9个性状。利用主成分分析和逐步回归分析法进行苗期抗旱综合评价,结果表明株系1949最为抗旱;回归分析和相关分析表明最大根长、根数、根鲜重和根系相对含水量对抗旱性影响显著,可作为苗期抗旱性鉴定综合指标。采用综合抗旱D值进行抗旱遗传分析,表明BILs群体的苗期抗旱性表现2对独立主基因+多基因控制。结果可知,东乡野生稻可作为水稻抗旱遗传改良的宝贵资源。

东乡野生稻细胞质源雄性不育系“东B11A”的选育

以东乡野生稻为母本与栽培稻品种“红优”、“HA7931 7-7”杂交 ,在其杂交后代中分离出雄性不育株 ,再与栽培稻品种“B1 1”杂交 ,并连续选不育株与“B1 1”回交 ,育成东乡野生稻质源新不育系“东B1 1A”。该不育系属早籼类型 ,在宜春 4～ 6月播种 ,播种至始穗历期 63～ 78d,主茎叶片数 1 2～ 1 3叶 ;株型较散 ,茎秆粗壮 ,穗大粒多 ,田间抗性较强 ;不育株率、不育度均达 1 0 0 % ;不育花粉中以典败为主 ;柱头发达 ,外露率达 81 .1 % ,异交结实率一般 5 0 %以上 ;配合力强 ,所配组合“东B1 1A/D1 8”、“东B1 1A/R40 2”和“东B1 1A/C1 42 9”

东乡野生稻育性恢复性的鉴定与遗传分析

以东乡野生稻水桃树、东塘上2个居群与不同细胞质来源的5个水稻不育系（B06S、珍汕97A、协青早A、中97A和粤泰A）配组，根据F1结实率的高低对东乡野生稻育性恢复性进行鉴定。建立了10个组合的P1、F1、P2、F2群体，利用混合模型理论的Akaike信息准则（AIC），在F2代中鉴定影响数量性状的主基因存在与否，主基因存在时通过分离分析估计主基因和微效基因的遗传效应以及所占总变异的分量。F1代的结实率变化范围为45．98％～76．57％，表明东乡野生稻具有一定的育性恢复性。F2代中该性状符合1对主基因＋多基因的遗传模式，主基因遗传率为56．63％～88．29％，多基因遗传率为2．74％～30．97％，总基因型遗传率为63．17％～94．01％。中9A／东塘上居群杂交组合的F2代中，主基因是加性遗传，无显性效应，其他9个组合主基因是完全显性遗传。

水稻越冬品系及越冬不育系的选育

利用农垦58航天诱变后代与东乡野生稻杂交系选出10个越冬品系,利用东乡野生稻与普通栽培稻A08天然异交后代中选育出3个越冬不育株系。对10个越冬品系和3个越冬不育株系的特征特性进行了分析,并对越冬品系和越冬不育株系的利用进行了展望。

东乡野生稻抗寒基因的利用与前景展望

东乡野生稻是普通野生稻（φＮ／２８°１４’），它具有特强的耐寒性、耐瘠性等有利基因资源，值得重视。本文阐述了东乡野生稻抗寒性的研究与利用，分析东乡野生稻耐寒基因资源利用的可行性和开发前景。