Preparation of DNA from Silica Gel Dried Mini_amount of Leaves of Oryza rufipogon for RAPD Study and Total DNA Bank Construction

Gene resources of Oryza rufipogon Griff. play a crucial role in rice breeding, and hence to study their conservation is of utter importance. The authors describe a method for preparation of DNA from mini_amount of the silica_gel_dried leaves of Oryza rufipogon . The high molecular weight DNAs of 1?168 individuals representing 44 populations have been obtained with high yields, which could be used for RAPD PCR and construction of total DNA bank of this species. The template DNA from silica_gel_dried leaves stored for one year at room temperature gave the same RAPD results as that from the newly prepared silica_gel_dried leaves. The optional template DNA concentrations for amplification ranged from 3.1 ng to 50 ng. In addition, the quality and quantity of the template DNAs that affect RAPD results are also discussed.

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.

广东高州普通野生稻(Oryza rufipogon Griff)的遗传多样性和居群遗传分化研究

利用 30对SSR引物对广东高州普通野生稻 3个群体进行了遗传多样性分析和居群遗传分化研究。结果表明 ,30对引物中只有 2 0对表现出多态 ,多态位点比率P为 6 6 7% ;在 2 0个多态位点中共检测出 81个等位变异 ,平均等位变异数(Ap)为 4 0 5个 ;3个群体总的遗传多样性 (Ht)为 0 6 1,其中 ,居群内的遗传多样性为居群间的遗传多样性的 3倍多 ,说明总的遗传多样性主要来自居群内 ;虽然居群间的遗传分化系数 (GST)较低 ,仅为 0 2 4 2 7,但当遗传相似系数临界值增加时 ,3个群体在聚类图上相对独立 ,说明 3个群体既存在着高度的遗传相似性 ,又具有一定程度的遗传分化 ,可以作为 3个居群进行原生境保护。

普通野生稻(Oryza rufipogon Grif.)抗稻褐飞虱新基因的鉴定与利用

稻褐飞虱(BPH,NilaparvatalugensSt#l)是中国和世界稻区最严重的水稻害虫之一,发现和利用新抗性基因和进行抗性种质创新对于稻褐飞虱抗性品种的培育具有重要的意义。本研究进行了1200多份普通野生稻(OryzarufipogonGriff)种质对多种BPH生物型的抗性鉴定,获得了30份抗性资源,其中6份对分布于世界主要稻区的稻褐飞虱生物型1和2、孟加拉、湄公河(越南)、九龙江(越南)、潘特纳加(印度)等6种生物型或其中5种具有广谱高抗性。遗传分析证明了这些种质对BPH生物型2和九龙江生物型的抗性受2对重复作用隐性基因控制,对潘特纳加生物型的抗性受1对隐性基因控制,表明抗源对不同褐飞虱生物型具有不同的遗传机制。普通野生稻材料2183存在的2对隐性基因很可能是新发现的基因,因为在这些染色体区域还没报道有BPH抗性基因,这两对基因暂命名为bph18(t)和bph19(t)。研究总共培育出143份抗性创新种质和6份抗性品系或高产优质杂交水稻组合,这些优良的抗性创新种质为培育抗性新品种建立了坚实的基础。

陵水普通野生稻(Oryza rufipogon)内生菌的固氮及溶磷特性

从陵水普通野生稻中分离了36株内生菌,经乙炔还原法确定22株菌为内生固氮菌,并测定了其溶磷特性.在22株内生固氮菌中,除菌株Ls2不具有溶磷能力外,其余21株菌溶磷能力差异较大,为(0.002±0.01)^(12.7±0.04)μgmL-1.将这些内生固氮菌接种籼型水稻华航1号,除菌株Ls23外,其余菌株均能使接种水稻苗的根、茎长度增加(在α=0.05水平上差异显著).以上研究结果表明,这些菌株在水稻菌肥研制方面具有较大的开发潜力.

广西普通野生稻(Oryza rufipogon Griff)表型性状和SSR多样性研究

以中国普通野生稻初级核心种质中广西普通野生稻部分中的 2 2 3份野生稻为材料 ,以平均分布于水稻 12条染色体上的 34对SSR引物和中国稻种资源目录中的表型性状分析广西普通野生稻SSR位点的等位变异、多样性的地理分布及不同生长习性间的多样性分布等。结果表明 ,每对引物检测到的多态性片段 7～ 4 8条 ,平均为 2 4 .91条 ,普通野生稻的等位变异数明显大于地方稻种 ,在所分析的SSR位点中杂合位点比例变化在 1.35 %～ 81.31%之间 ,平均为 32 .0 1% ,与自花授粉的栽培稻相比具有较高的杂合率 ;北纬 2 2°～ 2 3°和 2 3°～ 2 4°范围内的两个区域内(一个包括隆安、扶绥和邕宁三县 ,另一个包括象州、来宾、武宣、玉林和贵港五个县 )所包含的普通野生稻数量多 ,遗传多样性大 ,在DNA水平上是广西普通野生稻的遗传多样性中心 ,而表型性状多样性中心是在北纬 2 1°～ 2 2°和2 2°～ 2 3°,其多样性分布与DNA水平不完全一致。在 4种生长习性间 ,DNA水平上的遗传多样性大小依次为匍匐型 ,倾斜型 ,半直立型和直立型 ,表型水平的多样性与DNA水平的多样性基本一致。

中国普通野生稻(Oryza rufipogon Griff.)原生境保护与未保护居群的遗传多样性比较

为了明确我国已建立的普通野生稻原生境保护居群的遗传多样性状况及其代表性,利用24对SSR引物对15个原生境保护居群的427份普通野生稻材料和在我国野生稻分布区内按照纬度划分后随机挑选的15个未保护居群的357份普通野生稻材料进行遗传多样性分析。结果表明,保护居群24个位点的平均有效等位基因数(Ae)为5.98,平均香农指数(I)为1.90,均大于未保护居群在24个位点的平均Ae(5.85)和I(1.86)值,但保护居群在24个位点的平均预期杂合度(He)为0.79,略小于未保护居群He值(0.80)。显著性检验结果显示,保护居群和随机挑选的未保护居群在24个位点上及居群水平上的Ae、I和He值差异不显著,表明保护居群可以代表我国普通野生稻的遗传多样性状况。保护居群的特有等位变异数(Sa)为40,远大于未保护居群的20,说明保护居群保护了更多的特殊基因,具有较高的保护价值。根据前人的研究结果,对应普通野生稻保护居群的地理信息,发现15个保护居群涵盖了我国普通野生稻分布区内所有已知的典型地理类型,表明我国普通野生稻原生境保护居群具有典型性。由此可以看出,我国已建立的15个普通野生稻原生境保护点的选择是科学合理的。根据对我国普通野生稻遗传结构的分析,建议下一步开展的普通野生稻原生境保护点建设应以广西南部及广东为主。

云南元江普通野生稻(Oryza rufipogon)群体籼粳分化的SSR分析

利用经测验可区分籼粳品种的19对SSR特异引物对来自云南元江普通野生稻自然居群的56个个体进行了SSR分析。19对引物中,有17对(占89.47%)在所有参试个体中仅能扩增出一种带型,而RM18和RM251能扩增出多态带型。RM4等16对(84.21%)引物扩增出的带与籼稻或粳稻特征带相同,其中11个位点偏粳而4个位点偏籼;RM18、RM202、RM205等3对引物扩增出的带型不同于籼稻或粳稻带型。结果表明,云南元江普通野生稻基因组DNA在所检测的19个位点上,多数位点(89.47%)上个体间无差异,84.21%的位点已有籼粳分化,13.79%的位点仍具有原始性。说明云南元江普通野生稻主体比较纯而原始,但已开始了籼粳的分化。

中国普通野生稻Oryza rufipogon Griff.的形态分类研究

选用中国7省571份,国外27份普通野生稻(Oryza rufipogon Griff简称普野)的10个形态性状进行了聚类分析并结合普野栖生地的生态考察,将中国普通野生稻划分为多年生与一年生两个普野群,七个普野型。作者根据普野植株在北京温室越冬与翌春植株再生情况及其繁殖方式并联系其生长习性与同工酶分析论证了,中国除多年生普野外还存在一年生普野,但其野生自然群体尚待考察与研究确认。本文还对中国栽培稻的原始型祖先种普野的形态、生态、酯酶同工酶谱型及其群体特点进行了讨论。

普通野生稻Oryza rufipogon Griff.生态分化的初探

为探讨普通野生稻 Oryza rufipogon Griff.的生态分化式样 ,对中国的 31个天然居群及其生境进行了考察。通过对其生活史特性、生长习性、抽穗光周期、百粒重和繁育系统的初步观察后得到如下结果 :1)在中国没有发现一年生的天然居群 ;2 )随着纬度的升高 ,普通野生稻的繁育系统有从无性生殖向有性生殖偏移的趋势 ;3)适应日照长度变化的结果使其始穗期有随着纬度的升高而提早的趋势 ;4 )茎的生长习性的多型性与周围栽培稻的基因流有关 ,在小生境中水分条件的差异亦可导致生长习性从匍匐到直立的渐变。但是 。

普通野生稻(Oryza rufipogon)DNA导入栽培稻后代主要性状的遗传变异

通过花粉管通道法将普通野生稻(Oryza rufipogon)DNA导入宁夏水稻品种宁粳16号和宁粳23号中,获得外源DNA导入系(以D表示),选择的导入系经4次连续自交获得D4代材料。选择其中36个株系在北京种植,进行农艺性状调查和变异类型分析。结果表明,这些导入系分别在株型、生育期、分蘖力、株高、每穗粒数、每穗粒重、穗长、千粒重等性状上发生变异;一些导入系的每穗实粒数、每穗粒重、穗长、千粒重和分蘖数等明显高于受体,表现出高产潜力。本文着重阐述导入系的每穗实粒数、千粒重、分蘖数等与产量相关性状的遗传变异。

广西来宾市五里塘普通野生稻(Oryza rufipogon Griff．)居群遗传多样性与核心种质研究

以位于北回归线穿越的广西中部西江流域的来宾市五里塘285份普通野生稻样本为材料,利用平均分布于水稻12条染色体上的24个微卫星标记进行遗传多样性分析。结果显示:24对微卫星标记引物共检测到73个等位基因,平均每对引物得到3个,平均多态信息含量(PIC)为0.4177,平均遗传杂合度(H)为0.4245,说明这些普通野生稻材料中有丰富的遗传多样性。通过逐步聚类分析,从285份材料中选出30份作为核心种质,该核心种质包含了24个微卫星标记能够检测到的所有基因突变位点,基本上可以代表该居群的遗传多样性。研究结果对居群的种质评估、遗传多样性保护和资源利用提供了参考依据。鉴于广西来宾市五里塘普通野生稻遗传多样性较丰富,建议对该原生地的现有居群进行重点保护和利用。

广西邕宁普通野生稻(Oryza rufipogon Griff.)种群遗传多样性及核心种质构建研究

[目的]研究广西邕宁普通野生稻种群的遗传多样性和核心种质构建,为普通野生稻种群保护提供依据。[方法]从水稻12条染色体上均匀选取24对SSR标记对243份样本进行遗传多样性分析。[结果]结果表明,24对引物均表现出多态性,多态位点比率为100%;24个多态位点共检测出103个等位变异,平均等位基因数A为4.291 7,平均有效等位基因数E是2.511 2,平均期望杂合度He为0.573 2,平均多态信息含量PIC为0.572 0。根据聚类结果分析,从243份材料中筛选出31份核心种质,包含了24个SSR位点检测到的所有的遗传变异,其平均期望杂合度He是0.595 8,平均多态信息含量PIC是0.586 3,基本上可以代表广西邕宁普通野生稻种群的遗传多样性。[结论]广西邕宁普通野生稻资源具有丰富的遗传多样性。

Identification of a Resistance Gene bls1 to Bacterial Leaf Streak in Wild Rice Oryza rufipogon Griff

Bacterial leaf streak （BLS） of rice, caused by Xanthomonas oryzae pv. oryzicola （Xoc） is a worldwide destructive disease. Development of resistant varieties is considered to be one of the most effective and eco-friendly ways to control the disease. However, only a few genes/QTLs having resistance to BLS have been identified in rice until now. In the present study, we have identified and primarily mapped a BLS-resistance gene, blsl, from a rice line DP3, derived from the wild rice species Oryza rufipogon Griff. A BC2F2 （9311/DP3//9311） population was constructed to map BLS-resistance gene in the rice line DP3. The segregation of the resistant and susceptible plants in BCzFz in 1：3 ratio （Z2=0.009, Z20 05,1=3.84, P〉0.05）, suggested that a recessive gene confers BLS resistance in DP3. In bulked segregant analysis （BSA）, two SSR markers RM8116 and RM584 were identified to be polymorphic in resistant and susceptible DNA bulks. For further mapping the resistance gene, six polymorphic markers around the target region were applied to analyze the genotypes of the BC2F2 individuals. As a result, the BLS-resistant gene, designated as blsl, was mapped in a 4.0-cM region flanked by RM587 and RM510 on chromosome 6.

Analysis on Genomic Structure Changes and Diversity of Introgression Lines in Dongxiang Wild Rice(Oryza rufipogon Griff.)

[Objective] The aim of the study was to make research on genomic struc- ture variation and variety analysis of Dongxiang wild rice. [Method] Introgression groups of BC1F6 were based on donor of Oryza rufipogon Griff. and receptor of O. sativa sp. indica Kate. Strains of 239 in the group were analyzed on Polymor- phism with the help of 25 couples of SSR primers distributed in 12 pairs of chromo- somes. [Result] Gene fragments of O. rufipogon Griff. were found penetrated in the 25 microsatellite sites and most of the groups kept the parents of Xieqinzao B or DNA sequence of O. rufipogon Griff. The average rate of recurrent homozygous bands was 78.13% in the ILs, but the highest was 94.98% （amplified by primer RM131） and the lowest was 60.25% （RM171）. The average rate of donor homozy- gous bands was 13.37%, but the highest was 32.64% （RM171） and the lowest was 2.93% （RM1095）. There were numerous heterozygous sites in the population and the average heterozygosis rate was 5.62%, while the highest was 10.04%（RM401）. Moreover, we found some parental fragments were lost and some novel fragments were not detected in either parent in BC1F6 population. The average rate of lost bands was 2.88%, while the highest was 13.39% （RM311） and the lowest was 0 （RM401）. The average rate of new bands was 1%. The average of Nei＇s gene di- versity （He） and Shannon＇s Information index （I） were 0.276 and 0.457 respectively in high generation of introgression lines. [Conclusion] The study demonstrated that distant hybridization led to extensive genetic and epigenetic variations in high gener- ation of introgression lines, which expanded the base of genetic variation and laid an important foundation for rice improvement and germplasm innovation.

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.

海南普通野生稻(Oryza rufipogon Griff.)Adh2基因多态性分析

海南岛的4个保护区和2个非保护区内普通野生稻与2种栽培稻(Nipponbare,9311)的Adh2基因序列在编码区共有单现突变9个,信息位点4个,同义突变8个,替代突变5个。内含子内有单现突变21个,信息位点29个,插入/缺失位点161个。Adh2基因在整个区域的多态性(πR,θR)均为0.011,内含子区域的多态性明显高于编码区域。在编码区域,同义位点多态性明显高于替代位点。中性测试表明虽然Adh2各区域的净化选择在统计上不具有显著性,但编码区的净化选择作用明显大于内含子区域。聚类分析表明海南普通野生稻可以划分为2大类,其中一类(QH,LD)与中国大陆普通野生稻和栽培稻更接近,另一类(WC,WA,WN,DZ)则更接近东南亚普通野生稻。

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.

Identification of a novel planthopper resistance gene from wild rice(Oryza rufipogon Griff.)

Rice planthoppers,including brown planthopper(BPH)and white-backed planthopper(WBPH),are the most destructive pests in Asian rice cultivation regions.Planthopper resistance genes that have been mapped and characterized advance our understanding of underlying resistance mechanisms and facilitate the breeding of resistant varieties,thereby contributing to an efficient pest management strategy.In this study,a novel resistance gene Bph38 derived from the wild rice species Oryza rufipogon Griff.was found to confer high resistance to BPH and WBPH.Conventional mapping was performed to identify regions associated with BPH and WBPH resistance,and two mapping efforts led to the same region on chromosome 4 flanked by markers RM16563 and RM16763.Bulked-segregant analysis and next-generation sequencing were performed using the same population to detect the resistance gene.Conventional mapping narrowed the region to a 12.3-Mb segment,and fine mapping using BC1 F2 recombinants identified a 79-kb segment flanked by markers YM112 and YM190.Near-isogenic lines(NILs)carrying Bph38 in the 9311(indica)and BR54(japonica)genetic backgrounds were developed by crossing and backcrossing with marker-assisted selection.The agronomic traits and BPH and WBPH resistance of the NILs were similar to those of the recurrent parents.Mandatory feeding and host-choice tests revealed that Bph38 showed both antibiotic and antixenotic effects in both insects,with stronger effects in indica-background lines.Further fine mapping and characterization of the major gene may result in map-based cloning of the gene and allow its application in breeding insectresistant rice varieties.

SSR Marker Analysis on indica-japonica Differentiation of Natural Population of Oryza rufipogon in Yuanjiang, Yunnan Province

By using 19 pairs of primers that could identify two subspecies （indica and japonica） of cultivated rice （Oryza sativa L.）, the indica-japonica differentiation of 56 individuals from the natural population of common wild rice （Oryza rufipogon Griff.） in Yuanjiang was analyzed by SSR （microsatellite DNAs, or simple sequence repeat）. Of the 19 pairs of primers, 17 pairs （89.47%） could amplify only one kind of band type among all of the individuals, but primers RM251 and RM18 could amplify polymorphic band types. The bands amplified by 16 pairs of primers （84.21%） were identical to the indica-japonica diagnostic bands of relevant locus in cultivated rice, including 11 japonica-like loci and 4 indica-like loci. The bands amplified by the other three pairs of primers （RM18, RM202, RM205） were different from indica or japonica diagnostic bands of cultivated rice. The results showed that according to 19 loci analyzed, 84.21% of SSR loci in genomic DNA of common wild rice in Yuanjiang displayed indica-japonica differentiation and 13.79% of the loci still kept primitive, and most of the detected loci were homoaenetic in the natural population.