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。

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。

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。

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 。

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.

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 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.

Genetic diversity of Oryza rufipogon Griff. in Hainan Province analyzed by ISSR and SSR markers

Assessment of genetic diversity is an essential component in germplasm characterization and conservation.There are three wild rice species in Hainan Province,including Oryza rufipogon Griff.In order to detect the genetic diversity of different populations of Oryza rufipogon in Hainan,ISSR(inter-simple sequence repeat)and SSR(simple sequence repeat)markers were used to investigate 180 accessions from six localities in Hainan.Fourteen ISSR primers amplified 185 alleles with 171(92.43%)polymorphic,the number of alleles ranged from 8 to 17,with an average of 13.14 alleles per locus.Thirty-eight pairs of SSR primers used in this study amplified 213 alleles with 190(89.20%)polymorphic,the number of alleles ranged from 2 to 14,with an average of 5.66 alleles per locus.Both ISSR and SSR analyses revealed a high level of genetic diversity in the wild populations.The population with the highest genetic diversity is Wanning(WN),and the population with lowest genetic diversity is Wenchang(WC).The results of a UPGMA cluster using the NTSYS program showed that each population has a low degree of genetic differentiation.Furthermore,the Mantel test revealed that the genetic similarities detected by ISSR and SSR were significantly correlated(r=0.8634,t=93.67)when detecting genetic diversity at the species level.The two molecular marker systems were able to determine the genetic diversity among Oryza rufipogon,and the two groups of indexes obtained by using the two markers have a high level of consistency.

普通野生稻（O.rufipogon）微核心种质中广亲和基因S5^n的检测与分析

普通野生稻微核心种质中蕴含丰富的优异基因资源,是用于水稻品种改良和基础研究的重要材料。为了筛选携带广亲和基因S5n的普通野生稻种质,利用前人开发的鉴定水稻S5n基因的InDel分子标记,对107份野生稻微核心种质进行了PCR扩增和读带分析。结果筛选出3份携带S5n广亲和基因的野生稻种质,均来自海南省,该基因可能在海南完成演化。野生稻广亲和材料有助于籼粳交育种材料的创制。

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.

普通野生稻的抗水稻白叶枯病(Xanthomonas oryzaepv.oryzae)新基因Xa-23(t)的鉴定和分子标记定位

经抗谱评价、抗性类型比较、遣传分析鉴定出一个来自普通野生稻的抗白叶枯病新基因 Xa-2 3( t) (暂名 )。结果表明它是迄今已知基因中抗谱最广 ,抗性导入效应很强的一个完全显性的全生育期抗性基因。用携有新基因 Xa- 2 3( t) 的一对近等基因系 ( JG30 6* / / Xa- 2 3( t) )构建 F2 群体中选出 160个单株用 SSR(微卫星 )标记进行基因定位 ,初步将该基因定位于水稻第 11染色体 SSR标记 OSR0 6和RM2 2 4附近 ,图距分别为 5.3c M和 2 7.7c M。

海南普通野生稻OrERF1的克隆和表达分析

根据EST(expressed sequence tags,EST)序列信息,从海南普通野生稻中克隆了一个AP2/ERF(apetala2/ethylene response factor)类转录因子OrERF1。该基因包含一个1 014 bp的完整阅读框,编码337个氨基酸。推导OrERF1氨基酸序列具有典型的AP2/ERF类转录因子结构特征,与水稻(Oryza sativa)、大麦(Hordeum vulgare)、玉米(Zea mays)和橙子(Citrus sinensis)中相应的AP2/ERF类蛋白具有较高的同源性。聚类分析表明OrERF1是ERF类转录因子。对OrERF1启动子序列进行分析,发现存在多种激素应答和胁迫响应的调控元件。Real-time PCR结果显示,低温、干旱和盐胁迫均能诱导OrERF1的表达。这些结果表明OrERF1可能在逆境响应过程中具有重要功能。

普通野生稻中NBS-LRR类基因的克隆及Southern杂交

利用已克隆植物的R基因NBS序列中保守基序设计简并引物进行PCR扩增是克隆NBS有效的方法。从广东普通野生稻HLW028中克隆出3条序列,经同源性分析均为NBS,序列号为DQ272573-DQ272575。从NCBI中下载普通野生水稻和功能已知的水稻NBS-LRR类基因,与本实验所提交的NBS进行聚类分析。这些NBS可分为5大类,其中DQ272574是一个新的NBS基因类型。从野生水稻中克隆NBS基因存在P-loop、RNBS-A、kin-2、RNBS-B、RNBS-C和PLAL的基序。Southern杂交结果表明,野生水稻HLW028和GZW035中具有与DQ272574同源的3个拷贝。

利用粳稻基因组DNA和C_ot-1 DNA探针对普通野生稻和亚洲栽培稻的比较分析

以粳稻日本晴基因组DNA和Cot-1 DNA为探针,分别对日本晴、籼稻广陆矮4号和普通野生稻的染色体组进行了基因组原位杂交(GISH)和Cot-1 DNA荧光原位杂交(FISH)分析,并对3种染色体组进行了同源聚类和比较研究.结果表明:粳稻基因组DNA和Cot-1 DNA探针信号在3种水稻染色体组中的分布状况和覆盖率相似,Cot-1 DNA的覆盖率分别为(47.13±0.18)%、(45.89±0.22)%、(44.24±0.21)%,3种水稻基因组同源性高,亲缘关系接近.Cot-1 DNA在3种水稻染色体上的杂交信号分布各有特点,中高度重复序列的变异在普通野生稻向栽培稻进化和亚洲栽培稻籼、粳分化过程中具有重要意义,中高度重复序列含量较低的2、5、8号染色体是水稻染色体组进化过程中相对活跃的成分.

Effect of Low Temperature and Sparse Light Conditions on Cold Tolerance of Different Rice Lines at Seedling Stage

Low temperature and sparse light in early spring is one of the factors causing reduction for rice production. So it is important to develop cold tolerance cultivars. In the present study, cold tolerance characters of 36 parents and 423 rice lines from 68 hybrid groups were investigated at seedling stage under low tempera- ture and sparse light conditions in field. There were 10 parent accessions with strong cold tolerance of level 1. Among them, 3 were common wild rice accessions; 4 were japonica rice cultivars; 2 were indica rice; 1 was offspring from hybrid be- tween indica and japonica.There were 33 lines with strong cold tolerance of level 1 from the hybrid groups of common wild rice,and 15 from the hybrid groups of IRBB5, and only 3 from the hybrid groups of BPHR96. There were abundant cold tolerance resources in rice germplasm. It was feasible to develop cold tolerance cul- tivars from the hybrids among common wild rice, japonica cultivars and indica culti- vars.

茶陵野生稻冷响应基因OrCr3的克隆及其生物信息学分析

OrCr3(Oryza rufipogon cold responsive gene 3)是一个从茶陵野生稻中发现的受低温诱导表达的基因.以茶陵野生稻为材料克隆了其cDNA及其启动子区域序列,生物信息学分析表明,该基因的完整ORF(open reading frame)长为1 110 bp,推测编码一个由369个氨基酸残基组成的山梨糖醇脱氢酶蛋白,理论相对分子质量为39.3 kD,pI值为6.03;山梨糖醇脱氢酶具有催化山梨糖醇向果糖转变的作用;对OrCr3的启动子区域进行分析,发现多种可能与逆境应答相关的顺式作用调控元件;该基因编码的蛋白在不同物种中存在高度相似性.以上结果说明OrCr3基因可能是一个保守的耐逆相关基因.

普通野生稻种子萌发的转录组动态研究

普通野生稻是亚洲栽培稻的祖先种,对其种子萌发过程的解析对野生稻资源的挖掘利用有重要促进作用。本研究在表型分析基础上利用转录组测序技术,对吸胀后0h、2h、4h、6h、8h、10h和12h的普通野生稻种子表达基因进行检测,并与栽培稻转录组数据进行比较分析。研究发现普通野生稻种子萌发过程中有11853个差异表达基因(Fold change≥2.0)。表达模式可分为5种类型,其中一组在快速吸涨期(PhaseⅠ)到吸涨滞缓期(PhaseⅡ)为独特的瞬时表达模式,并富含转录因子(占该组基因的10.33%)。和栽培稻种子表达基因比较结果表明,植物同源结构域(PHD)和热休克因子(HSF)家族基因在普通野生稻中延后表达,其中5个表达模式不同的HSF家族基因在普通野生稻中得到qRT-PCR验证。进一步分析发现HSF基因通过热激元件(HSE)顺式调控32个差异表达基因的表达,推测它们在普通野生稻种子萌发过程中发挥重要作用。本研究初步明确了普通野生稻种子萌发过程的差异表达基因及转录因子,它们可作为野生稻优异基因利用的参考资源。

Diversity and high nitrogenase activity of endophytic diazotrophs isolated from Oryza rufipogon Griff.

Diversity and nitrogenase activity of endophytic diazotrophs colonized in the wild rice Oryza rufipogon Griff grown in Boluo, Huilai County in Guangdong Province and Lingshui County in Hainan Province were studied. Thirty-seven isolates obtained from Oryza rufipogon were identified as putative endophytic nitrogen-fixing bacteria by ARA (acetylene reduction assay) test and further confirmed by PCR amplification of nifH gene fragments. All obtained strains have ARA activity and the same sized nifH gene fragments. Above the similarity level of 80%, the obtained isolates were assigned as Group Ⅰ to Ⅷ by the clustering of IS-PCR fingerprints. The SDS-PAGE whole-cell protein patterns were similar to those of IS-PCR fingerprints. Components and contents of fatty acid methyl esters (FAMEs) were used to differentiate the representative strains (Ls13, Ls8, BL1, BL12, HL6, Ls4) from Group Ⅰ to Group Ⅵ. The six representative strains showed significant difference in contents and components of cellular fatty acid methyl ester. 16S rDNA sequencing analysis showed that strains of Group Ⅰ to Ⅶ were located in Enterobacteraceae (γ-proteobacteria). Strains of Group Ⅰ and Group II were closely related to Klebsiella sp.; Strain Ls8 of Group Ⅱ was a little far away from the genus of Pantoea (homology level 96% with Pantoea agglomerans), which may represent a new species or genus in Enterobacteraceae; Strains of Groups IV and V belonged to different Enterobacter sp.; Strain Ls4 and Ls 9 representing Group Ⅵ were close to Citrobacter amalonaticus with 98% sequence similarity; Strain Ls15 of Group Ⅶ showed 98% sequence identity with Pantoea sp.; Strains of Group Ⅷ were assigned to the genus Ideonella (β-proteobacteria). Based on the above results, endophytic diazotrophs isolated from O. rufipogon showed great diversity and some diazotrophs showed high nitrogenase activity with 42.52 μmol/mL·h C2H4. Inoculation to rice tests indicated that the isolated endophytic diazotrophs significantly promoted the rice growth.

广西普通野生稻种质资源抗褐飞虱评价与SSR遗传多样性分析

【目的】探究76份广西普通野生稻种质对褐飞虱的抗性与遗传多样性,为优异抗性水稻种质资源的合理利用提供参考依据。【方法】对来自临桂、贵港、邕宁、博白、横州、扶绥、玉林和防城港8个居群的76份广西普通野生稻种质资源进行苗期抗褐飞虱鉴定,利用筛选出的62对SSR引物对供试材料进行遗传多样性分析,采用Structure ver 2.3.4进行群体遗传结构分析,并构建聚类图,利用GenAlEx 6.5分析居群间的遗传分化系数和基因流,并进行不同类群的分子方差分析。【结果】广西普通野生稻种质资源褐飞虱抗性鉴定表明,表现为高抗、抗、中抗、中感的材料分别有7、34、31和4份。遗传多样性分析中,76份材料中共检测到669个等位基因,平均每个位点含10.79个等位基因,等位基因数变幅为4~19个,其中稀有等位基因348个,占总数的52.02%,平均每个位点含5.613个;主要等位基因频率变异范围在0.158~0.855,平均为0.373;Nei’s基因多样性指数(He)变异范围为0.260~0.894,平均为0.754;多态信息含量(PIC)的变异范围为0.248~0.885,平均值为0.727。8个居群He表现为防城港>邕宁>贵港>博白>扶绥>横县>玉林>临桂,种质褐飞虱抗性与居群纬度和He无显著相关性(P>0.05),居群纬度与居群He的相关系数r为-0.822,达显著水平(P<0.05)。遗传结构分析显示,K=3时,供试种质可分为3个类群,与聚类分析结果相似;8个居群两两之间有85.71%的居群间表现为中等程度的遗传分化;基因流分析表明,防城港、博白、邕宁居群之间及贵港与扶绥之间基因交流频繁。AMOVA分析表明,遗传变异来源主要为个体内与个体间,群组间的变异比例较小。【结论】76份广西普通野生稻种质对褐飞虱表现出较好抗性与丰富的遗传多样性,结合现代分子生物学技术鉴定新的褐飞虱抗性基因和优异等位基因,并转育创新抗性新种质,可用于培育多抗优质水稻品种。