Characterization of Interspecific Hybrids Between Oryza sativa L. and Three Wild Rice Species of China by Genomic In Situ Hybridization

In the genus Oryza, interspecific hybrids are useful bridges for transferring the desired genes from wild species to cultivated rice （Oryza sativa L.）. In the present study, hybrids between O. sativa （AA genome） and three Chinese wild rices, namely O. rufipogon （AA genome）, O. officinalis （CC genome）, and O. meyeriana （GG genome）, were produced. Agricultural traits of the F1 hybrids surveyed were intermediate between their parents and appreciably resembled wild rice parents. Except for the O. sativa × O. rufipogon hybrid, the other F1 hybrids were completely sterile. Genomic in situ hybridization （GISH） was used for hybrid verification. Wild rice genomic DNAs were used as probes and cultivated rice DNA was used as a block. With the exception of O. rufipogon chromosomes, this method distinguished the other two wild rice and cultivated rice chromosomes at the stage of mitotic metaphase with different blocking ratios. The results suggest that a more distant phylogenetic relationship exists between O. meyeriana and O. sativa and that O. rufipogon and O. sativa share a high degree of sequence homology. The average mitotic chromosome length of O. officinalis and O. meyeriana was 1.25- and 1.51-fold that of O. sativa, respectively. 4＇,6＇-Diamidino- 2-phenylindole staining showed that the chromosomes of O. officinalis and O. meyeriana harbored more heterochromatin, suggesting that the C and G genomes were amplified with repetitive sequences compared with the A genome. Although chromocenters formed by chromatin compaction were detected with wild rice-specific signals corresponding to the C and G genomes in discrete domains of the F1 hybrid interphase nuclei, the size and number of O. meyeriana chromocenters were bigger and greater than those of O. officinalis. The present results provide an important understanding of the genomic relationships and a tool for the transfer of useful genes from three native wild rice species in China to cultivars.     

云南野生稻中Xa21基因外显子II的分离及序列分析

Xa21是已经分离克隆的一个具有广谱抗性的水稻白叶枯病抗性基因,根据已克隆的白叶枯病抗性基因Xa21外显子II序列设计特异性引物对云南三种野生稻及其它稻种进行PCR扩增。结果表明只有普通野生稻（景洪普通野生稻和元江普通野生稻）及长雄野生稻中扩增到了长400 bp的目的片段,而疣粒野生稻和药用野生稻及栽培稻中均没有扩增到目的片段。通过序列比较发现所克隆的序列同长雄野生稻的氨基酸序列变化是随机的。     

Isolation and Sequence Analysis of the Xa21 Gene Wxon II Homologus from Different Species of Wild Rice in Yunnan.]

The Xa21 gene previously cloned from the wild rice species Oryzae longistaminata confers broad-spectrum resistance to rice leaf blight caused by different strains of Xanthomonas oryzae pv. oryzae. Here we attempted to determine the existence of Xa21 homologs in other wild rice species and rice cultivars and the sequence differences between the homologs. We synthesized specific primers based on the reported Xa21 sequence to amplify homologs of the gene exon II from several rice cultivars and three wild rice species in Yunnan Province, China. The fragments cloned from various types of O. rufipogon Griff from Jinghong and Yuanjiang, Yunnan Province, were highly homologous to the reported Xa21 gene exon II. However, the fragment was not found in O. officinalis Wall. and O. meyeriana Baill. Sequence analysis suggested that differences in nucleotides were located randomly in the fragments we cloned.     

云南野生稻叶茎根组织结构特性的比较研究

采用徒手切片法对云南3种野生稻和栽培稻的叶片、茎秆及根的组织结构进行比较研究,以明确野生稻的内部结构,为进一步揭示其结构与云南野生稻的生长势旺盛、营养吸收能力强、抗某些病虫害能力强的关系奠定基础。结果表明,(1)云南野生稻与栽培稻叶片主脉、茎秆及根的组织结构差异显著,其中景洪疣粒野生稻、景洪药用野生稻与栽培稻的差异最明显。(2)在叶主脉结构中,景洪疣粒野生稻无气腔结构,维管束数量少、面积小;景洪药用野生稻、3个普通野生稻材料存在多个维管束和气腔结构,维管束、束内导管直径及气腔面积较栽培稻大,而栽培稻中的气腔均为2个。(3)在茎秆结构方面,景洪疣粒野生稻茎秆最小,维管束数量最少,其茎壁内的维管束排列方式与栽培稻不同;景洪药用野生稻和普通野生稻茎秆及茎壁较栽培稻粗厚,维管束数量也较栽培稻多,普通野生稻的茎壁中有通气组织。(4)在根的组织结构中,3种野生稻的导管数量较多,导管直径及中柱面积较栽培稻大,外皮层出现了具有凯氏带功能的凯氏点等。     

珍稀濒危植物--云南药用野生稻自然生态群的新发现及其特性

野生稻包括云南药用野生稻(Oryza officinalis Wall.ex Watt.,染色体组CC,染色体数2n=24)是宝贵的基因资源库,自然分布急剧减少。2001年,在云南耿马孟定遮甸新发现了1个药用野生稻分布点,该地药用野生稻植株高度可塑性大,可以根据密度和荫蔽情况自动大幅度调节株高;比其它生态群的结实率高;而其自然发病和人工接种鉴定发现抗稻瘟病能力中等偏上,抗白叶枯病害能力强,抗螟虫和稻飞虱能力也很强。品质分析结果表明该居群药用野生稻蛋白质含量比其它野生稻的高,而直链淀粉含量偏低,说明品质较好。本发现对采取有效措施进行药用野生稻原生地保护,以及试验结果对药用野生稻有利基因的发掘和利用都有重要参考价值。     

Variation in the loss of seed dormancy during after-ripening of wild and cultivated rice species

BACKGROUND AND AIMS: The aim of this paper was to verify the variation in the loss of seed dormancy during after-ripening and the interspecific and interpopulation variability in the degree of dormancy of seven wild and two cultivated rice species comprising 21 populations and two cultivars. METHODS: Four wild rice species from South America, Oryza glumaepatula, O. latifolia, O. grandiglumis and O. alta, and two O. sativa cultivars were tested in one experiment. In a second experiment, five wild species, O. punctata, O. eichingeri, O.rufipogon, O. latifolia and O. glumaepatula, and one cultivated species (O. glaberrima) were evaluated. Initial germination tests were performed soon after the seeds were harvested and subsequently at 2-month intervals, for a total of six storage periods in the first experiment and three in the second. All tests were conducted in the dark at a temperature of 27 degrees C. KEY RESULTS: Different patterns of after-ripening among populations within and between species were observed. CONCLUSIONS: The cultivated species (O. sativa and O. glaberrima) and, amongst the wild species, the tetraploids O. latifolia, O. grandiglumis and the diploids O. eichingeri and O. punctata, had weak dormancy, losing it completely 2 months after harvest, while O. rufipogon and O. glumaepatula exhibited pronounced dormancy. The latter showed different patterns of after-ripening between populations indigenous to the Amazon region and those originating in the Paraguay River system. Seeds of Solimoes (Amazon) and Japura origin showed weak dormancy whereas those of Paraguay origin showed deep dormancy. Ecological differences among natural habitats may be involved in such differentiation.     

云南野生稻不同染色体组型和外植体材料的离体培养研究

云南野生稻不同外植体愈伤组织诱导能力差别较大。花粉培养中愈伤组织诱导率差异在0％～11．8％之间,用成熟胚诱导愈伤组织,其诱导率在18．0％～35．2％之间,茎叶培养则在12．0％～25．0％之间。云南野生稻不同外植体诱导的愈伤组织再分化为绿苗的分化率在8．3％～100．0％之间。疣粒稻组培特性最好,东乡普通野生稻和景洪普通野生稻次之,药用稻最难组培。本文建立了疣粒、东乡、景洪普野3种野生稻的离体无性系,为长期保存云南野生稻资源奠定了基础。     

云南野生稻离子吸收效率及其动力学特征研究

分析和检测4种,共6个不同类型的野生稻吸收H2PO4^-、K^＋、Mg^2＋、NO3^-的动力学特征以及培养液pH值变化的研究结果表明,景洪普通野生稻吸收K^＋较强;药用野生稻和元江普通野生稻次之。小粒野生稻吸收磷的能力最强。元江普通野生稻吸收Mg^2＋和NO3^-的能力强。药用野生稻吸收Mg^2＋较强;直立型景洪普通野生稻有较强的吸收NO3^-的能力。     

中国野生稻原生境保护方法研究

在充分调查我国野生稻种质资源现状的基础上,分析了野生稻的濒危状况及其致濒因素,制定了野生稻原生境保护的选点原则,结合江西东乡、广东高州、云南元江和广西武宣等4个野生稻原生境保护示范点建设及各个保护点的社会、经济和环境条件,提出了围墙、围栏、植物篱笆和农民参与性等不同的保护方法,并就原生境保护点的开发性保护管理模式进行了探讨。     

茶陵野生稻苗期耐冷性与抗氧化系统的关系

为探讨茶陵野生稻苗期耐冷的生理机制,以耐冷性强弱不同的栽培稻及东乡野生稻为对照,研究了茶陵野生稻苗期冷胁迫后抗氧化系统的变化.结果表明,茶陵野生稻经冷胁迫后,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性以及抗坏血酸(AsA)、还原型谷胱甘肽(GSH)含量增加幅度大于冷敏感对照,而稍小于或相当于耐冷对照;超氧阴离子(O2)产生速率和丙二醛(MDA)含量低于冷敏感对照品种,而稍高于或相当于耐冷对照.说明茶陵野生稻苗期耐冷性与其抗氧化系统冷胁迫后的适应性变化密切相关.     

云南野生稻籽粒淀粉合成关键酶活性测定

为研究云南3种野生稻直链淀粉合成机制并利用其直链淀粉含量较低的优良品质,以云南3种野生稻和4种当地栽培稻为材料,研究野生稻灌浆期籽粒4种淀粉合成关键酶(包括ADPG焦磷酸化酶、可溶性淀粉合成、颗粒凝结型淀粉合成酶、淀粉分支酶)活性变化。结果表明,野生稻中4种淀粉合成酶的变化趋势与栽培稻相似,但活性有较大差别。颗粒凝结型淀粉合成酶的活性与直链淀粉含量呈正相关,说明在野生稻中同样是由颗粒凝结型淀粉合成酶控制直链淀粉的合成。同时发现在同一灌浆期,同种材料中可溶性淀粉合成酶和淀粉分支酶的活性变化呈相反趋势,推测这两种酶之间可能在淀粉合成过程中存在某种反馈调节机制。     

普通野生稻和亚洲栽培稻线粒体DNA的RFLP分析

通过７个探针、１７种内切酶探针组合对１１８份普通野生稻和７６份亚洲栽培稻的线粒体ＤＮＡ（ｍｔＤＮＡ）ＲＦＬＰ分析表明,籼粳分化是亚洲栽培稻线粒体基因组分化的主流,７６个栽培稻中,３６个品种ｍｔＤＮＡ为籼型,４０个品种ｍｔＤＮＡ为粳型。普通野生稻ｍｔＤＮＡ以籼型为主（８６份）,粳型较少（７份）,１份类型难以确定,还有２４份没有籼粳分化。     

栽培稻与其野生近缘种的可交配性研究

通过人工授粉方法研究栽培稻与二倍体和四倍体野生稻之间的可交配性.以栽培稻为对照,用光学显微镜观察不同野生稻花粉在同一栽培种柱头上的萌发生长情况.结果表明,在栽培稻柱头上普通野生稻(AA)花粉萌发最好,与对照萌发情况相近.药用野生稻(CC)萌发差,表现为柱头上花粉附着量少,开始萌发时间迟,萌发量少,花粉管扭曲、缠绕、伸长慢等.四倍体野生稻未观察到有萌发现象.说明普通野生稻与栽培稻亲缘关系近,可交配性好;药用野生稻与栽培稻可交配性差;四倍体野生稻与栽培稻可交配性极差.由此推断,转基因水稻与普通野生稻通过花粉途径发生基因漂移的可能性很大,而与药用野生稻和其他基因组野生稻发生基因漂移的可能性很小.     

Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races

Several diazotrophic species of Azoarcus spp. occur as endophytes in the pioneer plant Kallar grass. The purpose of this study was to screen Asian wild rice and cultivated Oryza sativa varieties for natural association with these endophytes. Populations of culturable diazotrophs in surface-sterilized roots were characterized by 16S rDNA sequence analysis, and Azoarcus species were identified by genomic fingerprints. A. indigens and Azoarcus sp. group C were detected only rarely, whereas Azoarcus sp. group D occurred frequently in samples of flooded plants: in 75% of wild rice, 80% of land races of O. sativa from Nepal and 33% of modern cultivars from Nepal and Italy. The putatively endophytic populations of diazotrophs differed with the rice genotype. The diversity of cultured diazotrophs was significantly lower in wild rice species than in modern cultivars. In Oryza officinalis (from Nepal) and O. minuta (from the Philippines), Azoarcus sp. group D were the predominant diazotrophic putative endophytes in roots. In contrast, their number was significantly lower in modern cultivars of O. sativa, whereas numbers and diversity of other diazotrophs, such as Azospirillum spp., Klebsiella sp., Sphingomonas paucimobilis, Burkholderia sp. and Azorhizobium caulinodans, were increased. In land races of O. sativa, the diazotrophic diversity was equally high; however, Azoarcus sp. was found in high apparent numbers. Similar differences in populations were also observed in a culture-independent approach comparing a wild rice (O. officinalis) and a modern-type O. sativa plant: in clone libraries of root-associated nitrogenase (nifH) gene fragments, the diazotrophic diversity was lower in the wild rice species. New lineages of nifH genes were detected, e.g. one deeply branching cluster within the anf (iron) nitrogenases. Our studies demonstrate that the natural host range of Azoarcus spp. extends to rice, wild rice species and old varieties being preferred over modern cultivars.     

导入反义蜡质基因改良水稻稻米的食味品质和营养品质

经PCR、Southern blot和稻米GUS检测,蜡质基因反义片段与gus构成的融合基因已整合到8株水稻基因组中,并能正确表达。通过稻米GUS染色追踪分析获得转反义蜡质基因纯合后代。将转基因植株的稻米送农业部稻米及制品质量监督检验测试中心进行糙米蛋白质含量和直链淀粉含量测定。结果显示：（1）在转反义蜡质基因纯合的超2-10粳稻后代中,大多数单株糙米在直链淀粉含量降低的同时,蛋白质含量有不同程度提高,糙米蛋白质含量和直链淀粉含量呈显著负相关关系;（2）对照糙米直链淀粉平均含量和糙米蛋白质平均含量分别为13.4%和9.5%。在转基因植株中,稻米直链淀粉含量最低的为11.4%,而蛋白质含量也相对最高,为13.5%。本试验结果表明在水稻中导入反义蜡质基因不仅能够降低水稻稻米的直链淀粉含量,还有可能提高水稻稻米的蛋白质含量。     

8个野生稻种的叶片硅体研究

用强酸溶液分离8个野生稻种叶片中的硅体并用光学显微镜和电子扫描显微镜进行观测研究.结果显示,(1)叶片含有3种类型的硅体,即不规则硅体、哑铃形硅体和扇形硅体,前2种硅体不能作为属内分类的依据,但短药野生稻哑铃形硅体的表面纹饰独特,可作为识别该物种的特征.(2)同一个物种的扇形硅体变异很大,统计学表明它总以其中的某一种类型占有优势,紧穗野生稻和短舌野生稻中小型硅体占优势,药用野生稻和普通野生稻中约有一半为中型硅体,另一半为小型硅体,其余4种野生稻包括斑点野生稻、澳洲野生稻、展颖野生稻和短药野生稻中绝大多数为中型硅体,即野生稻的扇形硅体以中、小型为主.(3)根据扇面长/扇柄长的比值,将扇形硅体划分为长柄型、中间形和短柄型3种形态,依此对8个稻种的硅体分类.结果表明,斑点野生稻、药用野生稻、紧穗野生稻和澳洲野生稻以长柄型为优势;普通野生稻、短舌野生稻、展颖野生稻和短药野生稻则以中间型为主,且扇形硅体的扇面长/扇柄长的比值的大小与野生稻染色体组型或原产地有关.     

普遍野生稻和亚洲栽培稻遗传多样性的研究

用 ４４个 ＲＦＬＰ标记对来自中国、印度、泰国等亚洲 １０个国家的普通野生稻（简称普野,下同）和来自多个国家的７５个栽培稻品种,从多态位点的比率、等位基因数、基因型数、平均杂合度及平均基因多样性等多个方面,比较了不同国家和不同地区的普通野生稻、栽培稻籼粳亚种及栽培稻与普野之间遗传多样性的差异。结果表明：中国普野的遗传多样性最大;其次是印度普野;南亚普野的平均基因多样性大于东南亚普野,而多态位点的比率、等位基因数及基因型数等却低于东南亚普野;栽培稻的遗传多样性明显小于普通野生稻。在所检测的４４个位点中,栽培稻的多态位点数仅为野生稻的３／４,等位基因数约为野生稻的６０％,基因型种类约为野生稻的１／２。栽培稻中籼稻的遗传多样性高于粳稻。在平均每个位点的实际杂合度上,以中国普野杂合度最高,普通野生稻是栽培稻的２倍。说明从野生稻演化成栽培稻的过程中,经过自然选择和人工选择,杂合度降低,等位基因减少,基因多样性下降。     

RAPD, RFLP and SSLP analyses of phylogenetic relationships between cultivated and wild species of rice.

RAPD, RFLP, nuclear SSLP and chloroplast SSLP analyses were carried out to clarify the phylogenetic relationships among A-genome species of rice. In total, 12 cultivars of Oryza sativa (4 Japonica, 3 Javanica and 5 Indica), one cultivar of O. glaberrima, and 17 wild accessions (12 O. rufipogon, 2 O. glumaepatula, 1 O. longistaminata, 1 O. meridionalis and 1 O. barthii) were used. Their banding patterns were scored and compared to evaluate the similarity between accessions. Genetic differentiation within and between taxa was examined based on the average similarity indices. Except for chloroplast SSLP analysis, the average similarities were higher within O. sativa than within O. rufipogon, and O. sativa Indica had greater intrasubspecific variation than Japonica and Javanica. Comparisons between cultivated and wild species showed that O. sativa was closely related to O. rufipogon, while O. glaberrima was closely related to O. barthii. This indicated that two cultivated species, O. sativa and O. glaberrima, originated from O. rufipogon and O. barthii, respectively. Domestication of O. sativa seemed to be diphyletic, since strong similarity was observed between O. sativa Japonica-Javanica and O. rufipogon from China and between O. sativa Indica and O. rufipogon from tropical Asia. In addition, dendrograms for RAPD, RFLP, and nuclear and chloroplast SSLP analyses were constructed to reveal the overall genetic relationships among A-genome species. In all analyses, O. sativa and O. glaberrima formed groups with O. rufipogon and O. barthii, respectively. However, their manners of clustering with other wild species were not the same. The results of RAPD and RFLP analyses indicate that O. glumaepatula was relatively close to the groups of O. sativa and O. glaberrima whereas O. longistaminata and O. meridionalis were highly differentiated from other A-genome species. On the other hand, clear interspecific relationships were not obtained by nuclear or chloroplast SSLP analyses.     

Polyphyletic origin of cultivated rice: based on the interspersion pattern of SINEs

The wild rice species Oryza rufipogon with wide intraspecific variation is thought to be the progenitor of the cultivated rice species Oryza sativa with two ecotypes, japonica and indica. To determine the origin of cultivated rice, subfamily members of the rice retroposon p-SINE1, which show insertion polymorphism in the O. sativa -O. rufipogon population, were identified and used to "bar code" each of 101 cultivated and wild rice strains based on the presence or absence of the p-SINE1 members at the respective loci. A phylogenetic tree constructed based on the bar codes given to the rice strains showed that O. sativa strains were classified into two groups corresponding to japonica and indica, whereas O. rufipogon strains were in four groups, in which annual O. rufipogon strains formed a single group, differing from the perennial O. rufipogon strains of the other three groups. Japonica strains were closely related to the O. rufipogon perennial strains of one group, and the indica strains were closely related to the O. rufipogon annual strains, indicating that O. sativa has been derived polyphyletically from O. rufipogon. The subfamily members of p-SINE1 constitute a powerful tool for studying the classification and relationship of rice strains, even when one has limited knowledge of morphology, taxonomy, physiology, and biochemistry of rice strains.     

Nuclear- and chloroplast-microsatellite variation in A-genome species of rice.

Simple sequence length polymorphism analysis was carried out to reveal microsatellite variation and to clarify the phylogenetic relationships among A-genome species of rice. Total DNA from 29 cultivars (23 Oryza sativa and 6 O. glaberrima) and 30 accessions of wild A-genome species (12 O. rufipogon, 5 O. glumaepatula, 2 O. longistaminata, 6 O. meridionalis, and 5 O. barthii) was used as a template for PCR to detect 24 nuclear and 10 chloroplast microsatellite loci. Microsatellite allelic diversity was examined based on amplified banding patterns. Microsatellites amplified clearly in all 59 accessions, with an average of 18.4 alleles per locus. The polymorphism information content (PIC) value ranged from 0.85 to 0.94, with an average of 0.89. At the species level, high average PIC values were observed in O. sativa (0.79) and O. rufipogon (0.80). For chloroplast microsatellites, the average number of alleles per locus and the average PIC value were 2.9 and 0.38, respectively. While the magnitude of diversity was much greater for nuclear microsatellites than for chloroplast microsatellites, they showed parallel patterns of differentiation for each taxonomic group. Using the ratio of common alleles (estimated as size of amplified fragments) as a similarity index, the average percentages of common microsatellite alleles were calculated between taxa. For both nuclear and chloroplast microsatellites, O. sativa showed the highest similarity values to O. rufipogon, and O. glaberrima was most similar to O. barthii. These data support previous evidence that these cultivars originated from the corresponding wild ancestral species.