重复DNA顺序pRRD9在稻属中的拷贝数测定

本文应用狭缝印渍杂交方法,把水稻基因组总DNA和含水稻中度重复顺序片段的质粒(pRRD9)DNA分别转移到尼龙膜上形成狭缝印渍、然后用~(32)P标记的 pRRD9插入片段进行杂交、根据各狭缝印渍的放射性强度,测定水稻(Oryza)一些栽培种和野生种基因组中重复DNA顺序的拷贝数,并就拷贝数与水稻进化关系及基因组型的联系进行讨论.     

重复DNA顺序pRRD9在稻属中的拷贝数测定

本文应用狭缝印渍杂交方法,把水稻基因组总DNA和含水稻中度重复顺序片段的质粒(pRRD9)DNA分别转移到尼龙膜上形成狭缝印渍、然后用³²P标记的 pRRD9插入片段进行杂交、根据各狭缝印渍的放射性强度,测定水稻(Oryza)一些栽培种和野生种基因组中重复DNA顺序的拷贝数,并就拷贝数与水稻进化关系及基因组型的联系进行讨论.     

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.     

The Genic Nature of Gamete Eliminator in Rice

The two cultivated rice species, Oryza sativa and Oryza glaberrima, are morphologically alike but are reproductively isolated from each other by hybrid sterility. The hybrid is male sterile but partially female fertile. Backcross experiments were conducted to introduce an alien factor controlling hybrid sterility from O. glaberrima (W025) into O. sativa (T65wx) and examine the genetic basis. An extracted sterility factor, closely linked to the wx locus, induced gametic abortion due to allelic interaction and was tentatively designated as S(t). The segregation patterns for infertility was explained by assuming that W025 and T65wx carried S(t) and S(t)a, respectively, and gametes with S(t)a aborted only in the heterozygote (S(t)/S(t)a) although the elimination of female gametes was incomplete. Thus, S(t) seemed to be intermediate between a gamete eliminator and pollen killer. However, S(t) was proven to be likely the same as S1 which was formerly reported as gamete eliminator in a different genetic background of O. sativa. In addition, a chromosomal segment containing S1 (or S(t] caused a marked suppression of crossing over around it, suggesting the presence of an inversion. Further, female transmission of S1a increased as the segment containing S1 became small by recombination. After S1 was further purified by successive backcrosses up to the BC15 generation, it became pollen killer. The present results give evidence that a profound sterility gene such as gamete eliminator can be made from accumulation of pollen killer and its modifier(s) when pollen killer and modifier(s) are linked, they behave as a gene complex in the hybrid.     

Genetic differentiation for nuclear,mitochondrial and chloroplast genomes in common wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.) and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The genetic differentiation of nuclear, mitochondrial (mt) and chloroplast (cp) genomes was investigated by Southern and PCR analysis using 75 varieties of cultivated rice ( Oryza sativa L.) and 118 strains of common wild rice (CWR, Oryza rufipogon Griff.) from ten countries of Asia. The distinguishing differences between the Indica and Japonica cultivars were detected both in the nuclear genome and the cytoplasmic genome, confirming that the Indica-Japonica differentiation is of major importance for the three different classes of genome in cultivated rice. This differentiation was also detected in common wild rice with some differences among the genome compartments and the various regions. For nuclear DNA variation, both Indica-like and Japonica-like types were observed in the Chinese CWR, with the latter more-frequent than the former. No Japonica-like type was found in South Asia, and only two strains of the Japonica-like type were detected in Southeast Asia, thus the Indica-like type is the major type among South and Southeast Asian CWR. For mtDNA, only a few strains of the Japonica-like type were detected in CWR. For cpDNA, the Japonica type was predominant among the CWR strains from China, Bangladesh and Burma, while the Indica type was predominant among the CWR strains from Thailand, Malaysia, Cambodia and Sri Lanka, and both types were found in similar frequencies among the Indian CWR. Altogether, however, the degree of Indica-Japonica differentiation in common wild rice was much-less important than that in cultivated rice. Cluster analyses for nuclear and mitochondrial DNA variation revealed that some CWR strains showed large genetic distances from cultivated rice and formed clusters distinct from cultivated rice. Coincidence in the genetic differentiation between the three different classes of genome was much higher in cultivated rice than in CWR. Among the 75 cultivars, about 3/4 entries were "homoeotype" showing congruent results for nuclear, mt and cpDNA regarding the Indica-Japonica differentiation. In CWR, the proportions of homoeotypes were 5.7%, 15% and 48.8% in China, South Asia and Southeast Asia, respectively. Based on the average genetic distance among all the strains of CWR and cultivated rice for nuclear and mitochondrial genomes, the variability of the nuclear genome was found to be higher than that of the mitochondrial genome. The global pattern based on all genomes shows much-more diversification in CWR than that in cultivated rice.     

QTL clusters reflect character associations in wild and cultivated rice

The genetic basis of character association related to differentiation found in the primary gene pool of rice was investigated based on the genomic distribution of quantitative trait loci (QTLs). Major evolutionary trends in cultivated rice of Asiatic origin (Oryza sativa) and its wild progenitor (O. rufipogon) are: (1) differentiation from wild to domesticated types (domestication), (2) ecotype differentiation between the perennial and annual types in wild races, and (3) the Indica versus Japonica type differentiation in cultivated races. Using 125 recombinant inbred lines (RILs) derived from a cross between an Indica cultivar of O. sativa and a strain of O. rufipogon carrying some Japonica-like characteristics, we mapped 147 markers, mostly RFLPs, on 12 chromosomes. Thirty-seven morphological and physiological quantitative traits were evaluated, and QTLs for 24 traits were detected. The mapped loci showed a tendency to form clusters that are composed of QTLs of the domestication-related traits as well as Indica/Japonica diagnostic traits. QTLs for perennial/annual type differences did not cluster. This cluster phenomenon could be considered "multifactorial linkages" followed by natural selection favoring co-adapted traits. Further, it is possible that the clustering phenomenon is partly due to pleiotropy of some unknown key factor(s) controlling various traits through diverse metabolic pathways. Chromosomal regions where QTL clusters were found coincided with the regions harboring genes or gene blocks where the frequency of cultivar-derived alleles in RILs is higher than expected. This distortion may be partly due to unconscious selection favoring cultivated plant type during the establishment of RILs.     

Genetic basis of hybrid breakdown in a Japonica/Indica cross of rice,Oryza sativa L

Reproductive barriers often arise in hybrid progeny between two varietal groups of Asian cultivated rice ( Oryza sativa L.), Japonica and Indica. Hybrid breakdown showing poor growth habit, and complete sterility was found in the backcrossed progeny derived from a cross between a Japonica variety, Asominori, and an Indica variety, IR24. We employed RFLP analysis in the segregating population to study the genetic basis underlying hybrid breakdown. It was found that the hybrid breakdown is caused by a set of two nuclear genes, which were symbolized as hwe1 and hwe2. The parental varieties, Asominori and IR24, carry hwe1(+) hwe1(+) hwe2hwe2 and hwe1hwe1hwe2(+) hwe2(+) genotypes, respectively, whereas the progenies that showed a weakness performance carry the double recessive genotype ( hwe1hwe1hwe2hwe2). Abnormality was not observed in the progenies that carry the other genotypes, indicating that a single dominant allele at either locus is necessary for normal growth. Based on linkage analysis with RFLP markers, the hwe1 locus was located between RFLP markers R1869 and S1437 on chromosome 12 and the hwe2 locus was located between R3192 and C1211 on chromosome 1. The genetic basis was reconfirmed using near-isogenic lines carrying the genes with reciprocal genetic backgrounds. The present study provides clear evidence, viewed by previous workers, that hybrid breakdown is attributed to complementary genes from both parents.     

Differentiation of Indica-Japonica rice revealed by insertion/deletion (InDel) fragments obtained from the comparative genomic study of DNA sequences between 93-11 (Indica) and Nipponbare (Japonica)

DNA polymorphisms from nucleotide insertion/deletions (InDels) in genomic sequences are the basis for developing InDel molecular markers.To validate the InDel primer pairs on the basis of the comparative genomic study on DNA sequences between an Indica rice 93-11 and a Japonica rice Nipponbare for identifying Indica and Japonica rice varieties and studying wild Oryza species,we studied 49 Indica,43 Japonica,and 24 wild rice accessions collected from ten Asian countries using 45 InDel primer pairs.Results indicated that of the 45 InDel primer pairs,41 can accurately identify Indica and Japonica rice varieties with a reliability of over 80%.The scatter plotting data of the principal component analysis (PCA) indicated that:(i) the InDel primer pairs can easily distinguish Indica from Japonica rice varieties,in addition to revealing their genetic differentiation;(ii) the AA-genome wild rice species showed a relatively close genetic relationship with the Indica rice varieties;and (iii)the non-AA genome wild rice species did not show evident differentiation into the Indica and Japonica types.It is concluded from the study that most of the InDel primer pairs obtained from DNA sequences of 93-11 and Nipponbare can be used for identifying lndica and Japonica rice varieties,and for studying genetic relationships of wild rice species,particularly in terms of the Indica-Japonica differentiation.     

长江三角洲主要超级稻CH4排放特征及其与植株生长特性的关系

采用盆栽试验研究了长江三角洲14个主要超级稻品种(6个粳型超级稻和8个籼型杂交超级稻)CH₄排放特征及其与植株生长特性之间的关系.结果表明: 粳型和籼型超级稻全生育期CH₄排放均呈双峰模式,排放峰值分别出现在分蘖盛期和孕穗期.粳型超级稻的平均CH₄排放总量比籼型超级稻高37.6%(P<0.01),品种间排放差异主要出现在生长后期.虽然两种类型超级稻的CH₄排放总量均与最大叶面积呈显著正相关,但CH₄排放与其他生长特性的关系因品种类型而异.在株高上,粳型超级稻CH₄排放总量与株高呈显著正相关,而籼型超级稻的相关不显著.在生产力上,籼型超级稻CH₄排放总量与其总生物量、籽粒产量和收获指数呈显著负相关,而粳型超级稻的相关不显著.籼型超级稻CH₄排放量低主要是由于其根系生物量显著高于粳型超级稻.     

长江三角洲主要超级稻CH4排放特征及其与植株生长特性的关系

采用盆栽试验研究了长江三角洲14个主要超级稻品种(6个粳型超级稻和8个籼型杂交超级稻)CH₄排放特征及其与植株生长特性之间的关系.结果表明: 粳型和籼型超级稻全生育期CH₄排放均呈双峰模式,排放峰值分别出现在分蘖盛期和孕穗期.粳型超级稻的平均CH₄排放总量比籼型超级稻高37.6%(P<0.01),品种间排放差异主要出现在生长后期.虽然两种类型超级稻的CH₄排放总量均与最大叶面积呈显著正相关,但CH₄排放与其他生长特性的关系因品种类型而异.在株高上,粳型超级稻CH₄排放总量与株高呈显著正相关,而籼型超级稻的相关不显著.在生产力上,籼型超级稻CH₄排放总量与其总生物量、籽粒产量和收获指数呈显著负相关,而粳型超级稻的相关不显著.籼型超级稻CH₄排放量低主要是由于其根系生物量显著高于粳型超级稻.     

Plant regeneration from protoplasts of Indica type rice and CMS rice

Cell suspensions were established from 12 rice genotypes including Indica type, Japonica type and cytoplasmic male sterile (cms) rice lines of both types. 133 green plants were regenerated from protoplasts isolated from an Indica type rice and 15 plants were obtained from a Japonica type cms rice. A cell suspension, from which protoplasts can be isolated (about 10⁶/ml) but from which cell division has not yet been induced, has been established from the most widely used Indica type cms rice which contains Wild Abortive (WA) cytoplasm.     

The extent of parental genotypic divergence determines maximal heterosis by increasing fertility in inter-subspecific hybrids of rice (Oryza sativa L.)

The magnitude of heterosis in F₁ hybrids is related not only to the performance of parents per se but also to the genetic diversity between two parents. The extent of genotypic divergence between hybrid rice parents was investigated at the molecular level, using two subsets of rice materials: a subset of doubled haploid (DH) lines derived from an Indica × Japonica cross (Gui630/02428) and another subset of Indica or Japonica lines representative of a broad spectrum of the Asian cultivated rice gene pool, including landraces, primitive cultivars, historically important cultivars, modern elite cultivars, super rice and parents of superior hybrids. 57 entries deliberately selected from the 81-DH lines (in total) were testcrossed to two widely used rice lines in China, photoperiod-sensitive genic male sterile (PGMS) N422s and thermo-sensitive genic male sterile (TGMS) Peiai64s. Results of the two sets of test-cross F₁ populations showed congruently that parental genotypic divergence has a relatively low impact on heterosis for the two yield components, i.e., panicle number and 1000-grain weight, but it has a great bearing on fertility parameters, i.e., filled grains per plant and seedset. Heterosis for grain yield in the two test-cross populations exhibited a sharp maximum when the proportion of Japonica alleles in the male parent was between 50 and 60%, so was the heterosis for fertility parameters correspondingly. Thus fertility parameters were the most sensitive and important factors which were influenced by the extent of parental genotypic divergence. Moreover, our results showed that parents with moderate extent of genotypic divergence played an important role in the use of inter-subspecific rice heterosis.     

Relative contributions of photochemical and non-photochemical routes to excitation energy dissipation in rice and barley illuminated at a chilling temperature

The mechanistic basis for differential sensitivities to chilling-induced photoinhibition among two rice ( Oryza sativa L.) cultivars (an Indica and a Japonica type) and one barley cultivar ( Hordeum vulgare L. cv. Albori) was examined. When leaf segments were exposed to moderate illumination at 4°C, a sustained decrease in the photochemical efficiency of photosystem (PS) II measured as the ratio of variable to maximal fluorescence (F_v/F_m) was observed for several hours. An analysis of fluorescence quenching revealed a sudden drop in PSII-driven electron transport rate (ETR) and a rapid rise in the reduction state of the primary electron acceptor Q_A upon exposure to chilling in moderate light. There was no appreciable difference in the level of non-photochemical quenching (NPQ) nor in the xanthophyll cycle activity between Japonica rice and barley. However, barley was capable of sustaining a higher ETR, thereby keeping a lower reduction state of Q_A throughout the chilling for 6 h. The Indica rice was characterized by the lowest ability to develop the xanthophyll cycle-associated NPQ, particularly the fast relaxing NPQ component (qf), accompanied by the highest reduction state of Q_A and photoinhibitory quenching (qI). It is concluded that the lower susceptibility of barley to chilling-induced photoinhibition than Japonica rice is attributable to its higher potential to dissipate excess light energy via a photochemical mechanism, whereas Indica rice is more sensitive to photoinhibition at a chilling temperature than Japonica rice, due primarily to its lower capacity to develop an efficient NPQ pathway.     

Polymorphism at the esterase isozyme locus Est10 associated with phylogenetic differentiation in rice

A new esterase isozyme locus, Est10, with 6 alleles including the null form, has been found in rice by using polyacrylamide gel electrophoresis. Thirty F(2) populations of all possible combinations between 5 different band morphs were studied. The segregation pattern indicated that bands 1, 2, 3, 4, and the null form (0) were allelic with each other. The alleles of Est10 were distributed at different frequencies among different varietal groups of rice and also between cultivated rice and its wild relatives (Oryza rufipogon Griff.). Alleles 1 and 2 were frequently found in Japonica and Indica types, respectively. Allele 3 showed a high frequency in Aus and Boro, both Indica types cultivated in South Asia. Allele 4 was frequent in wild rice O. rufipogon. Judging from the linkage between Est10 and RFLP marker RG220 and isozyme marker Est5, Est10 is located on chromosome 1. The importance of this locus in evolutionary studies of rice is discussed.     

Differentiation and classification of parental lines and favorable genic interactions affecting F1 fertility in distant crosses of rice (Oryza sativa L.)

 This study was intended to investigate the extent of genetic differentiation in parental lines of rice hybrids and to analyze the genetic basis underlying the fertility phenomenon in distant crosses. Two subsets of rice material (111 entries in total) were used, including 81 doubled-haploid (DH) lines and 30 Indica and Japonica rice varieties or lines (as a control). The DH lines was derived from a heterotic Indica/Japonica cross (Gui630/02428) by anther culture. The materials in the control represent a broad spectrum of the Asian cultivated rice gene pool including landraces, primitive cultivars, historically important cultivars, modern elite cultivars, super rice and parents of superior hybrids. In accordance with the NC II design, 57 out of the DH lines were test-crossed to two important wide compatibility lines: photoperiod-sensitive genetic male sterile (PGMS) line N422s and thermo-sensitive genetic male sterile (TGMS) line Peiai64s. The F₁s and their parents, 182 entries in total, were examined for the performance of seven traits in a replicated field trial. All the rice materials was surveyed for polymorphisms using 92 RFLP markers selected from two published molecular marker linkage maps. Genotypes of the F₁ hybrids at the molecular-marker loci were deduced from the parental genotypes. The analysis showed that there were two types of genetic differentiation in the two subsets of rice material; that is, qualitative differentiation in the control and quantitative differentiation in the DH lines. In addition, favorable genic interactions (both intra- or inter-locus) contributed to better increase the fertility in hybrids of distant crosses through incorporation of a wide-compatibility line as the female parent. Favorable genic interactions can be applied in hybrid rice breeding programs by selecting parents with an appropriate extent of genetic differentiation. Received: 5 June 1997 / Accepted: 10 September 1997     

Regeneration of fertile plants from isolated zygotes of rice (Oryza sativa)

A simple mechanical method has been developed which allows the routine isolation of unfertilized and fertilized egg cells from ovules of Japonica and Indica rice varieties. In the experiments described, the majority of the egg cells and zygotes survived the isolation procedure when the donor plants were in a vigorous state. About 40% of the surviving zygotes underwent sustained development when cultured in Millicell inserts with a non-morphogenic rice feeder-cell culture. Nearly all zygote-derived callus cultures regenerated multiple shoots, which could be subsequently rooted with high efficiency. Zygote-derived plantlets matured to fertile plants when transplanted to soil. So far, about 80 independent plants each from the Japonica variety 'Taipei309' and the Indica variety 'IR58' have been regenerated. The potential of this single-cell regeneration system for marker gene-free transformation is discussed. Received: 26 November 1998 / Revision received: 15 March 1999 / Accepted: 21 March 1999     

东乡野生稻与栽培稻正反交种间杂种F_1的雄配子发生与发育

该文以东乡野生稻与栽培稻（美国光壳稻P1003）的正反交种间杂种F1（正交为光壳稻P1003×东乡野生稻;反交为东乡野生稻×光壳稻P1003）为试材,研究其各个减数分裂时期的染色体行为特征、染色体交换重组及雄配子发育特点。结果表明：正反交杂种F1花粉母细胞细胞核减数分裂的同步性较高,细胞质为连续型胞质分裂类型。在细胞核分裂的过程中,核仁在前期I到中期I逐渐消失。染色体在前期I到中期I逐渐收缩,变得更加清晰可见。在终变期和中期I,90.54%以上的花粉母细胞能形成12个二价体（含少数棒状二价体和8字型二价体）,部分花粉母细胞（正交9.46%,反交7.65%）出现少量的单价体、三价体和四价体。后期I观察到1.27%–1.35%的花粉母细胞含有1至数条落后染色体。最终有92.6%–94.8%的小孢子能发育成形态正常、染色能力强的成熟花粉粒。另外,在正交杂种F1的粗线期中存在2个核仁,而反交杂种F1及其双亲的粗线期只有1个核仁。这些研究结果可为作物品种改良、种质资源创新以及种间亲缘关系研究提供细胞学证据。     

In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants.     

不同类型甘蓝的可交配性研究

野生甘蓝是改良栽培甘蓝的重要资源,在漫长进化中,部分野生甘蓝类型间,以及与栽培甘蓝间杂交存在一定的生殖障碍。本研究选用8个栽培变种和7个野生类型品种,共35份甘蓝材料进行随机杂交,不同类型甘蓝的可交配性存在显著差异,甘蓝间的遗传差异、杂交方向以及生活习性等因素对甘蓝类植物可交配性存在显著影响。研究数据表明,以野生甘蓝为母本可以显著提高与栽培甘蓝杂交的效率。