Microsatellite diversity within Oryza sativa with emphasis on indica-japonica divergence

The molecular evolution of cultivated rice Oryza sativa L. has long been a subject of rice evolutionists. To investigate genetic diversity within and differentiation between the indica and japonica subspecies, 22 accessions of indica and 35 of japonica rice were examined by five microsatellite loci from each chromosome totalling 60 loci. Mean gene diversity value in the indica rice (H=0.678) was 1.18 times larger than in the japonica rice (H=0.574). Taking the sampling effect into consideration, average allele number in the indica rice was 1.40 times higher than that in the japonica rice (14.6 vs 10.4 per variety). Chromosome-based comparisons revealed that nine chromosomes (1, 2, 3, 4, 5, 8, 9, 10 and 11) harboured higher levels of genetic diversity within the indica rice than the japonica rice. An overall estimate of F(ST) was 0.084-0.158, indicating that the differentiation is moderate and 8.4-15.8% of the total genetic variation resided between the indica and japonica groups. Our chromosome-based comparisons further suggested that the extent of the indica-japonica differentiation varied substantially, ranging from 7.62% in chromosome 3 to 28.72% in chromosome 1. Cluster analyses found that most varieties formed merely two clusters for the indica and japonica varieties, in which two japonica varieties and five indica varieties were included in the counterpart clusters, respectively. The 12 chromosome-based trees further showed that 57 rice varieties cannot be clearly clustered together into either the indica or japonica groups, but displayed relatively different clustering patterns. The results suggest that the process of indica japonica differentiation may have proceeded through an extensive contribution by the alleles of the majority in the rice genome.     

Genetic diversity analysis of traditional and improved Indonesian rice (Oryza sativa L.) germplasm using microsatellite markers

The archipelago of Indonesia has a long history of rice production across a broad range of rice-growing environments resulting in a diverse array of local Indonesian rice varieties. Although some have been incorporated into modern breeding programs, the vast majority of these landraces remain untapped. To better understand this rich source of genetic diversity we have characterized 330 rice accessions, including 246 Indonesian landraces and 63 Indonesian improved cultivars, using 30 fluorescently-labeled microsatellite markers. The landraces were selected across 21 provinces and include representatives of the classical subpopulations of cere, bulu, and gundil rices. A total of 394 alleles were detected at the 30 simple sequence repeat loci, with an average number of 13 alleles per locus across all accessions, and an average polymorphism information content value of 0.66. Genetic diversity analysis characterized the Indonesian landraces as 68% indica and 32% tropical japonica, with an indica gene diversity of 0.53 and a tropical japonica gene diversity of 0.56, and a Fst of 0.38 between the two groups. All of the improved varieties sampled were indica, and had an average gene diversity of 0.46. A set of high quality Indonesian varieties, including Rojolele, formed a separate cluster within the tropical japonicas. This germplasm presents a valuable source of diversity for future breeding and association mapping efforts.     

Molecularmarker diversity and hybrid sterility in indica-japonica rice crosses

 The partial sterility of hybrids between the indica and japonica rice subspecies of Asian cultivated rice is a serious constraint for utilizing inter-subspecific heterosis in hybrid rice breeding. In this study, we have investigated the relationship between molecular-marker polymorphism and indica-japonica hybrid fertility using a diallel set involving 20 rice accessions including 9 indica and 11 japonica varieties. Spikelet fertility of the resulting 190 F₁s and their parents was examined in a replicated field trial. Intra-subspecific hybrids showed much higher spikelet fertility than inter-subspecific hybrids except in crosses involving wide-compatibility varieties. The parents were surveyed for DNA polymorphism using 96 RFLP and ten SSR markers, which revealed extensive genetic differentiation between indica and japonica varieties. A large number of markers detected highly significant effects on hybrid fertility. The chromosomal locations for many of the positive markers coincided well with previously identified loci for hybrid sterility. The correlation between hybrid fertility and parental distance was low in both intra- and inter-subspecific crosses. The results suggest that the genetic basis of indica-japonica hybrid sterility is complex. It is the qualitative, rather than the quantitative, difference between the parents that determines the fertility of hybrids. Received: 3 January 1997/Accepted: 17 January 1997     

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。     

香稻资源遗传多样性的比较

利用60个水稻SSR标记, 对来自国内外的370份香稻材料的遗传多样性进行了比较分析。结果共检测到361个等位基因, 每个位点的等位基因变幅为2~10个, 平均Nei基因多样性指数(He)为0.663, 变幅为0.104(RM308)~0.885(RM2634)。籼粳亚种间的遗传多样性具有明显差异, 籼稻的等位基因数和Nei基因多样性指数均高于粳稻。地方品种的遗传多样性高于选育品种, 选育品种等位基因数仅为地方品种的86.5%。分子方差分析表明, 香稻材料中总变异的43.08%是由于亚种间的遗传差异引起的。不同稻区的遗传分化程度总体介于1.69%~14.40%之间。其中, 华南与西南、华中与西南地方品种间遗传差异的分化程度达显著水平。聚类分析将参试材料明显分为籼粳两大类, 同时地域相同(稻区)、相邻省份的香稻材料基本归为同一类群。     

Molecular diversity and multilocus organization of the parental lines used in the International Rice Molecular Breeding Program

One hundred and ninety three parental lines obtained from 26 countries for an international rice molecular breeding program were evaluated using 101 well-distributed simple sequence repeat (SSR) markers. An overall genetic diversity of 0.68 and an average of 6.3 alleles per locus were revealed, indicating a high level of genetic variation in these lines. Cluster analysis of the 193 accessions showed three major groups and nine subgroups. Group I corresponded to the classical indica subspecies, whereas groups II and III belong to the japonica subspecies. Indica and japonica differentiation accounted for only 6.5% of the total variation in the entire sample and 93.5% was due to within-subspecies diversity. Differentiation among eco-geographic regions accounted for 24% of the diversity within the subspecies. Larger amounts of the eco-geographical differentiation were resolved within japonica than within indica. The largest indica-japonica differentiation based on the single locus level was detected by markers on chromosomes 9 and 12, while the smallest differentiation was detected by markers on chromosomes 4 and 8. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between indica and japonica and among the main geographic regions. The multilocus analysis in genetic diversity showed a higher proportion of variation caused by predominant non-random associations of different loci within and among the classified subspecies and geographic subdivisions. The results suggest that selection for eco-geographical adaptation on multilocus associations was largely responsible for the maintenance of the extensive variation in the primary gene pool of rice.Communicated by Q. ZhangS.B. Yu and W.J. Xu contributed equally to the work     

RAPD Determinations on the Genetic Differentiation of Chinese Common Wild Rice

Ninety accessions which included Chinese common wild rice (Oryza rufipogon) from 8 provinces and traditional cultivars from lower and middle basins of Yangtze River, southeast of China and Yunnan Province as well as some commercial varieties were analyzed by RAPD with 24 primers. A scattered figure suggesting the indica-japonica and wild-domestication differentiations among 90 rice accessions was generated based on RAPD data. The results indicated that Chinese common wild rice, indica and japonica accessions were divided into 3 groups respectively. Chinese common wild rice were somewhat closer to the japonica type than the indica type.     

Classification of rice germplasm. II. Discrimination of indica from japonica via analysis of amplicon length polymorphisms

The polymerase chain reaction (PCR) was used to survey DNA sequence variation among 12 indica and 10 japonica rice cultivars. Of the 143 primer pairs used, 37 detected amplicon length polymorphism (ALP) and 11 revealed PCR banding patterns paralleled with the indica/japonica differentiation. Thus the 11 primer pairs were used to discriminate the two rice subspecies. A collection of 116 accessions representing the breadth of rice germplasm was analyzed for ALP at the 11 loci. Rice accessions with scores of 0.3 or more were classified as indica while those with –0.3 or less were classified as japonica. Those with scores from –0.3 to 0.3 were considered intermediate. With this criterion, 70 accessions were classified as indica, 35 accessions as japonica, and 11 accessions as intermediate. The concept and the approach used here for rice should be equally applicable for classifying other plant species. Received: 1 July 1997 / Revision received: 4 December 1997 / Accepted: 29 December 1997     

Subspecies-specific intron length polymorphism markers reveal clear genetic differentiation in common wild rice （Oryza rufipogon L.） in relation to the domestication of cultivated rice （O. sativa L.）

It is generally accepted that Oryza rufipogon is the progenitor of Asian cultivated rice （O. sativa）. However, how the two subspecies of O. sativa （indica and japonica） were domesticated has long been debated. To investigate the genetic differentiation in O. rufipogon in relation to the domestication of O. sativa, we developed 57 subspecies-specific intron length polymorphism （SSILP） markers by comparison between 10 indica cultivars and 10 japonica cultivars and defined a standard indica rice and a standard japonica rice based on these SSILP markers. Using these SSILP markers to genotype 73 O. rufipogon accessions, we found that the indica alleles and japonica alleles of the SSILP markers were predominant in the O. rufipogon accessions, suggesting that SSILPs were highly conserved during the evolution of O. sativa. Cluster analysis based on these markers yielded a dendrogram consisting of two distinct groups： one group （Group I） comprises all the O. rufipogon accesions from tropical （South and Southeast） Asia as well as the standard indica rice; the other group （Group II） comprises all the O. rufipogon accessions from Southern China as well as the standard japonica rice. Further analysis showed that the two groups have significantly higher frequencies of indica alleles and japonica alleles, respectively. These results support the hypothesis that indica rice and japonica rice were domesticated from the O. rufipogon of tropical Asia and from that of Southern China, respectively, and suggest that the indica-japonica differentiation should have formed in O. rufipogon long before the beginning of domestication. Furthermore, with an O. glaberrima accession as an outgroup, it is suggested that the indica-japonica differentiation in O. ruffpogon might occur after its speciation from other AA-genome species.     

应用微卫星标记鉴别水稻籼粳亚种

应用70个微卫星标记分析了3个籼稻测验种和3个粳稻测验种的多态性,发现其中36个标记可以区分籼粳测验种。再以18个籼粳品种进一步筛选,找到了分布于12条染色体的21个籼粳特异性微卫星标记。在这21个标记中,20个在籼粳亚种间带型相异,其中7个在亚种内带型一致,13个在亚种内带型不一致;1个标记在12个籼稻品种和1个粳稻品种检测到相同的带型,其余11个粳稻品种具有另一种带型。微卫星标记和RFLP标记检测籼粳亚种不仅具有一致性,而且还有互补性。 Abstract:Six indica and japonica testers were assayed using 70 microsatellite markers.Thirty-six markers distinguishing indicas from japonicas were detected.By further-screening among 18 indica and japonica varieties,21 markers distributed on 12 rice chromosomes were found to be indica-japonica differentiated.No indica varieties shared same patterns with any japonica varieties at 20 marker loci,of which identical patterns were observed within subspecies at 7 loci while within-subspecies variations were observed at 13 loci.At the remaining locus,12 indica and 1 japonica varieties had the same allele,while other 11 japonica varieties had another allele.It also showed that SSLP was not only consistent,but also complementary,to RFLP for the subspecies identification.     

Genotypic and phenotypic characterization of genetic differentiation and diversity in the USDA rice mini-core collection

A rice mini-core collection consisting of 217 accessions has been developed to represent the USDA core and whole collections that include 1,794 and 18,709 accessions, respectively. To improve the efficiency of mining valuable genes and broadening the genetic diversity in breeding, genetic structure and diversity were analyzed using both genotypic (128 molecular markers) and phenotypic (14 numerical traits) data. This mini-core had 13.5 alleles per locus, which is the most among the reported germplasm collections of rice. Similarly, polymorphic information content (PIC) value was 0.71 in the mini-core which is the highest with one exception. The high genetic diversity in the mini-core suggests there is a good possibility of mining genes of interest and selecting parents which will improve food production and quality. A model-based clustering analysis resulted in lowland rice including three groups, aus (39 accessions), indica (71) and their admixtures (5), upland rice including temperate japonica (32), tropical japonica (40), aromatic (6) and their admixtures (12) and wild rice (12) including glaberrima and four other species of Oryza. Group differentiation was analyzed using both genotypic distance Fst from 128 molecular markers and phenotypic (Mahalanobis) distance D(2) from 14 traits. Both dendrograms built by Fst and D(2) reached similar-differentiative relationship among these genetic groups, and the correlation coefficient showed high value 0.85 between Fst matrix and D(2) matrix. The information of genetic and phenotypic differentiation could be helpful for the association mapping of genes of interest. Analysis of genotypic and phenotypic diversity based on genetic structure would facilitate parent selection for broadening genetic base of modern rice cultivars via breeding effort.     

东北地区杂草稻与栽培稻的遗传多样性及籼粳分化

利用88对籼粳特异性分子标记对收集于我国东北三省的35份杂草稻和36份栽培稻遗传基础及籼粳分化进行研究,结果表明上述标记能够高效地鉴别稻属资源的籼粳属性,共检测到156个等位基因,平均有效等位基因(Na)为1.773。遗传多样性分析表明,东北地区杂草稻多样性水平略高于当地栽培稻,其中杂草稻的等位基因数(Na)、杂合度(He)、基因多样性(Hsk)以及多态性信息含量(PIC)分别为1.659、0.006、0.076和0.085,而东北栽培稻分别为1.557、0.004、0.060和0.067。遗传结构和聚类分析结果表明,东北地区杂草稻与栽培稻具有较近的亲缘关系,均存在一定程度的籼粳分化。进一步对籼粳血缘进行相对量化分析发现,杂草稻的籼型基因型频率(F_i=0.050)略高于当地栽培稻(F_i=0.043)。东北三省籼型基因型频率变化趋势为:辽宁杂草稻(0.062)辽宁栽培稻(0.058)吉林栽培稻(0.048)黑龙江杂草稻(0.041)吉林杂草稻(0.024)黑龙江栽培稻(0.020)。     

云南地方稻种持久抗瘟性的研究

1996～1999年,在不同纬度、不同海拔和不同稻作生态类型的重病区设立5个持久抗性稻瘟病鉴定圃.试验材料为云南的74份地方稻种资源,其中粳稻56份,籼稻18份(含野生稻3份).通过多个抗性组分进行了系统研究,初步表明大白谷(粳、墨江县)、毫弄早(籼、勐海县)、毫玉浪(籼、勐海县)、疣粒野生稻(野、西双版纳自治州)等具有持久抗瘟性能;其中疣粒野生稻高抗细菌性条斑病,对白叶枯病抗性为0级,接近免疫,中抗稻瘟病.通过对品种多抗性组分分析和品种抗性系统聚类分析,提出在不同生态类型时、空动态的病叶片上的产孢量和病斑表型可作为简易、快速鉴定持久抗瘟性指标.     

中国普通野生稻遗传分化的RAPD研究

多数学者已认定亚洲栽培稻（ＯｒｙｚａｓａｔｉｖａＬ．）的祖先是普通野生稻（Ｏ．ｒｕｆｉｐｏｇｏｎ）。然而栽培稻的籼、粳分化是发生在驯化之前还是在驯化之后,也即普通野生稻是否存在籼、粳分化的问题,是十几年来稻作起源研究中争论的热点之一。Ｓｅｃｏｎｄ［１］用多个同工酶位点的分析结果得出结论,普通野生稻在驯化为栽培稻之前就已经发生了籼、粳分化,即有籼型普通野生稻和粳型普通野生稻之分。Ｍｏｒｉｓｈｉｍａ和Ｇａｄｒｉｎａｂ［２］用２４个形态和生理性状及１２个同工酶位点和杂交亲合力等方法证明普通野生稻没有发…     

Elemental Content in Brown Rice by Inductively Coupled Plasma Atomic Emission Spectroscopy Reveals the Evolution of Asian Cultivated Rice

The phylogenetic relationship for classification traits and eight mineral elements in brown rice (Oryza sativa L.) from Yunnan Province in China was carried out using microwave assisted digestion followed by inductively coupled plasma atomic emission spectroscopy, and the analytical procedures were carefully controlled and validated. In general, the results show that the mean levels of K, Ca, Mg, Fe and Cu in brown rice for 789 accessions of rice landraces was distinctly lower than that of improved cultivars. They further demonstrate that Ca plays an important role in the differentiation of subspecies indica-japonica, especially to enhance adaptation of cold stress, and that five mineral elements in brown rice enhance the eurytopicity from landrace to improved cultivar. Hierarchical cluster analysis, using average linkage from SPSS software based on eight mineral elements in brown rice, showed that Yunnan rice could be grouped into rice landrace and improved cultivar, with the rice landrace being further clustered into five subgroups, and that, interestingly, purple rice does not cluster with either of the groups. Our present data confirm that indica is the closest relative of late rice and white rice, and that they constitute rice landraces together, whereas japonica is the closest relatives of non-nuda, early-mid and glutinous rice. It is further shown that japonica, non-nuda, early-mid, glutinous, white and red rice might be more primitive than indica, nuda, late, non-glutinous and purple rice, respectively.     

栽培稻种子含氮量的基因型差异

采用田间试验研究了421个常规籼稻、63个常规粳稻和82个籼型杂交稻品种种子含氮量的基因型差异。结果表明,栽培稻种子含氮量变幅在0.85%~2.55%,平均为1.39%。种子平均含氮量最高的是籼型杂交稻,高达1.46%,显著高于常规粳稻(1.39%)和常规籼稻(1.37%)。不同类型的栽培稻种子含氮量呈正态分布,但频数分布最高的区间存在一定的差异。以50%品种的种子含氮量(累积百分数从25%~75%)为依据,常规籼稻、常规粳稻、籼型杂交稻分别集中在1.21%~1.52%、1.21%~1.51%、1.36%~1.53%。LSR测验表明,籼型杂交稻极差显著小于常规籼稻和常规粳稻,但常规粳稻与常规籼稻的差异未达显著水平,即籼型杂交稻种子含氮量比常规籼稻和常规粳稻的分布更加集中。不同类型栽培稻内的种子含氮量极差较大,可分为若干组,且各组间达极显著差异。     

TAC1, a major quantitative trait locus controlling tiller angle in rice

A critical step during rice (Oryza sativa) cultivation is dense planting: a wider tiller angle will increase leaf shade and decrease photosynthesis efficiency, whereas a narrower tiller angle makes for more efficient plant architecture. The molecular basis of tiller angle remains unknown. This research demonstrates that tiller angle is controlled by a major quantitative trait locus, TAC1 (Tiller Angle Control 1). TAC1 was mapped to a 35-kb region on chromosome 9 using a large F(2) population from crosses between an indica rice, IR24, which displays a relatively spread-out plant architecture, and an introgressed line, IL55, derived from japonica rice Asominori, which displays a compact plant architecture with extremely erect tillers. Genetic complementation further identified the TAC1 gene, which harbors three introns in its coding region and a fourth 1.5-kb intron in the 3'-untranslated region. A mutation in the 3'-splicing site of this 1.5-kb intron from 'AGGA' to 'GGGA' decreases the level of tac1, resulting in a compact plant architecture with a tiller angle close to zero. Further sequence verification of the mutation in the 3'-splicing site of the 1.5-kb intron revealed that the tac1 mutation 'GGGA' was present in 88 compact japonica rice accessions and TAC1 with 'AGGA' was present in 21 wild rice accessions and 43 indica rice accessions, all with the spread-out form, indicating that tac1 had been extensively utilized in densely planted rice grown in high-latitude temperate areas and at high altitudes where japonica rice varieties are widely cultivated.     

Genetic differentiation of wild relatives of rice as assessed by RFLP analysis

To study genetic diversity and relationships of wild relatives of rice, 58 accessions of Oryza rufipogon, Oryza nivara, Oryza sativa f. spontanea and the cultivated Oryza sativa, representing a wide range of their distribution, were analyzed using the restriction fragment length polymorphism (RFLP) technique. All 30-used RFLP probes detected polymorphisms among the Oryza accessions, with an average of 3.8 polymorphic fragments per probe. Considerable genetic diversity was scored among the Oryza accessions with a similarity coefficient ranging from 0.28 to 0.93; but the cluster analysis of the accessions did not show an apparent grouping based on the species classification, instead they were scattered randomly in different groups. Noticeably, the Oryza accessions from the same geographic region, or near-by geographic regions, tended to be clustered in the same groups. The indica rice varieties showed relatively high genetic diversity and were scattered in different groups of their wild relatives, but the japonica varieties showed a relatively low variation and formed an independent group. It is concluded from the molecular analytical result that: (1) the four Oryza taxa have a remarkably close relationship and their independent species status need to be carefully reviewed; (2) geographic isolation has played a significant role in the differentiation of the Oryza accessions; therefore, a wide geographic range needs to be covered in collecting wild rice germplasm for ex situ conservation; and (3) the conventional conclusion of indica rice being directly domesticated from its ancestral wild species, and japonica rice being derived from indica, gains support from our data.