Experimental validation of inter-subspecific genetic diversity in rice represented by the differences between the DNA sequences of ＇Nipponbare＇ and ＇93-11＇

The pedigrees of three sequenced rice cultivars were analyzed to show that a majority of the genetic composition of ＇Nipponbare＇ originates from japonica cultivars while the minority originates from indica cultivars. In contrast, ＇93-11＇ is derived mainly from indica cultivars with a smaller contribution from japonica cultivars. All ancestors of ＇Guang lu ai 4＇ appeared to be indica lines. A set of molecular markers （46 InDels and 53 SSRs） polymorphic between ＇Nipponbare＇ and ＇93-11＇ were examined in 46 typical indica and 47 typical japonica cultivars selected from 443 accessions according to Cheng＇s index. All cultivars were divided into indica and japonica groups without overlapping when clustered by Cheng＇s index, InDels and SSRs. Much higher InDel and SSR diversity between groups than within groups implies that the marker polymorphisms between ＇Nipponbare＇ and ＇93-11＇ represent a large proportion of inter-subspecific diversity. About 85% of indica cultivars and more than 90% of japonica cultivars were confirmed to have the same PCR banding patterns as ＇93-11＇ and ＇Nipponbare＇, respectively. Some polymorphic loci between ＇Nipponbare＇ and ＇93-11＇ cannot be validated in other indica and japonica cultivars, either as subspecies-specific but not predominant alleles, or alleles not specific between the two groups. It was concluded that molecular markers developed from sequence polymorphism between ＇Nipponbare＇ and ＇93-11＇ often represent inter-subspecific diversity, although some exceptions were sensitive to either particular marker loci or particular cultivars.     

Genetic structure and eco-geographical differentiation of cultivated Hsien rice (Oryza sativa L. subsp. indica) in China revealed by microsatellites

Indica is not only an important rice subspecies widely planted in Asia and the rest of the world,but it is also the genetic background of the majority of hybrid varieties in China.Studies on genetic structure and genetic diversity in indica germplasm resources are important for the classification and utilization of cultivated rice in China.Using a genetically representative core collection comprising 1482 Chinese indica landraces,we analysed the genetic structure,geographic differentiation and diversity.Model-based structure analysis of varieties within three ecotypes revealed nine eco-geographical types partially accordant with certain ecological zones in China.Differentiation of eco-geographical types was attributed to local ecological adaption and physical isolation.These groups may be useful for developing heterotic groups of indica.To facilitate the identification of different ecotypes and eco-geographical types,we identified characteristic SSR alleles of each ecotype and eco-geographical type and a rapid index of discrimination based on characteristic alleles.The characteristic alleles and rapid discrimination index may guide development of heterotic groups,and selection of hybrid parents.     

Advances in the understanding of inter-subspecific hybrid sterility and wide-compatibility in rice

Hybrid sterility is a major form of postzygotic reproductive isolation and frequently occurs in hybrids between divergent populations, such as the indica and japonica subspecies of Asian cultivated rice （Oryza sativa L.）. It has been a major barrier for utilization of the strong heterosis expressed in hybrids between indica and japonica. A large number of loci for rice inter-subspecific hybrid sterility have been identified by genetic analysis. Cytological studies revealed that male and female gamete abortions and reduced affinity between the uniting gametes all occurred in indica-japonica hybrids, suggesting the complexity of the causes for inter-subspecific hybrid sterility. Two genes conditioning embryo-sac and pollen sterility respectively in indica-japonica hybrids have been cloned recently, providing opportunities for molecular characterization of the indica-japonica hybrid sterility and wide-compatibility. Future studies should aim at cloning more genes for indica-japonica hybrid sterility, characterizing the underlying molecular mechanism, and utilization of the findings for the development of inter-subspecific hybrids to increase rice productivity.     

Identification and classification of subspecies of Asian cultivated rice and their hybrids

Having reviewed the major classification systems proposed by various scholars across the world, it is found thatindica andjaponica underO. sativa L. are two major directions thoroughly differentiated from the Asian cultivated rice, forming the framework of the classification structure. A system withindica andjaponica as the only two subspecies is therefore reiterated. There are various ways to determine the indica-japonica identity of hybrid rice, but the “combined morphological trait index” (CMT index) method is more efficient and easier to handle, although the isozyme analysis, molecular marker analysis and grain quality assay methods are also feasible.     

Genetic diversity, geographic differentiation and evolutionary relationship among ecotypes of Glycine max and G. soja in China

The knowledge of origin and evolution of cultivated soybeans is one of the basic issues in both biology and agronomy of the crop. In order to investigate the nuclear and cytoplasmic genetic diversity, geographic differentiation and genetic relationship among geographic ecotypes of cultivated （Glycine max） and wild （G. soja） soybeans, the allelic profiles at 60 nuclear simple-sequence repeat （nuSSR） loci and 11 chloroplastic SSR （cpSSR） loci evenly distributed on whole genome of 393 landraces and 196 wild accessions from nation-wide growing areas in China were analyzed. （i） The genetic diversity of the wild soybean was obviously larger than that of the cultivated soybean, with their nuSSR and cpSSR alleles as 1067 vs. 980 and 57 vs 44, respectively. Of the 980 nuclear alleles detected in the cultivated soybean, 377 new ones （38.5%） emerged, while of the 44 chloroplastic alleles in the cultivated soybean, seven new ones （15.9%） emerged after domestication. （ii） Among the cultivated geographic ecotypes, those from southern China, including South-Central China, Southwest China and South China possessed relatively great genetic diversity than those from northern China, while among the wild geographic ecotypes, the Middle and Lower Changjiang Valleys wild ecotype showed the highest genetic diversity. （iii） The analysis of molecular variance, association analysis between geographic grouping and molecular marker clustering and analysis of specific-present alleles of ecotypes demonstrated that the geographic differentiation of both cultivated and wild soybeans associated with their genetic differentiation, or in other words, had their relevant genetic bases. （iv） The cluster analysis of all accessions clearly showed that the wild accessions from Middle and Lower Changjiang Valleys and South-Central ＆ Southwest China had relatively small genetic distances with all cultivated accessions. The UPGMA dendrogram among geographic ecotypes further showed that the genetic distances between all cultivated ecotypes and the Middle and Lower Changjiang Valleys wild ecotype were smaller than those with other wild ones, including their local wild counterparts. Therefore, it is inferred that the wild ancestors in southern China, especially those from Middle and Lower Changjiang Valleys might be the common ancestor of all the cultivated soybeans.     

Efficient indica and japonica rice identification based on the InDel molecular method: Its implication in rice breeding and evolutionary research

An efficient molecular method for the accurate and efficient identification of indica and japonica rice was created based on the polymorphisms of insertion/deletion (InDel) DNA fragments obtained from the basic local alignment search tool (BLAST) to the entire genomic sequences of indica (93-11) and japonica rice (Nipponbare). The 45 InDel loci were validated experimentally by the polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) in 44 typical indica and japonica rice varieties, including 93-11 and Nipponbare. A neutrality test of the data matrix generated from electrophoretic banding patterns of various InDel loci indicated that 34 InDel loci were strongly associated with the differentiation of indica and japonica rice. More extensive analyses involving cultivated rice varieties from 11 Asian countries, and 12 wild Oryza species with various origins confirmed that indica and japonica characteristics could accurately be determined via calculating the average frequency of indica- or japonica-specific alleles on different InDel loci across the rice genome. This method was named as the “InDel molecular index” that combines molecular and statistical methods in determining the indica and japonica characteristics of rice varieties. Compared with the traditional methods based essentially on morphology, the InDel molecular index provides a very accurate, rapid, simple, and efficient method for identifying indica and japonica rice. In addition, the InDel index can be used to determine indica or japonica characteristics of wild Oryza species, which largely extends the utility of this method. The InDel molecular index provides a new tool for the effective selection of appropriate indica or japonica rice germplasm in rice breeding. It also offers a novel model for the study of the origin, evolution, and genetic differentiation of indica and japonica rice adapted to various environmental changes.     

Geographical genetic diversity and divergence of common wild rice （O. rufipogon Griff.） in China

Using 36 SSR markers and 889 accessions of common wild rice in China, the genetic diversity and the divergence among different geographical populations are investigated. Guangdong Province has the largest number of alleles, which account for 84% of the total alleles detected in the study, followed by Guangxi Province. The Nei＇s gene diversity indices, from high to low, are in the sequence of Hainan, Guangdong, Guangxi, Fujian, Hunan, Jiangxi, and Yunnan provinces. Two genetic diversity centers of Chinese common wild rice are detected on the basis of geographic analysis, i.e., the region covering Boluo, Zijin, Lufeng, Haifeng, Huidong and Huiyang counties of Guangdong Province and the region covering Yongning, Longan, Laibin and Guigang counties of Guangxi Province. The common wild rice in Yunnan, Hunan, Jiangxi, and Fujian provinces are diverged into respectively independent populations with relatively large genetic distances, whereas, those in Hainan, Guangdong and Guangxi provinces have relatively low genetic divergence. Under the condition of geographic separation, natural selection is considered as one of the primary forces contributing to the divergence of common wild rice in China.     

Molecular diversity of the 5S rRNA gene in cultivated and wild rice species

We have used the polymerase chain reaction to analyze variation in the size of 5S ribosomal gene spacer sequence. Eighty accessions, including 65 cultivated rice and 15 wild rice, were analyzed. Among them seven size classes of 5S DNA spacer were observed. Classification asindica orjaponica on the basis of 5S DNA spacer patterns generally agrees with classification based on morphological studies, indicating that the length polymorphism of 5S DNA spacer could be used as a molecular marker for taxonomic and phylogenetic analysis. Supported by the National Natural Science Fundation of China Yi Qingming: born in Apr. 1938, Professor     

Molecular diversity of the 5S rRNA gene in cultivated and wild rice species

We have used the polymerase chain reaction to analyze variation in the size of 5S ribosomal gene spacer sequence. Eighty accessions, including 65 cultivated rice and 15 wild rice, were analyzed. Among them seven size classes of 5S DNA spacer were observed. Classification asindica orjaponica on the basis of 5S DNA spacer patterns generally agrees with classification based on morphological studies, indicating that the length polymorphism of 5S DNA spacer could be used as a molecular marker for taxonomic and phylogenetic analysis. Supported by the National Natural Science Fundation of China Yi Qingming: born in Apr. 1938, Professor     

Genetic analysis and gene mapping of a narrow leaf mutant in rice ( Oryza sativa L.)

A narrow leaf mutant was obtained after T-DNA transformation conducted on a rice variety Zhonghua 11. Several abnormal morphological characteristics, including semi-dwarf, delayed flowering time, narrow and inward rolling leaves, and lower seed-setting, were observed. The rate of net photosynthesis (under saturate light) of flag leaves in the mutant was significantly lower than that of the wild type. Moreover, the leaf transpiration rate and stomatal conductance in the mutant flag leaf were lower than those of the wild type at the grain filling stage. It was found that the mutant phenotype was not caused by the T-DNA insertion. Genetic analysis showed that the mutant was controlled by a single recessive gene, designated as nal3(t). A genetic linkage map was constructed using a large F₂ mapping population derived from a cross between nal3(t) and an indica variety Longtefu B with 6 polymorphic markers on chromosome 12 identified from 366 SSR markers by the BAS method. Gene nal3(t) was mapped between the markers RM7018 and RM3331. Fine mapping of nal3(t) locus was conducted with 22 newly developed STS markers based on the sequence diversity around the region harboring nal3(t) between Nipponbare and 93–11, and nal3(t) was finally mapped to a 136-kb region between the STS markers NS10 and RH12-8. Supported by National High Technology Research and Development Program of China (863 Program) (Grant No. 2006AA10A102), National Natural Science Foundation of China (Grant No. 30600349) and Natural Science Foundation of Zhejiang Province (Grant No. Y306149)     

Studies of new EST-SSRs derived from Gossypium barbadense

Existing cotton EST-SSR markers are mostly derived from Gossypium arboreum and Gossypium hir-sutum, but EST-SSR markers from Gossypium barbadense are scarce. One hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G. barbadense cv. Pima3-79. Among the SSRs, trinucleotide AAG appeared at a high frequency of 11.76%. 36 accessions (consisting of 13 diploids of the A genome, 11 diploids of the D genome and 12 allotetraploids of the AD genome) were employed to test new EST-SSRs. 76 EST-SSRs were successfully amplified, and 313 polymorphic fragments were yielded, with an average of 4.11 fragments per primer pair. The PIC ranged from 0.17 to 0.95 with an average of 0.53. Based on Jaccard’s genetic similarity coefficient, these 36 accessions were clustered into three groups. 21 EST-SSRs exhibited polymorphisms in BC1 population ((Emian22 × Pima3-79) × Emian22), 24 polymor- phic loci were generated, while 22 of the 24 polymorphic loci were integrated with our interspecific BC1 backbone genetic linkage map, and anchored in 12 chromosomes. This study effectively proved that EST-SSRs from G. barbadense are valuable for genetic diversity analysis and genetic mapping.     

Genetic diversity and construction of core collection in Chinese wheat genetic resources

Genetic diversity among 5029 accessions representing a proposed Chinese wheat core collection was analyzed using 78 pairs of fluorescent microsatellite （SSR） primers mapped to 21 chromosomes. A stepwise hierarchical sampling strategy with priority based on 4×10^5 SSR data-points was used to construct a core collection from the 23090 initial collections. The core collection consisted of 1160 accessions, including 762 landraces, 348 modern varieties and 50 introduced varieties. The core accounts for 23.1% of the 5029 candidate core accessions and 5% of the 23090 initial collections, but retains 94.9% of alleles from the candidate collections and captures 91.5% of the genetic variation in the initial collections. These data indicate that it is possible to maintain genetic diversity in a core collection while retaining fewer accessions than the accepted standard, i.e., 10% of the initial collections captured more than 70% of their genetic diversity. Estimated genetic representation of the core constructed by preferred sampling （91.5%） is much higher than that by random sampling （79.8%）. Both mean genetic richness and genetic diversity indices of the landraces were higher than those of the modern varieties in the core. Structure and principal coordinate analysis revealed that the landraces and the modern varieties were two relatively independent subpopulaUons. Strong genetic differentiation associated with ecological environments has occurred in the landraces, but was relatively weak in the modern culUvars. In addition, a mini-core collection was constructed, which consisted of 231 accessions with an estimated 70% representation of the genetic variation from the initial collections. The mini-core has been distributed to various research and breeding institutes for detailed phenotyping and breeding of genetic introgression lines.     

Origin of the Chinese cultivated rice (Oryza sativa L.)

The following new research progresses are summarized and discussed, which are related to 3 main problems in origin of rice cultivation in China: 1. new hypothesis of rice cultivation in China— middle Yangtze River and upper Huai River regions; 2. primitive cultivated rice and the strengthen period of domestication; 3. genetic diversity centers of cultivated rice in China; 4. China and South Asia might be two independent systems of origin and differentiation of Asian cultivated rice; 5. morphological classification of common wild rice of China; 6. primitive progenitor of common wild rice; 7. direct progenitor of cultivated rice; 8. present or not the annual wild rice in China; 9. differentiation or not the common wild rice into Indica and Japonica; 10. origin and differentiation of Indica and Japonica.     

A draft sequence of the rice (Oryza sativa ssp. indicd) genome

The sequence of the rice genome holds fundamental information for its biology, including physiology, genetics, development, and evolution, as well as information on many beneficial phenotypes of economic significance. Using a “whole genome shotgun” approach, we have produced a draft rice genome sequence ofOryza sativa ssp.indica, the major crop rice subspecies in China and many other regions of Asia. The draft genome sequence is constructed from over 4.3 million successful sequencing traces with an accumulative total length of 2214.9 Mb. The initial assembly of the non-redundant sequences reached 409.76 Mb in length, based on 3.30 million successful sequencing traces with a total length of 1797.4 Mb from anindica variant cultivar93-11, giving an estimated coverage of 95.29% of the rice genome with an average base accuracy of higher than 99%. The coverage of the draft sequence, the randomness of the sequence distribution, and the consistency of BIG-ASSEMBLER, a custom-designed software package used for the initial assembly, were verified rigorously by comparisons against finished BAC clone sequences from bothindica andjapanica strains, available from the public databases. Over all, 96.3% of full-length cDNAs, 96.4% of STS, STR, RFLP markers, 94.0% of ESTs and 94.9% unigene clusters were identified from the draft sequence. Our preliminary analysis on the data set shows that our rice draft sequence is consistent with the comman standard accepted by the genome sequencing community. The unconditional release of the draft to the public also undoubtedly provides a fundamental resource to the international scientific communities to facilitate genomic and genetic studies on rice biology. These authors contributed equally to this work.     

Study on heterosis of intersubspecies between indica and japonica rice （Oryza sativa L.） using chromosome segment substitution lines

Great heterosis exists in the inter-subspecific crossesbetween indica and japonica rice cultivars[1,2] and the ex-ploitation of this heterosis has long been considered as apromising method to further increase rice yield potential[3].Previous studies indicated that there were large differencesbetween the two subspecies with respect to morphology,isozyme, polymorphism of molecular markers and DNAstructure due to long time genetic differentiation[4,5]. Thosedifferences were identified to be clos…     

Generating of rice OsCENH3-GFP transgenic plants and their genetic applications

In order to investigate rice functional centromeres, OsCENH3-GFP chimeric gene was constructed and transformed into the indica rice variety, Zhongxian 3037, mediated by Agrobacturium. The integration of the exogenous genes in the transgenic plants was confirmed by PCR and Southern blotting. The transgenic plants grow normally during their whole life time, just like Zhongxian 3037. No significant defects were detected in either mitosis or meiosis of the transgenic plants. The overlapping of GFP signals and anti-CENH3 foci in both mitotic and meiotic cells from T0 and T1 generation plants indicated that GFP had been successfully fused with CENH3, so the GFP signals can well represent the CENH3 locations on each chromosome. To evaluate the applicability of the transgenic plants to other genetic studies, fluorescence in situ hybridization （FISH） using rice centromeric tandem repetitive sequence CentO as the probe was conducted on the zygotene chromosomes of pollen mother cells （PMCs）. It has been revealed that the GFP signals are overlapping with CentO FISH signals, showing that CentO is one of the key elements constituting rice functional centromeres. Immunofluorescent staining using anti-o-tublin antibody and anti-PAIR2 antibody on the chromosomes during mitosis and meiosis stages of the transgenic plants further reveals that OsCENH3-GFP transgenic plants can be widely used for studying rice molecular biology, especially for tagging functional centromeres in both living cells and tissues.     

Ancient DNA sequences of rice from the low Yangtze reveal significant genotypic divergence

Rice (Oryza sativa) was first domesticated in the lower and middle Yangtze regions of China, and rice remains have been found in many Chinese archaeological sites. Until now, only phenotypic archeobotanical evidence, such as the spikelet bases of ancient grains, has been used to speculate on the domestication process and domestication rate of rice. In this study, we sequenced 4 genomic segments from rice remains in Tianluoshan, a site of the local Hemudu Neolithic culture in the low Yangtze and two other archaeological sites (??2400 and 1200 BC, respectively). We compared our sequences with those of the current domesticated and wild rice (O. rufipogon) populations. At least two genotypes were found in the remains from each site, suggesting a heterozygotic state of the rice seeds. One ancient genotype was not found in the current domesticated population and might have been lost. The rice remains belonged to the japonica group, and most if not all were japonica-type, suggesting that the remains might be at an early stage of indica-japonica divergence or an indica-japonica mixture. We also identified sequences with significant similarity to those from species of Sapindales, Zygophyllales, and Brassicales, which is consistent with the identification of other plant remains in the Tianluoshan site and the common rice field weeds such as mustards in southern China.     

籼-粳稻特异插入／缺失分子标记揭示的稻属植物遗传分化

籼-粳分化在亚洲栽培稻(Oryza sativa L.)的驯化过程中非常重要,但其分化机制仍不清楚.有的学者认为水稻籼-粳分化是亚洲栽培稻在驯化过程中适应不同生境的结果,也有人认为籼-粳分化在水稻的野生祖先种中就已经存在.为了研究普通野生稻的籼-粳分化,并解析稻属(Oryza)植物的籼-粳遗传变异,利用34对籼-粳特异插入/缺失分子标记(InDel)引物,研究了50份典型籼稻(O.sativa L.subsp.Indica Kato)和粳稻(O.sativa L.subsp.japonica Kato)样本以及来源于35个国家的348份稻属其他种材料.结果表明,亚洲栽培稻存在明显的籼-粳分化,普通野生稻复合体(O.rufipogon complex)中存在"偏籼"和"偏粳"类型,而稻属的其他种均未出现籼-粳分化,普通野生稻复合体中"偏籼"和"偏粳"类型的地理分布格局与籼稻和粳稻的地理分布格局相吻合.考虑到大部分普通野生稻复合体的样本取自邻近有栽培稻种植的普通野生稻群体,推测得出部分普通野生稻样本中表现出的"偏籼"和"偏粳"类型可能是栽培稻的籼稻品种和粳稻品种在普通野生稻的长期协同进化过程中基因交流的结果.     

Molecular evolution of rice S 5 n and functional comparison among different sequences

The wide compatibility gene, S ₅ ⁿ , can overcome embryo sac sterility between indica and japonica subspecies of rice. Therefore, it is very important to characterize the features of the S ₅ ⁿ sequence to reveal the origin and evolution of S ₅ ⁿ . In this paper, 26 cultivated rice haplotypes and 22 wild rice accessions harboring S ₅ ⁿ were used to sequence S ₅ ⁿ . The results showed that 15 genotypes among the 48 materials were fully consistent with control cultivar 02428 (CK). The other 33 accessions had different degrees of variation in the S ₅ ⁿ sequence. Variations in the coding region mainly occurred in the second exon and eight materials showed a 10-bp deletion at 1710?C1719 bp, including wild (O. nivara) and cultivated rice, such as IRW501 and Yuetai B. S ₅ ⁿ sequences were not biased and evolved neutrally. The 48 materials could be divided into 4 categories using a phylogenetic tree of the amino acid sequences. Most of the wild rice clustered together, and the cultivated rice clustered into another group. Eight cultivated rice and O. nivara (wild rice) clustered in another group, which were found to lack 10 consecutive bases in exon 2. Eight rice varieties with high numbers of differences in their S ₅ ⁿ coding regions were crossed with testers (typically indica and japonica) to produced test cross F₁ populations. The F₁s were examined for their ability to overcome indica-japonica hybrid sterility. The result showed that the embryo sac fertility of S ₅ ⁿ -containing hybrids increased significantly compared with control hybrids, but there were no differences among the materials with divergent sequences, indirectly proving that S ₅ ⁿ is a non-functional gene.