Identifying neutral allele Sb at pollen-sterility loci in cultivated rice with Oryza rufipogon origin

Pollen sterility is commonly found in the intra-specific hybrids of indica and japonica rice, which is one of the main constrains for the utilization of heterosis between indica and japonica. Six loci controlling the pollen sterility of F1 between indica and japonica have been identified from previous studies. Neutral alleles at each locus are potential to overcome the F1 pollen sterility associated with the locus. Therefore, exploitation and utilization of neutral alleles are of significant importance. The present research was based on fine mapping of the F1 pollen-sterility gene Sb and the abundant genetic diversity of Oryza rufipogon Griff. indigenous to Gaozhou, Guangdong Province （referred to as Gaozhou wild rice）. Crosses were made using Taichung65 （with the genotype of Sb^jSb^j and referred to as El） and its near-isogenic line of F1 pollen sterility gene Sb（with the genotype of Sb^iSb^i, E2） as female parents, and 12 different accessions of Gaozhou wild rice as male parents. F1 pollen fertility was examined to identify the materials having the neutral alleles at the F1 pollen-sterility locus. Segregation of 4 molecular markers tightly linked with the Sb locus was analyzed in the F2 populations derived from the FlS carrying the neutral gene. The pollen fertility related to the 3 genotypes of the molecular markers was also checked by statistical test to determine whether it was consistent with the hypothesis. The results showed that the pollen fertility of two F1s from one accession of Gaozhou wild rice （GZW099） with E1 and E2 was （89.2±21.07）% and （85.65±1.05）%, respectively. Both of them were fertile and showed no significant difference by t-test. Segregation of the 3 genotypes of the 4 molecular markers followed the expected Mendelian ratio （1：2：1） in the F2 populations. There was no significant difference for the averaged pollen fertility of the plants related to the 3 genotypes, suggesting that no interaction exists between the alleles at the Sb locus in GZW099 and Taichung65 or E2. Evidentially, GZW099 carried the neutral gene （named Sb″Sb″） at the Sb locus, which provides valuable theoretical basis and resources for further studying and overcoming the sterility of indica-japonica hybrids.     

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.     

Genetic structure and diversity of Oryza sativa L. in Guizhou, China

Preserving many kinds of rice resources and rich variations, Guizhou Province is one of the districts with the highest genetic diversity of cultivated rice （Oryza sativa L.） in China. In the current research, genetic diversity and structure of 537 accessions of cultivated rice from Guizhou were studied using 36 microsetellite markers and 39 phenotypic characters. The results showed that the model-based genetic structure was the same as genetic-distance-based one using SSRs but somewhat different from the documented classification （mainly based on phenotype） of two subspecies. The accessions being classified into indica by phenotype but japonica by genetic structure were much more than that being classified into japonica by phenotype but indica by genetic structure. Like Ding Ying＇s taxonomic system of cultivated rice, the subspecific differentiation was the most distinct differentiation within cultivated rice. But the differentiation within indica or japonica population was different： japonica presented clearer differentiation between soil-watery ecotypes than indica, and indica presented clearer differentiation between seasonal ecotypes than japonica. Cultivated rices in Guizhou revealed high genetic diversity at both DNA and phenotypic levels. Possessing the highest genetic diversity and all the necessary conditions as a center of genetic diversity, region Southwestern of Guizhou was suggested as the center of genetic diversity of O. sativa L. from Guizhou.     

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     

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.     

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.     

A statistical method for genetic mapping of sterility genes that exhibit epistasis in remote hybridization of plants using molecular markers in an F2 population

Estimation of the position and effect of sterility genes is an important problem to be solved to understand the sterility mechanism in remote hybridization in plants.In this study,a maximum likelihood (ML) method was used for estimation of the position and effect of sterility genes that exhibit epistasis in an F 2 population using the distorted segregation of markers.The ML solutions for recombination fraction and viability were obtained via an expectation-maximization algorithm.The results of Monte Carlo simulations showed that the estimates of recombination fraction and viability were consistent with their true values.The bias and standard deviation of parameters indicated that a larger sample size,closer linkage and lower viability of sterile genes led to better estimates of the parameters involved.A subset of marker data of the F 2 population derived from a single cross between the rice japonica cultivar Nipponbare and the indica cultivar Kasalath was analyzed using this method.Eight sterility genes were identified on chromosomes 1,3,6,8 and 10,and significant epistasis was detected among four pairs of sterility genes.     

Mining rice new germplasm containing S5n gene by functional molecular marker and sequencing

Inter-subspecific hybrids between indica and japonica varieties yield strong biological heterosis, but it is difficult to utilize the hybrids directly in commercial production due to sterility of F1. A special gene S5^n may overcome the hybrids sterility, which is caused by the interaction between S5 loci. Recently, S5^n had been cloned, and it was revealed containing a large DNA deletion sequence that made the gene nonfunctional, compared to S5^i or S5^j. We designed a pair of primers flanking the deletion sequence of Ssn, and then applied to distinguish the varieties with S5^n or non-S5^n, convincing result suggested that the primers could be served as functional molecular marker to efficiently identify the new germplasm with S5^n, Using the functional marker, we surveyed 197 varieties from China National Micro-core Rice Collection, and found ten of which represented S5^n including following varieties： Haobuka, Sanbangqishiluo, Mubanggu, Xiaohonggu, Mowanggu＇neiza, Laozaogu, Fanhaopi, Feie＇nu02, Baoxie-7B, Teqingxuanhui. Among them, two varieties Sanbangqishiluo and Laozaogu was previously reported to contain S5^n gene. Further sequence analysis on the DNA sequence covering both sides of deletion in S5^n of the 10 varieties confirmed that the detected sequences in above varieties was identical with those of varieties containing S5^n, such as 02428 and Linglun. These results suggested that the gene in S5 locus of the ten varieties was also nonfunctional and it proved the presence of S5^n gene.     

Effects of elevated [CO2] on stem and root lodging among rice cultivars

Studies showed that elevated [CO2] would improve photosynthetic rates and enhance yields of rice;however,few studies have focused on the response of rice lodging,which is a major cause of cereal yield loss and quality reduction,under elevated [CO2].In this study,we examined the effects of elevated [CO2] on stem and root lodging using 4 rice cultivars(86Y8,japonica hybrid;LYP9,2-line indica hybrid;variety 9311,type of indica inbred rice,and SY63,3-line indica hybrid) grown under two [CO2] levels:400 and 680 μmol mol-1.Our results indicated that under elevated [CO2],the stem-lodging risk(SLR) of 9311 decreased,while in SY63 the SLR increased,86Y8 and LYP9 were not significantly affected;the risk of root lodging was reduced for all cultivars,because root biomass(instead of root number) and bending strength were increased significantly,and then the increase of anti-lodging ability is far higher than that of self-weight mass moment for all cultivars.These findings suggested that higher [CO2] can enhance the risk of stem-lodging for cultivars with strong-[CO2]-responses,but may not aggravate the root lodging for all rice cultivars.     

Genetic analysis and fine mapping of the pubescence gene GL6 in rice (Oryza sativa L.)

The pubescence of the leaf blade surface is an important agronomic characteristic for rice morphology and significantly influences rice growth as well as physiological characteristics. This characteristic was analyzed in F1 and F2 plants derived by crossing cultivar 75-1-127 with the indica cultivar Minghui 63, as well as the glabrous cultivar Lemont and indica cultivar 9311. Results indicated that the pubescence of the leaf blade surface was a dominant trait and controlled by a single gene. The GL6 gene was primarily mapped on rice chromosome 6 with recessive F2 population derived from 75-1-127/Minghui 63 by combining bulked segregation analysis and recessive class analysis using the Mapmaker3.0/MapDraw software. The genetic distances between the simple sequence repeat markers RM20491 and RM20547 were 7.2 and 2.2 cM, respectively. The GL6 gene was fine mapped in the interval between InDel-106 and InDel-115 at genetic distances of 0.3 and 0.1 cM, respectively. The large, recessive F2 population was derived from 75-1-127/Minghui 63. A high-resolution genetic and physical map of GL6 was constructed. Derived from the map-based sequences published by the International Rice Genome Sequencing Project, the GL6 gene was localized at an interval of 79 (japonica) and 116.82 kb (9311) bracketed by InDel-106 and InDel-115 within the BAC accession numbers AP008403 and AP005760. Seven annotated genes (japonica) and eight annotated genes (9311) were present. The basis was further set for GL6 cloning and function analysis.     

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.     

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.     

High-quality reference genes for quantifying the transcriptional responses of Oryza sativa L. (ssp. indica and japonica) to abiotic stress conditions

Rice (Oryza sativa L.) is important to food security and is also an excellent model plant for numerous cereal crops. A functional genomics study in rice includes characterization of the expression dynamics of genes by quantitative real-time PCR (qPCR) analysis; this is a significant key for developing rice varieties that perform well in the face of adverse climate change. The qPCR analysis requires the use of appropriate reference genes in order to make any quantitative interpretations meaningful. Here, the new potential reference genes were selected from a huge public database of rice microarray experiments. The expression stability of 14 candidates and 4 conventional reference genes was validated by geNorm PLUS and NormFinder software. Seven candidates are superior to the conventionally used reference genes in qPCR and three genes can be used reliably for quantitating the expression of genes involved in abiotic stress responses. These high-quality references EP (LOC_Os05g08980), HNR (LOC_Os01g71770), and TBC (LOC_Os09g34040) worked very well in three indica genotypes and one japonica genotype. One of indica genotypes including the Jasmine rice, KDML105 developed in Thailand for which no reference genes have been reported until now.     

Physical location of riceGm-6, Pi-5(t) genes inO. officinalis with BAC-FISH

A fluorescence in situ hybridization (FISH) procedure was adopted to physically map two rice BAC clones 24E21 and 4F22 linked to Gm-6 and Pi-5(t) in O. offi-cinalis. FISH results showed that the two BAC clones were located at 4L. The percentage distance from the centromere to the hybridization sites was 72 + 2.62 for 24E21 and 54+ 5.43 for 4F22, the detection rates were 52.70% and 61.2%. The results obtained from the BAC and plasmid clones, RG214 and RZ565 of cultivated rice and O. officinalis were the same. This suggested that the markers, RG214 and RZ565 of cultivated rice and O. officinalis were in the same BAC clones. The homologous sequences of Gm-6 and Pi-5(t) in O. officinalis were positions that signals existed on the 4L. Many signals were observed when no Cot-1 DNA blocked. This also showed that repetitive sequences were some ho-molgous between cultivated rice and O. officinalis. The identification of chromosome 4 of O. officinalis is based on Jena et al. (1994). In our study, we discussed the possibility of physical map in O. officinalis with rice BAC clones.     

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…     

Relationships between D1 protein, xanthophyll cycle and photodamage-resistant capacity in rice (Orysa sativa L.)

Relationships between D1 protein, xanthophyll cycle and subspecific difference of photodamage-resistant capacity have been studied inO. japonica rice varieties 02428 and 029 (photoinhibition-tolerance) andO. indica rice varieties 3037 and Palghar (photoinhibition-sensitivity) and their reciprocal cross F₁ hybrids after photoinhibitory treatment. It was shown that PS II photochemical efficiency (F _v /F _m) decreased, and xanthophyll cycle from violaxanthin (V), via anaxanthin (A), to zeaxanthin (Z) was enhanced and non-photochemical quenching (qN) increased accordingly in SM-pretreated leaves of rice when the synthesis of D1 protein was inhibited, and that there was a decrease inqN and, as a result, more loss of D1 protein and a big decrease inF _v/F _m in DTT-pretreated leaves when xanthophyll cycle was inhibited.O. japonica subspecies had a higher maintaining capacity of D1 protein and a decrease ofF _v/F _m in a more narrow range, and exhibited more resistance against photodamage, as compared withO. indica subspecies. The above physiological indexes in reciprocal cross F₁ hybrids, though between the values of their parents, were closer to maternal lines than to paternal lines. Experimental results support the concept that the turnover capacity for D1 protein is an important physiological basis of photoinhibition-tolerance, and will provide the physiological basis for selection of the photoinhibition-tolerant parents and develop a new approach to breed hybrids with high photosynthetic efficiency.     

Genetic analysis and mapping of rice （Oryza sativa L.） male-sterile （OsMS-L） mutant

A rice male-sterile mutant OsMS-L of japonica cultivar 9522 background, was obtained in M4 population treated with ^60Co γ-Ray. Genetic analysis indicated that the male.sterile phenotype was controlled by a single recessive gene. Results of tissue section showed that at microspore stage, OsMS-L tapetum was retarded. Then tapetal calls expanded and microspores degenerated. No matured pollens were observed in OsMS-L anther locus. To map OsMS-L locus, an F2 population was constructed from the cross between the OsMS-L (japonica) and LongTeFu B(indica). Firstly, the OsMS-L locus was roughly mapped between two SSR markers, RM109 and RM7562 on chromosome 2. And then eleven polymorphic markers were developed for further fine fine-mapping. At last the OsMS-L locus was mapped between the two lnDel markers, Lhsl0 and Lhs6 with genetic distance of 0.4 cM, respectively. The region was delimited to 133 kb. All these results were useful for further cloning and functional analysis of OsMS-L.     

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.     

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.