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.     

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.     

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.     

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.     

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.     

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

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.     

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.     

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.     

Identification and gene mapping of a narrow and upper-albino leaf mutant in rice (Oryza sativa L.)

The leaf blade consists of color and shape traits. Studies of leaf-blade development are important for improvement of rice yield and quality because it is an essential organ for photosynthesis. A narrow and upper-albino leaf mutant (nul1) was identified from among progeny of the indica restorer line Jinhui10 raised from seeds treated with ethyl methane sulfonate. Under field conditions, the mutant displayed narrow and upper-albino leaf blades with significantly decreased photosynthetic pigment contents throughout their development. The narrow-leaf trait is caused by a decreased number of small veins. In contrast to the wild type, the growth period was extended by approximately 8 d and agronomic traits, such as effective panicle number, percentage seed set and 1000-grain weight, declined significantly in the nul1 mutant. Genetic analysis suggested that the narrow and upper-albino leaf characteristics showed coseparation and were controlled by one recessive gene. The Nul1 gene was mapped onto chromosome 7 between the Indel marker Ind07-1 and the Simple Sequence Repeat marker RM21637. The physical distance between the markers was 75 kb and eight genes were annotated in this region based on the rice Nipponbare genome sequence. These results provide a foundation for cloning and function analysis of Nul1.     

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.     

QTL analysis of the rice seedling cold tolerance in a double haploid population derived from anther culture of a hybrid betweenindica andjaponica rice

A doubled haploid population, derived from anther culture of F₁ hybrid between a typicalindica cv. and ajaponica cv. has been used to investigate the seedling cold tolerance (SCT) in growth cabinet. By dynamically analyzing every day’s survival percentages of the parents and DH lines under 7-d cold plus 9-d normal temperature condition, the quantitative trait loci (QTLs) for SCT have been mapped based on a molecular linkage map constructed from this population. The results show that two parents had significant differences in SCT and the segregation of SCT in DH lines was basically a continuous distribution with most serious injury on the 6th d of the cold treatment. A total of 4 QTLs for SCT have been identified on chromosomes 1, 2, 3 and 4 respectively. The additive effects of qSCT-1, qSCT-2 and qSCT-3 have been contributed by thejaponica cv JX17, but that of qSCT-4 has been contributed by theindica cv ZYQ8. The mechanism of SCT seems complicated since the above 4 QTLs detected at different stages during the treatment. Further study on the genotypes for these SCT QTLs in the DH lines shows transgressive segregation. It is demonstrated that the lines with stronger SCT over JXI7 have 3–4 loci for SCT. Integration of these QTLs into an appropriate variety may lead to a successful rice breeding program for cold tolerance.     

Differential response of root morphology to potassium deficient stress among rice genotypes varying in potassium efficiency

Disparity in the root morphology of six rice （Oryza sativa L.） genotypes varying in potassium （K） efficiency was studied with three K levels： 5 mg/L （low）, 10 mg/L （moderate） and 40 mg/L （adequate） in hydroponic culture. Morphological parameters included root length, surface area, volume and count of lateral roots, as well as fine （diameter〈0.2 mm） and thick （diameter〉0.2 mm） roots. The results indicate that the root growth of all genotypes was reduced under low K, but moderate K deficiency increased the root length of the efficient genotypes. At deficient and moderate K levels, all the efficient rice genotypes developed more fine roots （diameter〈0.2 mm） than the inefficient ones. Both fine root count and root surface area were found to be the best parameters to portray K stress in rice. In accordance with the root morphology, higher K concentrations were noted in shoots of the efficient genotypes when grown at moderate and deficient K levels, indicating that root morphology parameters are involved in root uptake for K and in the translocation of K up to shoots. K deficiency affected not only the root morphology, but also the root ultra-structure. The roots of high-efficient genotypes had stronger tolerance to K deficient stress for root membrane damage, and could maintain the developed root architecture to adapt to the low K growth medium.     

Introduction of antisensewaxy gene into main parent lines ofindica hybrid rice

Amylose content in rice endosperm is one of the key determinants of rice eating and cooking quality, and the poor quality ofindica hybrid rice is closely related to the high amylose level in rice grains. In order to improve the grain quality of theindica hybrid rice by genetic engineering, an antisense fragment of ricewaxy gene, driven by the 5′-franking sequences of the ricewaxy gene, was successfully introduced into three major parent lines ofindica hybrid rice, all contain a high amylose level in the grains, viaAgrobacterium, and more than 100 hygromycinresistant plants were regenerated. The analysis of PCR amplification and Southern blots indicated that the T-DNA containing the antisensewaxy gene had been integrated into the genome of transgenic rice plants. Most of the primary transgenic rice plants grew normally, and the mature seeds from these transgenic plants were performed for analysis of the amylose content. The results showed that the amylose content in the endosperm of some grains was reduced and the lowest reached 7.02% in one homozygous transgenic line, 72.4% lower than that of the wild type. The influence of the altered amylose content on the gelatinization temperature and gel consistency was also observed in several homozygous transgenic rice plants. The two authors contributed equally to this work.