Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

Rice is a leading grain crop and the staple food for over half of the world population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.     

Physical mapping of the rice genome with YAC clones

Construction of a rice physical map covered by YAC clones which have been arranged over half of the genome length is presented here. A total of 1285 RFLP and RAPD markers almost evenly distributed on the rice genetic map could select 2974 YAC clones and 2443 clones of them were located on their original positions. Rice YACs carrying 350 kb average insert fragments of 2443 clones could cover 222 megabase length of the rice genome, corresponding to 52% of the whole genome size (4.3 Mb). Chromosome landing with many YAC clones on the high-density genetic map loci efficiently integrated the genetic map with a physical map. This is the first step to generate a comprehensive genome map of rice. An integrated genome map should be an indispensable tool to figure out genome structure as well as to clone trait genes by map-based cloning.     

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.     

An integrated physical and genetic map of the rice genome

Rice was chosen as a model organism for genome sequencing because of its economic importance, small genome size, and syntenic relationship with other cereal species. We have constructed a bacterial artificial chromosome fingerprint–based physical map of the rice genome to facilitate the whole-genome sequencing of rice. Most of the rice genome (~90.6%) was anchored genetically by overgo hybridization, DNA gel blot hybridization, and in silico anchoring. Genome sequencing data also were integrated into the rice physical map. Comparison of the genetic and physical maps reveals that recombination is suppressed severely in centromeric regions as well as on the short arms of chromosomes 4 and 10. This integrated high-resolution physical map of the rice genome will greatly facilitate whole-genome sequencing by helping to identify a minimum tiling path of clones to sequence. Furthermore, the physical map will aid map-based cloning of agronomically important genes and will provide an important tool for the comparative analysis of grass genomes.     

Next-generation sequencing applications for wheat crop improvement

? Bread wheat (Triticum aestivum; Poaceae) is a crop plant of great importance. It provides nearly 20% of the world's daily food supply measured by calorie intake, similar to that provided by rice. The yield of wheat has doubled over the last 40 years due to a combination of advanced agronomic practice and improved germplasm through selective breeding. More recently, yield growth has been less dramatic, and a significant improvement in wheat production will be required if demand from the growing human population is to be met. ? Next-generation sequencing (NGS) technologies are revolutionizing biology and can be applied to address critical issues in plant biology. Technologies can produce draft sequences of genomes with a significant reduction to the cost and timeframe of traditional technologies. In addition, NGS technologies can be used to assess gene structure and expression, and importantly, to identify heritable genome variation underlying important agronomic traits. ? This review provides an overview of the wheat genome and NGS technologies, details some of the problems in applying NGS technology to wheat, and describes how NGS technologies are starting to impact wheat crop improvement.     

Genomics in cereals: from genome-wide conserved orthologous set (COS) sequences to candidate genes for trait dissection

Recent updates in comparative genomics among cereals have provided the opportunity to identify conserved orthologous set (COS) DNA sequences for cross-genome map-based cloning of candidate genes underpinning quantitative traits. New tools are described that are applicable to any cereal genome of interest, namely, alignment criterion for orthologous couples identification, as well as the Intron Spanning Marker software to automatically select intron-spanning primer pairs. In order to test the software, it was applied to the bread wheat genome, and 695 COS markers were assigned to 1,535 wheat loci (on average one marker/2.6 cM) based on 827 robust rice–wheat orthologs. Furthermore, 31 of the 695 COS markers were selected to fine map a pentosan viscosity quantitative trait loci (QTL) on wheat chromosome 7A. Among the 31 COS markers, 14 (45%) were polymorphic between the parental lines and 12 were mapped within the QTL confidence interval with one marker every 0.6 cM defining candidate genes among the rice orthologous region.     

Mapping in the realm of polyploidy: The wheat model

Wheat is an allopolyploid containing three distinct but genetically related (homoeologous) genomes, A, B and D. Because of polyploid inheritance and large genome size (16×10¹² bp), the wheat genome is thought to be intractable to map-based cloning of agronomic and other genes of interest. We propose a targeted geneti mapping strategy that combines linkage and physical mapping and may facilitate map-based cloning. High-density linkage maps are either generated in wheat or in diploid Triticum tauschii, the donor of the D genome to wheat. Molecular marker-based chromosome maps are constructed, using an array of deletion lines in wheat. The conventional genetic linkage maps are aligned with chromosome maps to construct cytogenetic ladder maps (CLMs). The CLMs allow region-specific mapping and convert genetic distances into physical distances. The information from CLMs suggests that many genes in wheat are present in clusters that are highly recombiogenic, small, and may be amenable to cloning by chromosome walking. Therefore, the effective genome size of wheat is relatively small in comparison to the whole genome. The utility of using the smaller genome of rice for mapping and homologous gene cloning is discussed.     

Molecular Transfer of Nematode Resistance Genes

Recombinant DNA techniques have been used to introduce agronomically valuable traits, including resistance to viruses, herbicides, and insects, into crop plants. Introduction of these genes into plants frequently involves Agrobacterium-mediated gene transfer. The potential exists for applying this technology to nematode control by introducing genes conferring resistance to nematodes. Transferred genes could include those encoding products detrimental to nematode development or reproduction as well as cloned host resistance genes. Host genes that confer resistance to cyst or root-knot nematode species have been identified in many plants. The best characterized is Mi, a gene that confers resistance to root-knot nematodes in tomato. A map-based cloning approach is being used to isolate the gene. For development of a detailed map of the region of the genome surrounding Mi, DNA markers genetically linked to Mi have been identified and analyzed in tomato lines that have undergone a recombination event near Mi. The molecular map will be used to identify DNA corresponding to Mi. We estimate that a clone of Mi will be obtained in 2-5 years. An exciting prospect is that introduction of this gene will confer resistance in plant species without currently available sources of resistance.     

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens

Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F₂ progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.     

水稻根系研究进展

水稻(Oryza sativa)是我国最重要的粮食作物之一, 在保障国家持续的粮食供给中扮演着重要角色。根系作为水稻生长发育必不可少的器官, 间接地决定着水稻地上部产量、品质、抗逆及广适性等诸多农艺性状的表现。近年来, 随着水稻根系法的不断改进和图位克隆技术的完善及广泛应用, 水稻根系研究也取得了较大进展, 并已成功定位、分离、克隆了一些控制水稻根系的相关基因。该文从水稻根系法、相关性、基因定位、克隆及功能解析等层面综述了国内外水稻根系的研究进展, 并阐述了水稻根系研究存在的问题和今后研究的重点。     

From genomics to functional markers in the era of next-generation sequencing

The availability of complete genome sequences, along with other genomic resources for Arabidopsis, rice, pigeon pea, soybean and other crops, has revolutionized our understanding of the genetic make-up of plants. Next-generation DNA sequencing (NGS) has facilitated single nucleotide polymorphism discovery in plants. Functionally-characterized sequences can be identified and functional markers (FMs) for important traits can be developed at an ever-increasing ease. FMs are derived from sequence polymorphisms found in allelic variants of a functional gene. Linkage disequilibrium-based association mapping and homologous recombinants have been developed for identification of “perfect” markers for their use in crop improvement practices. Compared with many other molecular markers, FMs derived from the functionally characterized sequence genes using NGS techniques and their use provide opportunities to develop high-yielding plant genotypes resistant to various stresses at a fast pace.     

Rice functional genomics research: progress and implications for crop genetic improvement

Rice is a staple food crop and has become a reference of monocot plant for functional genomic research. With the availability of high quality rice genome sequence, there has been rapid accumulation of functional genomic resources, including: large mutant libraries by T-DNA insertion, transposon tagging, and chemical mutagenesis; global expression profiles of the genes in the entire life cycle of rice growth and development; full-length cDNAs for both indica and japonica rice; sequences from resequencing large numbers of diverse germplasm accessions. Such resource development has greatly accelerated gene cloning. By the end of 2010, over 600 genes had been cloned using various methods. Many of the genes control agriculturally useful traits such as yield, grain quality, resistances to biotic and abiotic stresses, and nutrient-use efficiency, thus have potential utility in crop genetic improvement. This review was aimed to provide a comprehensive summary of such progress. We also presented our perspective for future studies.     

Development of a new transformation-competent artificial chromosome (TAC) vector and construction of tomato and rice TAC libraries

Recent research has shown that BIBAC (binary bacterial artificial chromosome) and TAC (transformation-competent artificial chromosome) vector systems are very useful tools for map-based cloning of agronomically important genes in plant species. We have developed a new TAC vector that is suitable for both dicot and monocot transformation. Using this new TAC vector, we constructed large-insert genomic libraries of tomato and rice. The tomato library contains 96,996 clones (28.3-38.5 kb insert size) and has 3.18 haploid genome equivalents. The rice TAC library has 32.7 kb average insert size and has 9.24 haploid genome equivalents. The quality of these two libraries was tested using PCR to verify genome coverage. Individual clones were characterized to confirm insert integrity by Southern analysis, end sequencing and genetic mapping. To investigate the potential application of these TAC libraries in map-based cloning, TAC constructs containing a 45 kb fragment were introduced into the rice genome via Agrobacterium-mediated transformation. Molecular analysis indicates that the 45 kb fragment was successfully transferred into the rice genome. Although rearrangements of the introduced DNA were detected, 50% of regenerated plants contained at least one intact copy of the 45 kb clone and associated vector sequences. These libraries provide us with a valuable resource to rapidly isolate important genes in tomato and rice.     

水稻广谱抗稻瘟病基因研究进展

稻瘟病是水稻生产中的最严重病害之一,由于稻瘟菌小种的高度变异性,垂直抗性基因难以持续控制稻瘟病的危害,因此,克隆和利用广谱持久抗瘟基因被认为是解决稻瘟病问题最经济有效的策略。本文从广谱抗源的筛选与利用,广谱抗瘟基因的定位、克隆与应用等方面对水稻广谱抗稻瘟病基因研究取得的进展进行了概述,并介绍了广谱抗性分子机理的最新研究进展。基于国内外稻瘟病抗性基因研究的现状及趋势,以及我国丰富的抗瘟水稻种质资源,克隆越来越多的广谱抗瘟基因具有重要的理论与应用价值。     

New in silico insight into the synteny between rice (Oryza sativa L.) and maize (Zea mays L.) highlights reshuffling and identifies new duplications in the rice genome

A unigene set of 1411 contigs was constructed from 2629 redundant maize expressed sequence tags (ESTs) mapped on the maizeDB genetic map. Rice orthologous sequences were identified by blast alignment against the rice genomic sequence. A total of 1046 (74%) maize contigs were associated with their corresponding homologues in the rice genome and 656 (47%) defined as potential orthologous relationships. One hundred and seventeen (8%) maize EST contigs mapped to two distinct loci on the maize genetic map, reflecting the tetraploid nature of the maize genome. Among 492 mono-locus contigs, 344 (484 redundant ESTs) identify collinear blocks between maize chromosomes 2 and 4 and a single rice chromosome, defining six new collinear regions. Fine-scale analysis of collinearity between rice chromosomes 1 and 5 with maize chromosomes 3, 6 and 8 shows the presence of internal rearrangements within collinear regions. Mapping of maize contigs to two distinct loci on the rice sequence identifies five new duplication events in rice. Detailed analysis of a duplication between rice chromosomes 1 and 5 shows that 11% of the annotated genes from the chromosome 1 locus are found duplicated on the chromosome 5 paralogous counterpart, indicating a high degree of re-organisations. The implications of these findings for map-based cloning in collinear regions are discussed.     

tA single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta

The rice blast resistance (R) gene Pi-ta mediates gene-for-gene resistance against strains of the fungus Magnaporthe grisea that express avirulent alleles of AVR-Pita. Using a map-based cloning strategy, we cloned Pi-ta, which is linked to the centromere of chromosome 12. Pi-ta encodes a predicted 928-amino acid cytoplasmic receptor with a centrally localized nucleotide binding site. A single-copy gene, Pi-ta shows low constitutive expression in both resistant and susceptible rice. Susceptible rice varieties contain pi-ta(-) alleles encoding predicted proteins that share a single amino acid difference relative to the Pi-ta resistance protein: serine instead of alanine at position 918. Transient expression in rice cells of a Pi-ta(+) R gene together with AVR-Pita(+) induces a resistance response. No resistance response is induced in transient assays that use a naturally occurring pi-ta(-) allele differing only by the serine at position 918. Rice varieties reported to have the linked Pi-ta(2) gene contain Pi-ta plus at least one other R gene, potentially explaining the broadened resistance spectrum of Pi-ta(2) relative to Pi-ta. Molecular cloning of the AVR-Pita and Pi-ta genes will aid in deployment of R genes for effective genetic control of rice blast disease.     

The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most serious diseases of rice. Here we describe the isolation and characterization of Pib, one of the rice blast resistance genes. The Pib gene was isolated by a map-based cloning strategy. The deduced amino acid sequence of the Pib gene product contains a nucleotide binding site (NBS) and leucine-rich repeats (LRRs); thus, Pib is a member of the NBS-LRR class of plant disease resistance genes. Interestingly, a duplication of the kinase 1a, 2 and 3a motifs of the NBS region was found in the N-terminal half of the Pib protein. In addition, eight cysteine residues are clustered in the middle of the LRRs, a feature which has not been reported for other R genes. Pib gene expression was induced upon altered environmental conditions, such as altered temperatures and darkness.     

图位克隆技术在农作物基因分离中的应用与评价

图位克隆(Map-based cloning)作为分离基因的有效方法, 已在小基因组作物的基因分离中得到了广泛应用和发展, 而在具有大量重复DNA序列的大基因组作物中的应用仍存在挑战。基于图位克隆在基因分离中的重要性, 文章就图位克隆技术的基本内容及发展做简要概述, 着重对图位克隆技术在大基因组作物中的应用进行分析和评价, 同时对它今后可能的发展方向进行了讨论, 以期为大基因组作物基因的分离提供借鉴。     

International Rice Genome Sequencing Project: the effort to completely sequence the rice genome

The International Rice Genome Sequencing Project (IRGSP) involves researchers from ten countries who are working to completely and accurately sequence the rice genome within a short period. Sequencing uses a map-based clone-by-clone shotgun strategy; shared bacterial artificial chromosome/P1-derived artificial chromosome libraries have been constructed from Oryza sativa ssp. japonica variety 'Nipponbare'. End-sequencing, fingerprinting and marker-aided PCR screening are being used to make sequence-ready contigs. Annotated sequences are immediately released for public use and are made available with supplemental information at each IRGSP member's website. The IRGSP works to promote the development of rice and cereal genomics in addition to producing genome sequence data.