Physical mapping,expression analysis and polymorphism survey of resistance gene analogues on chromosome 11 of rice

Rice is the first cereal genome with a finished sequence and a model crop that has important syntenic relationships with other cereal species. The objectives of our study were to identify resistance gene analogue (RGA) sequences from chromosome 11 of rice, understand their expression in other cereals and dicots by in silico analysis, determine their presence on other rice chromosomes, and evaluate the extent of polymorphism and actual expression in a set of rice genotypes. A total of 195 RGAs were predicted and physically localised. Of these, 91.79% expressed in rice, and 51.28% expressed in wheat, which was the highest among other cereals. Among monocots, sugarcane showed the highest (78.92%) expression, while among dicots, RGAs were maximally expressed in Arabidopsis (11.79%). Interestingly, two of the chromosome 11-specific RGAs were found to be expressing in all the organisms studied. Eighty RGAs of chromosome 11 had significant homology with chromosome 12, which was the maximum among all the rice chromosomes. Thirty-one per cent of the RGAs used in polymerase chain reaction (PCR) amplification showed polymorphism in a set of rice genotypes. Actual gene expression analysis revealed post-inoculation induction of one RGA in the rice line IRBB-4 carrying the bacterial blight resistance gene Xa-4. Our results have implications for the development of sequence-based markers and functional validation of specific RGAs in rice. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Supplementary tables pertaining to this article are available on the Journal of Biosciences Website at     

Fine mapping and candidate gene analysis of LM3, a novel lesion mimic gene in rice

A rice lesion mimic mutant, lm3, was obtained by the mutagenesis of an indica cultivar, 93-11, using γ-ray radiation. Brownish lesions appeared on the leaves of lm3 at the young seedling stage and persisted until the ripening stage. The lm3 mutant was characterised by a shorter plant height and delayed heading compared with the wild-type 93-11. A genetic analysis indicated that the lesion mimic phenotype was controlled by a single recessive gene. Using simple sequence repeat (SSR) markers, the target gene LM3 was first located between marker RM5748 and RM14906 on chromosome 3. We then developed Insertion-Deletion (InDel) markers to fine-map LM3, and the locus was localised to a 29 kb region defined by two InDel markers, In12571 and In12600. Five ORFs were predicted in the candidate region, and DNA sequencing detected a single-nucleotide polymorphism (SNP) in the coding region of LOC Os03g21900. The SNP in the fourth exon (C in 93-11; T in lm3) of LOC_Os03g21900 results in the substitution of a proline (P) with a serine (S) at the 140^th amino acid of the deduced uroporphyrinogen decarboxylase protein. We did not detect polymorphisms in the other predicted ORF regions between lm3 and 93-11. These results suggest that LOC_Os03g21900 is the most likely candidate gene for LM3.     

Comparative physical mapping between wheat chromosome arm 2BL and rice chromosome 4

Physical maps of chromosomes provide a framework for organizing and integrating diverse genetic information. DNA microarrays are a valuable technique for physical mapping and can also be used to facilitate the discovery of single feature polymorphisms (SFPs). Wheat chromosome arm 2BL was physically mapped using a Wheat Genome Array onto near-isogenic lines (NILs) with the aid of wheat-rice synteny and mapped wheat EST information. Using high variance probe set (HVP) analysis, 314 HVPs constituting genes present on 2BL were identified. The 314 HVPs were grouped into 3 categories: HVPs that match only rice chromosome 4 (298 HVPs), those that match only wheat ESTs mapped on 2BL (1), and those that match both rice chromosome 4 and wheat ESTs mapped on 2BL (15). All HVPs were converted into gene sets, which represented either unique rice gene models or mapped wheat ESTs that matched identified HVPs. Comparative physical maps were constructed for 16 wheat gene sets and 271 rice gene sets. Of the 271 rice gene sets, 257 were mapped to the 18-35?Mb regions on rice chromosome 4. Based on HVP analysis and sequence similarity between the gene models in the rice chromosomes and mapped wheat ESTs, the outermost rice gene model that limits the translocation breakpoint to orthologous regions was identified.     

Fine mapping of a male sterility gene, vr1, on chromosome 4 in rice

xs1 is a male sterile rice mutant derived from a spontaneous mutation. Pollen development in the xs1 mutant proceeds normally until the vacuolation stage, at which time xs1 pollen fails to vacuolate and no viable pollen is produced. Genetic analysis indicates that the xs1 mutant phenotype is controlled by a single recessive gene, designated vacuolation retardation 1 (vr1), which was mapped to rice chromosome 4. In order to fine-map the vr1 locus, two large mapping populations were generated and several SSR and InDel markers were developed from publicly available rice genomic sequences. By employing a strategy of chromosome-walking, the vr1 gene was finally located within a genetic interval of 0.27 cM, flanked by the markers FID30 and FS15, with distances of 0.11 and 0.16 cM, respectively, and co-segregating with the marker FC4-2. Based on the japonica rice genome sequence, the vr1 locus is estimated to cover a 48-kb region containing eight putative genes. Our results will facilitate the cloning and functional characterization of the vr1 gene.     

一个新的水稻叶绿素缺失黄叶突变体的特征及基因分子定位

从粳稻"嘉花1号"60Coγ射线辐照的后代中筛选到一个叶绿素缺失黄叶突变体(yl11),与野生型"嘉花1号"相比该突变体表现为全生育期植株叶片呈黄色,叶绿素含量以及净光合速率明显下降,叶绿体发育不完善,并且伴随着株高等主要农艺性状的变化。遗传分析表明,该突变性状受一对隐性核基因(yl11)控制。该突变体与籼稻"培矮64S"杂交生产的F2、F3群体中的分离出突变体型920个单株作为定位群体,利用SSR和InDel分子标记将yl11基因定位在水稻第11染色体长臂上的MM2199和ID21039分子标记之间,其物理距离约为110kb,目前该区域内没有发现与水稻叶绿素合成/叶绿体发育相关已知功能基因。研究结果为今后对该基因的克隆和功能分析奠定了基础。     

Genetic mapping with dispersed repeated sequences in the rice blast fungus: mapping the SMO locus

Summary The SMO genetic locus in strains of the fungus Magnaporthe grisea that infect weeping lovegrass, directs the formation of correct cell shapes in asexual spores, infection structures, and asci. We have identified and characterized a Smo^– strain of M. grisea that infects rice. The smo mutation in this strain segregates as a single gene mutation and is allelic to previously identified smo alleles. A marked reduction in pathogenicity co-segregates with the Smo^– phenotype, suggesting that the SMO locus plays a role in rice pathogenicity. A family of dispersed repeated DNA sequences, called MGR, have been discovered in the nuclear DNA of M. grisea rice pathogens. Genetic crosses between Smo^– rice pathogens and Smo⁺ non-rice pathogens were used to follow the segregation of the SMO locus and individual MGR sequences. Using DNA blot analysis with cloned MGR hybridization probes, we mapped the SMO locus to a chromosomal region flanked by two closely linked MGR sequences. We demonstrated that the copy number of MGR sequences could be reduced in subsequent crosses to non-rice pathogens of M. grisea, and that new MGR sequences did not occur following meiosis indicating that these sequences are stable in the genome. We conclude that restriction fragment polymorphism mapping with cloned MGR sequences as hybridization probes is an effective way to map genes in the rice blast fungus.     

Physical mapping of duplicated genomic regions of two chromosome ends in rice.

Two genomic regions duplicated in distal ends of the short arms of chromosomes 11 and 12 in rice (Oryza sativa L.) were characterized by YAC ordering with 46 genetic markers. Physical maps covering most of the duplicated regions were generated. Thirty-five markers, including 21 rice cDNA clones, showed the duplicated loci arrayed strictly in the same order along the two specific genomic regions. Regardless of their different genetic distances, the two duplicated segments may have a similar and minimum physical size with an expected length of about 2.5 Mb. However, differences of RFLP frequency for the duplicated DNA copies and recombination frequency for a given homoeologous area between the two regions were observed, indicating that these changes in genome organization occurred after the duplication. Our results establish a good model system for resolving the relationships between gene duplication, expression of duplicated genes, and the frequency of meiotic recombination in small chromosomal regions.     

Genetic mapping of hypervariable minisatellite sequences in rice (Oryza sativa L.)

Minisatellites, or DNA fingerprinting sequences, have been utilized in animal linkage studies for several years but have not been used as markers for plant genome mapping. In animal genome mapping they have resulted in limited success because they are evenly dispersed in some species but are often clustered near telomeric regions, as observed on human chromosomes. The purpose of the present study was to generate DNA fingerprints utilizing several rice-derived minisatellites containing different core sequences and numbers of repeat units, followed by assessing their potential for use as genetic markers when mapped to a rice recombinant inbred line (RIL) population. Sites of segregating minisatellite loci were mapped onto 11 of the 12 rice RIL linkage maps. The implications for the use of rice minisatellite core sequences as genetic markers on linkage maps in rice are discussed. Received: 1 March 1999 / Accepted: 22 June 1999     

QTL mapping of protein content in rice using single chromosome segment substitution lines

Protein content (PC) is an important component of rice nutritional quality. In order to better understand the genetic basis of this trait and increase related breeding efficiency, 21 single chromosome segment substitution (SCSS) lines grown in four sites over two growing seasons (regarded as eight environments) were used to associate PC with particular chromosome segments. Segments from 15 chromosomes were found to contain quantitative trait loci (QTLs) for PC in at least one environment. These included segments from chromosome 3 and 8, in which QTLs for PC had not previously been identified. The segment of chromosome 8 in CSSL-48 had the largest positive effect across all environments. The interaction between substitution and environment was highly significant. Some substitutions had large effects in one environment, but no effect in another (i.e. CSSL-08 and CSSL-17), while some substitutions significantly increased PC in one environment but decreased it in another (i.e. CSSL-41 and CSSL-43). By biplot and clustering analysis, the eight environments were grouped into two contrasting environment types, that is, Hainan and Jiangsu. The segment of chromosome 8 in CSSL-48 had PC-enhancing QTLs in both of the environment types. The segments in CSSL-34 had QTLs which increase PC in the Jiangsu environment but have no effect in the Hainan environment. For enhancing PC, CSSL-48 could be explored in breeding for wide adaptation across all environments, while CSSL-12, CSSL-14, CSSL-17, CSSL-41 and CSSL-43, and that in CSSL-34 could be explored in breeding for specific adaptation to the Hainan and Jiangsu environments, respectively. Near isogenic lines are under development to validate the QTLs with large effects in a range of genetic backgrounds relevant to Jiangsu and Hainan breeding programs. Secondary mapping populations are also being developed for further localising the responsible QTLs in CSSL-14, CSSL-34 and CSSL-48.     

Isolation and regional mapping of DNA sequences unique to human chromosome 21.

To isolate DNA sequences unique to chromosome 21 we have used a recombinant-DNA library, constructed from a mouse-human somatic-cell hybrid line containing chromosome 21 as the only human chromosome. Individual recombinant phage containing human DNA inserts were identified by their hybridization to total human DNA sequences and by their failure to hybridize to total mouse DNA sequences. A repeat-free human DNA fragment was then subcloned from each of 14 such recombinant phage. An independent somatic-cell hybrid was used to assign all 14 subcloned fragments to chromosome 21. Thirteen of the fragments have been regionally mapped using a somatic-cell hybrid containing a human 21 translocation chromosome. Two probes map proximal to the 21q21.2 translocation breakpoint, and 11 probes map distal to this breakpoint, placing them in the region 21q21.2-21q22. One of seven probes used to screen for restriction-fragment-length polymorphisms recognized polymorphic DNA fragments when hybridized to genomic DNA from unrelated individuals. These 14 unique probes provide useful tools for studying the structure and function of human chromosome 21 as well as for investigating the molecular biology of Down syndrome.     

Regional mapping of unique DNA sequences from human chromosome 3 derived from a flow-sorted chromosome library

Eight single-copy DNA probes specific for human chromosome 3 were isolated by screening a human chromosome 3-derived genomic library. Southern blot analyses of DNAs isolated from a panel of somatic cell hybrids allowed us to regionally assign all probes to subregions on chromosome 3. Three clones were localized to the short arm of chromosome 3 (3p21----pter), two to the long arm (3q21----qter), and three to the 3q21----3p21 subregion. Six of these DNA sequences map to regions overlapping a segment of chromosome 3 (3p14----p23) frequently deleted in small cell lung cancer cells. Restriction fragment length polymorphism analyses indicate that at least three of the eight single-copy probes studies show MspI or BglII polymorphisms. This library is a useful source of chromosome 3-specific probes.     

Regional mapping of six cloned DNA sequences on human chromosome 7.

The regional localization of six cloned DNA sequences on human chromosome 7 was assessed by molecular hybridization to human/rodent cell hybrid DNAs. The allelic distribution and familial segregation of two frequent polymorphisms revealed by two probes are presented.     

Differentiation and evolutionary relationships in Erythrinus erythrinus (Characiformes, Erythrinidae): comparative chromosome mapping of repetitive sequences

Erythrinus erythrinus presents extensive karyotypic diversity, with four karyomorphs (A–D) differing in the number of chromosomes, karyotype structure or sex chromosomes systems. Karyomorph A has 2n = 54 chromosomes in males and females without heteromorphic sex chromosomes, while karyomorph C has 2n = 52 chromosomes in females and 2n = 51 chromosomes in males, due a X₁X₁X₂X₂/X₁X₂Y sex chromosome system. Three allopatric populations of the karyomorph A and one population of the karyomorph C were now in deep investigated by molecular cytogenetic analyses, using repetitive DNAs as probes. The results reinforced the relatedness among populations of the karyomorph A, despite their large geographic distribution. Karyomorph C, however, showed a remarkably difference in the genomic constitution, especially concerning the amount and distribution of the 5S rDNA and Rex3 sequences on chromosomes. In addition, although karyomorphs C and D share several features, exclusive chromosomal markers show the derivative evolutionary pathway between them. Thus, besides the classical chromosomal rearrangements, the repetitive DNAs were useful tools to reveal the biodiversity, relatedness and differentiation of this fish group. The chromosomal set strongly corroborates that E. erythrinus corresponds to a species complex instead of a single biological entity.     

Genetic analysis and molecular mapping of a novel gene for zebra mutation in rice （Oryza sativa L.）

A novel zebra mutant, zebra-15, derived from the restorer line JinhuilO （Oryza sativa L. ssp. indica） treated by EMS, displayed a distinctive zebra leaf from seedling stage to jointing stage. Its chlorophyll content decreased （55.4%） and the ratio of Chla/Chlb increased （90.2%） significantly in the yellow part of the zebra-15, compared with the wild type. Net photosynthetic rate and fluorescence kinetic parameters showed that the decrease of chlorophyll content significantly influenced the photosynthetic efficiency of the mutant. Genetic analysis of F2 segregation populations derived from the cross of XinonglA and zebra-15 indicated that the zebra leaf trait is controlled by a single recessive nuclear gene. Ninety-eight out of four hundred and eighty pairs of SSR markers showed the diversity between the XinonglA and the zebra-15, their F2 population was then used for gene mapping. Zebra-15 （Z-15） gene was primarily restricted on the short arm of chromosome 5 by 150 F2 recessive individuals, 19.6 cM from marker RM3322 and 6.0 cM from marker RM6082. Thirty-six SSR markers were newly designed in the restricted location, and the Z-15 was finally located between markers nSSR516 and nSSR502 with the physical region 258 kb by using 1,054 F2 recessive individuals.     

Identification, mapping, and phylogenetic analysis of three novel chicken CC chemokines

We have identified three novel chicken CC chemokine genes among cDNA clones derived from lipopolysaccharide-stimulated cells of the chicken macrophage cell line HD11. Two of these chemokines show DNA sequence homology to the mammalian genes SCYA20 (MIP-3alpha) and SCYA5 (RANTES), while the third shows similar levels of homology to several mammalian CC chemokines. Sequencing of genomic DNA showed that all three chicken chemokines possess the three-exon structure and conserved intron positions typical of mammalian CC chemokines. Genetic mapping of the three chicken chemokines locates them in three chromosomal regions which correspond to regions containing homologous chemokines in humans. Phylogenetic analysis of the currently known chicken and human chemokines suggests that individual chicken and human chemokines derive from common ancestral genes in patterns that reflect their genomic positions, indicating that the diversity of chemokine genes pre-dated avian-mammalian divergence. Since the function of the chemokines is principally to act as intermediates between stimulated cells and specific subsets of responding immune cells, this suggests that the complex organization of the immune system and diversity of responding cells were largely in place at that time.     

Genetic mapping of expressed sequences in onion and in silico comparisons with rice show scant colinearity

The Poales (which include the grasses) and Asparagales [which include onion (Allium cepa L.) and other Allium species] are the two most economically important monocot orders. Enormous genomic resources have been developed for the grasses; however, their applicability to other major monocot groups, such as the Asparagales, is unclear. Expressed sequence tags (ESTs) from onion that showed significant similarities (80% similarity over at least 70% of the sequence) to single positions in the rice genome were selected. One hundred new genetic markers developed from these ESTs were added to the intraspecific map derived from the BYG15-23×AC43 segregating family, producing 14 linkage groups encompassing 1,907 cM at LOD 4. Onion linkage groups were assigned to chromosomes using alien addition lines of Allium fistulosum L. carrying single onion chromosomes. Visual comparisons of genetic linkage in onion with physical linkage in rice revealed scant colinearity; however, short regions of colinearity could be identified. Our results demonstrate that the grasses may not be appropriate genomic models for other major monocot groups such as the Asparagales; this will make it necessary to develop genomic resources for these important plants. Electronic Supplementary Material Supplementary material is available for this article at     

Slot blot method for the quantification of DNA sequences and mapping of chromosome rearrangements: Application to chromosome 21

As an alternative to the methods of gene dosage based on either RFLP studies or Southern blots using specific and reference probes, we designed a "slot blot" method for the evaluation of the copy number of unique chromosome 21 sequences. Varying amounts of denatured DNA from a normal control, a trisomy 21 patient, and the subject to be analyzed were loaded on the same membrane. Successive hybridizations with reference probes and chromosome 21 probes were then carried out. Intensities of the signals on autoradiograms were quantified by densitometric scanning. Graphic and statistical analysis of the linear regressions between reference and chromosome 21 probe signals were performed, and the conclusion that the DNA from the studied subject had two or three copies for a given chromosome 21 sequence was assessed by statistical comparison of the slopes. As a test for the validation of this method, 10 coded blood DNAs from five normal controls and from five trisomy 21 patients were analyzed, by using two reference (COL1A1 and COL1A2) and two chromosome 21 (D21S11 and D21S17) probes. Among the 10 DNAs analyzed, it was possible to diagnose, with 100% accuracy, normal controls and trisomic 21 individuals. Application of this methodology to the mapping of partial chromosome 21 rearrangements is presented.