Comparative genomic mapping between a 754 kb region flanking DREB1A in Arabidopsis thaliana and maize

Comparative mapping between model plant species for which the complete genome sequence is known and crop species has been suggested as a new strategy for the isolation of agronomically valuable genes. In this study, we tested whether comparative mapping between Arabidopsisand maize of a small region (754 kb) surrounding the DREB1A gene in Arabidopsis could lead to the identification of an orthologous region in maize containing the DREB1A homologue. The genomic sequence information available for Arabidopsis allowed for the selection of conserved, low-copy genes that were used for the identification of maize homologues in a large EST database. In total, 17 maize homologues were mapped. A second BLAST comparison of these genes to the recently completed Arabidopsis sequence revealed that 15 homologues are likely to be orthologous as the highest similarity score was obtained either with the original Arabidopsis gene or with a highly similar Arabidopsis gene localized on a duplication of the investigated region on chromosome 5. The map position of these genes showed a significant degree of orthology with the Arabidopsis region. Nevertheless, extensive duplications and rearrangements in the Arabidopsisand maize genomes as well as the evolutionary distance between Arabidopsis and maize make it unlikely that orthology and collinearity between these two species are sufficient to aid gene prediction and cloning in maize.     

Molecular analysis of the dhp1+ gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST 2 and RAT 1 genes of Saccharomyces cerevisiae

We report here the first cloning of a chalcone flavonone isomerase gene (CHI) from maize. Northern blot experiments indicate that the maize CHI gene (ZmCHI1) is regulated in the pericarp by the P gene, a myb homologue. The ZmCHI1 gene encodes a 24.3 kDa product 55% and 58% identical to CHI-A and CHI-B from Petunia, respectively. This maize CHI gene has four exons and an intron-exon structure identical to the CHI-B gene of Petunia hybrida. RFLP mapping data indicate that some inbred lines contain two additional CHI-homologous sequences, suggesting an organization more complex than that found in Petunia or bean. The possibility that the additional CHI-homologous sequences are responsible for the lack of CHI mutants in maize will be discussed.     

Gametophytic expression of genes controlling endosperm development in maize

Summary An indirect approach was adopted to select viable mutants affecting the male gametophytic generation in maize. This approach consists of a selection of endosperm defective mutants followed by a test for gametophytic gene expression, based on the distortion from mendelian segregation and on the measurement of pollen size and pollen sterility. The material used consisted of 34 endosperm defective viable mutants introgressed in B37 genetic background. Complementation tests indicated that the mutation in the collection of mutants affected different genes controlling endosperm development. The study of the segregation in F2 revealed four classes of de (defective endosperm) mutants: (1) mutants in which the mutation does not affect either gametophytic development or function; (2) mutants in which the effect on the gametophyte affects pollen development processes; (3) mutants showing effects on both pollen development and function, and (4) mutants where only pollen tube growth rate is affected. Positive and negative interactions between pollen and style were detected by means of mixed pollination (pollen produced by de/de plants and pollen from an inbred line used as a standard and carrying genes for colored aleurone), on de/de and de/ + plants. Positive interactions were interpreted as methabolic complementation between defective pollen and normal styles.     

Barley putative hypersensitive induced reaction genes: genetic mapping,sequence analyses and differential expression in disease lesion mimic mutants

The hypersensitive response (HR) is one of the most-efficient forms of plant defense against biotrophic pathogens, and results in localized cell death and the formation of necrotic lesions; however, the molecular components of pathways leading to HR remain largely unknown. Barley (Hordeum vulgare ssp. vulgare L.) cDNAs for putative hypersensitive-induced reaction (HIR) genes were isolated based on DNA and amino-acid homologies to maize HIR genes. Analyses of the cDNA and genomic sequences and genetic mapping found four distinct barley HIR genes, Hv-hir1, Hv-hir2, Hv-hir3 and Hv-hir4, on chromosomes 4(4H) bin10, 7(5H) bin04, 7(5H) bin07 and 1(7H) bin03, respectively. Hv-hir1, Hv-hir2 and Hv-hir3 genes were highly homologous at both DNA and the deduced amino-acid level, but the Hv-hir4 gene was similar to the other genes only at the amino-acid sequence level. Amino-acid sequence analyses of the barley HIR proteins indicated the presence of the SPFH protein-domain characteristic for the prohibitins and stomatins which are involved in control of the cell cycle and ion channels, as well as in other membrane-associated proteins from bacteria, plants and animals. HIR genes were expressed in all organs and developement stages analyzed, indicating a vital and non-redundant function. Barley fast-neutron mutants exhibiting spontaneous HR (disease lesion mimic mutants) showed up to a 35-fold increase in Hv-hir3 expression, implicating HIR genes in the induction of HR.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Wenzel     

A recombination hotspot in the maize A1 intragenic region

Summary We find that recombination between two alleles of the maize A1 locus that contain transposon insertions at known molecular positions can occur at 0.04–0.08 cM per kbp (centimorgan per kilobase pair), which is two orders of magnitude higher than the recombination rate for the whole maize genome. It is however, close to the rates found within the bronze locus, another maize structural gene for which both genetic and molecular data are available. This observation supports the idea that the genome consists of regions that are highly recombinogenic — in some cases, at least, structural genes — interspersed with regions that are less recombinogenic.     

Mapping genes essential for embryo development in Arabidopsis thaliana.

Summary We have previously isolated and characterized over 90 recessive mutants of Arabidopsis thaliana defective in embryo development. These emb mutants have been shown to differ in lethal phase, extent of abnormal development, and response in culture. We demonstrate in this report the value and efficiency of mapping emb genes relative to visible and molecular markers. Sixteen genes essential for embryo development were mapped relative to visible markers by analyzing progeny of selfed F₁ plants. Embryonic lethals are now the most common type of visible marker included on the linkage map of Arabidopsis. Backcrosses were used in several cases to orient genes relative to adjacent markers. Three genes were located to chromosome arms with telotrisomics by screening for a reduction in the percentage of aborted seeds produced by F₁ plants. A restriction fragment length polymorphism (RFLP) mapping strategy that utilizes pooled EMB/EMB F₂ plants was devised to increase the efficiency of mapping embryonic lethals relative to molecular markers. This strategy was tested by demonstrating that the biol locus of Arabidopsis is within 0.5 cM of an existing RFLP marker. Mapping embryonic lethals with both visible and molecular markers may therefore help to identify large numbers of genes with essential functions in Arabidopsis.     

Linkage mapping of maize and barley myo-inositol 1-phosphate synthase DNA sequences: correspondence with a low phytic acid mutation

 We sequenced and genetically mapped the myo-inositol 1-phosphate synthase (MIPS) genes of maize (Zea mays L.) and barley (Hordeum vulgare L). Our objective was to determine whether the genetic map positions of these MIPS loci correspond with the location of the low phtyic acid 1 (lpa1) mutations that were previously identified in maize and barley. Seven MIPS-homologous sequences were mapped to positions on maize chromosomes 1S, 4L, 5S, 6S, 8L, 9S and 9L, and a similar number of divergent MIPS sequences were amplified from maize. To the extent that we can compare across different genetic mapping populations, the position of the MIPS gene on maize chromosome 1S is identical to the location of the maize lpa1 mutation. However, only one MIPS sequence was identified in barley and this gene was mapped to a locus on chromosome 4H that is separate from the barley lpa1 mutation on chromosome 2H. Although several RFLP markers linked to the barley MIPS gene on chromosome 4H also detect loci near barley lpa1 on chromosome 2H, our experiments failed to reveal a second MIPS gene that could be associated with the barley lpa1 mutation. Therefore, genetic mapping results from this study support the MIPS candidate-gene hypothesis for maize lpa1, but do not support the MIPS candidate-gene-hypothesis for barley lpa1. These opposing results contradict the hypothesis that maize lpa1 and barley lpa1 are mutations of orthologous genes, which is suggested by the similar biochemical phenotypes of these mutants. Yet, comparisons of RFLP mapping studies show loci that are homologous between maize chromosome 1S, barley chromosome 4H and barley chromosome 2H, including regions flanking the respective MIPS and/or lpa1 loci. This putative relationship, between the regions flanking the lpa1 mutations on maize 1S and barley 2H, also supports the assertion that these mutations are orthologous despite contradictory results between our maize and barley candidate-gene experiments. Received: 24 August 1998 / Accepted: 19 December 1998     

Candidate gene identification of existing or induced mutations with pipelines applicable to large genomes

Bulked segregant analysis (BSA) is used to identify existing or induced variants that are linked to phenotypes. Although it is widely used in Arabidopsis and rice, it remains challenging for crops with large genomes, such as maize. Moreover, analysis of huge data sets can present a bottleneck linking phenotypes to their molecular basis, especially for geneticists without programming experience. Here, we identified two genes of maize defective kernel mutants with newly developed analysis pipelines that require no programing skills and should be applicable to any large genome. In the 1970s, Neuffer and Sheridan generated a chemically induced defective kernel (dek) mutant collection with the potential to uncover critical genes for seed development. To locate such mutations, the dek phenotypes were introgressed into two inbred lines to take advantage of maize haplotype variations and their sequenced genomes. We generated two pipelines that take fastq files derived from next‐generation (nextGen) paired‐end DNA and cDNA sequencing as input, call on several well established and freely available genomic analysis tools to call SNPs and INDELs, and generate lists of the most likely causal mutations together with variant index plots to locate the mutation to a specific sequence position on a chromosome. The pipelines were validated with a known strawberry mutation before cloning the dek mutants, thereby enabling phenotypic analysis of large genomes by next‐generation sequencing.     

Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Brown‐midrib maize (bm1) – a mutation affecting the cinnamyl alcohol dehydrogenase gene

Brown-midrib (bm) mutants of maize have modified lignin of reddish-brown colour. Although four independent bm loci are known, only one of the mutant genes has been previously identified. We report here that maize bm1, one of the less characterised mutants, shows severely reduced CAD activity in lignified tissues, resulting in the production of a modified lignin. Both the total lignin content and the structure of the polymer are altered by the mutation. We further describe the isolation and characterisation of the maize CAD cDNA and mapping of the CAD gene. CAD maps very closely to the known location of bm1 and co-segregates with the bm1 locus in two independent recombinant inbred populations. These data strongly support the premise that maize bm1 directly affects expression of the CAD gene.     

A double‐mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions

Mitogen‐activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single‐mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double‐mutants are created from a large library of single‐mutant lines. Here we describe a new collection of 275 double‐mutant lines derived from a library of single‐mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high‐throughput double‐mutant generating pipeline using a system for growing Arabidopsis seedlings in 96‐well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double‐mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single‐mutant line. Seeds for this double‐mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double‐mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling.     

A maize Ds transposable element containing a dihydrofolate reductase gene transposes in Nicotiana tabacum and Arabidopsis thaliana

Summary A mouse dihydrofolate reductase gene (DHFR), encoding an enzyme conferring methotrexate (MTX) resistance, under the control of the cauliflower mosaic virus (CaMV) 35 S promoter, was inserted within a maize nonautonomous Ds transposable element. The presence of at least one element (Ds-DHFR) can easily be monitored using methotrexate selection in plants. This chimeric element is able to transpose at a frequency similar to its unmodified progenitor in transgenic tobacco callus containing an autonomous Ac element. The orientation of the selectable marker cassette in the Ds element does not affect relative excision frequencies. Approximately two-thirds of these elements can be detected after excision while the remaining one-third cannot. The Ds-DHFR element is useful in elucidating the mechanism by which Ac/Ds transposition occurs, and allows for a rapid identification of mutants in which methotrexate resistance cosegregates with a mutant phenotype.     

Genetic characterization of Tetrahymena thermophila mutants unable to secrete capsules

Under appropriate conditions, Alcian Blue-induced exocytosis of Tetrahymena mucocysts leads to formation of a capsule that surrounds the cell. This phenomenon is an example of regulated secretion, a mechanism of fundamental significance in eukaryotic cells. In order to dissect genetically the mechanism of mucocyst biogenesis and regulated exocytosis, mutants unable to form capsules (Caps–) were isolated. In this paper we report a genetic characterization of Caps– mutants in this collection. The mutations in mutants SB255 and SB281 behave as single recessive Men-delian mutations. The mutation in SB251 is restricted to the macronucleus, and could not be further characterized by the genetic methods we used. Complementation tests suggest the existence of at least 2 genes, named exoA and exoB; additional mutant loci are likely to be included in the mutant collection. Deletion mapping using nulli-somic strains showed that exoA and exoB are located on the left arm of chromosome 4. The exo-3 mutation, which behaves as recessive and complements with exoA1 in SB255 and exoB2 in SB281, maps to chromosome 3. These Caps– mutants may be useful for the elucidation of the developmental pathway of mucocyst biogenesis and the control of regulated secretion in eukaryotic cells. © 1992 Wiley-Liss, Inc.     

Genetic and mutational tools for investigating the genetics and molecular biology of trichothecene production inGibberella pulicaris (Fusarium sambucinum)

Gibberella pulicaris (Fusarium sambucinum) is a promising organism for studying the genetics and regulation of trichothecene biosynthesis; conditions for obtaining fertile crosses have been defined (Desjardins & Beremand, 1987) and crosses between natural variants have provided some information about the number, location, arrangement, and role of genes which determine trichothecene production (Desjardins & Beremand, 1987; Beremand & Desjardins, 1988). The development of some additional experimental tools and methodologies required for the further genetic analysis of trichothecene production inG. pulicaris are described in the present study. A highly fertile, isogenic line was constructed forG. pulicaris strain R-6380. The ability to readily generate mutants in this strain was also demonstrated. Both biochemical and morphological mutants were obtained following UV-mutagenesis. The inheritance of some of these mutations through meiosis indicated that they will be useful genetic markers for crosses and mapping studies. Since strain R-6380 is also transformable (Salch & Beremand, 1988), it is an excellent choice for transmission and molecular genetic studies involving trichothecene production.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Characterization of a region of the X chromosome of Drosophila including multi sex combs (mxc), a Polycomb group gene which also functions as a tumour suppressor

Genetic analysis of the 8D3;8D8-9 segment of the Drosophila melanogaster X chromosome has assigned seven complementation groups to this region, three of which are new. A Polycomb group (Pc-G) gene, multi sex combs (mxc), is characterized and mutant alleles are described. Besides common homeotic transformations characteristic of Pc-G mutants that mimic the ectopic gain of function of BX-C and ANT-C genes, mxc mutants show other phenotypes: they zygotically mimic, in males and females, the characteristic lack of germ line seen in progeny of some maternal effect mutants of the so-called posterior group (the grandchildless phenotype). Loss of normal mxc function can promote uncontrolled malignant growth which indicates a possible relationship between Pc-G genes and tumour suppressor genes. We propose that gain-of-function of genes normally repressed by the wild-type mxc product could, in mxc mutants, give rise to an incoherent signal which would be devoid of meaning in normal development. Such a signal could divert somatic and germ line developmental pathways, provoke the loss of cell affinities, but allow or promote growth.