Mapping of the genes for dioecism and monoecism in Spinacia oleracea L.: evidence that both genes are closely linked

Spinach is basically a dioecious species, with occasional monoecious plants in some populations. Sexual dimorphism in dioecious spinach plants is controlled by an allelic pair termed X and Y located on the short arm of the longest chromosome (x = 6). Ten AFLP markers, closely linked to the X/Y locus, were identified using bulked segregant analysis, four of which were revealed to co-segregate with Y in the present mapping population. We mapped the AFLP markers and two known male-specific DNAs to a 13.4 cM region encompassing the locus. These markers will be the basis for positional cloning of the sex-determination gene. We also showed that a single, incompletely dominant gene is responsible for the highly staminate monoecious character. The gene was found to be located at a distance of 4.3 cM from microsatellite marker SO4, which mapped 1.6 cM from the X/Y locus. This indicates that the monoecious gene seems not to be allelic to but closely linked to the X/Y gene pair. SO4 will enable breeders to efficiently select highly male monoecious plants for preferential use as the pollen parent for hybrid seed production.     

The pectic disaccharides lepidimoic acid and β-d-xylopyranosyl-(1→3)-d-galacturonic acid occur in cress-seed exudate but lack allelochemical activity

Background and aims Cress-seed (Lepidium sativum) exudate exerts an allelochemical effect, promoting excessive hypocotyl elongation and inhibiting root growth in neighbouring Amaranthus caudatus seedlings. We investigated acidic disaccharides present in cress-seed exudate, testing the proposal that the allelochemical is an oligosaccharin—lepidimoic acid (LMA; 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnose).Methods Cress-seed exudate was variously treated [heating, ethanolic precipitation, solvent partitioning, high-voltage paper electrophoresis and gel-permeation chromatography (GPC)], and the products were bioassayed for effects on dark-grown Amaranthus seedlings. Two acidic disaccharides, including LMA, were isolated and characterized by electrophoresis, thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy, and then bioassayed.Key Results Cress-seed exudate contained low-M_r, hydrophilic, heat-stable material that strongly promoted Amaranthus hypocotyl elongation and inhibited root growth, but that separated from LMA on electrophoresis and GPC. Cress-seed exudate contained ∼250 µm LMA, whose TLC and electrophoretic mobilities, susceptibility to mild acid hydrolysis and NMR spectra are reported. A second acidic disaccharide, present at ∼120 µm, was similarly characterized, and shown to be β-d-xylopyranosyl-(1→3)-d-galacturonic acid (Xyl→GalA), a repeat unit of xylogalacturonan. Purified LMA and Xyl→GalA when applied at 360 and 740 µm, respectively, only slightly promoted Amaranthus hypocotyl growth, but equally promoted root growth and thus had no effect on the hypocotyl:root ratio, unlike total cress-seed exudate.Conclusions LMA is present in cress seeds, probably formed by rhamnogalacturonan lyase action on rhamnogalacturonan-I during seed development. Our results contradict the hypothesis that LMA is a cress allelochemical that appreciably perturbs the growth of potentially competing seedlings. Since LMA and Xyl→GalA slightly promoted both hypocotyl and root elongation, their effect could be nutritional. We conclude that rhamnogalacturonan-I and xylogalacturonan (pectin domains) are not sources of oligosaccharins with allelochemical activity, and the biological roles (if any) of the disaccharides derived from them are unknown. The main allelochemical principle in cress-seed exudate remains to be identified.     

Discovery of a SAR11 growth requirement for thiamin's pyrimidine precursor and its distribution in the Sargasso Sea

Vitamin traffic, the production of organic growth factors by some microbial community members and their use by other taxa, is being scrutinized as a potential explanation for the variation and highly connected behavior observed in ocean plankton by community network analysis. Thiamin (vitamin B₁), a cofactor in many essential biochemical reactions that modify carbon–carbon bonds of organic compounds, is distributed in complex patterns at subpicomolar concentrations in the marine surface layer (0–300 m). Sequenced genomes from organisms belonging to the abundant and ubiquitous SAR11 clade of marine chemoheterotrophic bacteria contain genes coding for a complete thiamin biosynthetic pathway, except for thiC, encoding the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) synthase, which is required for de novo synthesis of thiamin''s pyrimidine moiety. Here we demonstrate that the SAR11 isolate ‘Candidatus Pelagibacter ubique'', strain HTCC1062, is auxotrophic for the thiamin precursor HMP, and cannot use exogenous thiamin for growth. In culture, strain HTCC1062 required 0.7 zeptomoles per cell (ca. 400 HMP molecules per cell). Measurements of dissolved HMP in the Sargasso Sea surface layer showed that HMP ranged from undetectable (detection limit: 2.4 pM) to 35.7 pM, with maximum concentrations coincident with the deep chlorophyll maximum. In culture, some marine cyanobacteria, microalgae and bacteria exuded HMP, and in the Western Sargasso Sea, HMP profiles changed between the morning and evening, suggesting a dynamic biological flux from producers to consumers.     

Simple Y-Autosomal Incompatibilities Cause Hybrid Male Sterility in Reciprocal Crosses Between Drosophila virilis and D. americana

Postzygotic reproductive isolation evolves when hybrid incompatibilities accumulate between diverging populations. Here, I examine the genetic basis of hybrid male sterility between two species of Drosophila, Drosophila virilis and D. americana. From these analyses, I reach several conclusions. First, neither species carries any autosomal dominant hybrid male sterility alleles: reciprocal F₁ hybrid males are perfectly fertile. Second, later generation (backcross and F₂) hybrid male sterility between D. virilis and D. americana is not polygenic. In fact, I identified only three genetically independent incompatibilities that cause hybrid male sterility. Remarkably, each of these incompatibilities involves the Y chromosome. In one direction of the cross, the D. americana Y is incompatible with recessive D. virilis alleles at loci on chromosomes 2 and 5. In the other direction, the D. virilis Y chromosome causes hybrid male sterility in combination with recessive D. americana alleles at a single QTL on chromosome 5. Finally, in contrast with findings from other Drosophila species pairs, the X chromosome has only a modest effect on hybrid male sterility between D. virilis and D. americana.SPECIATION occurs when populations evolve one or more barriers to interbreeding (Dobzhansky 1937; Mayr 1963). One such barrier is intrinsic postzygotic isolation, which typically evolves when diverging populations accumulate different alleles at two or more loci that are incompatible when brought together in hybrid genomes; negative epistasis between these alleles renders hybrids inviable or sterile (Bateson 1909; Dobzhansky 1937; Muller 1942). Classical and recent studies in diverse animal taxa have provided support for two evolutionary patterns that often characterize the genetics of postzygotic isolation (Coyne and Orr 1989a). The first, Haldane''s rule, observes that when there is F₁ hybrid inviability or sterility that affects only one sex, it is almost always the heterogametic sex (Haldane 1922). Over the years, many researchers have tried to account for this pattern, but only two ideas are now thought to provide a general explanation: the “dominance theory,” which posits that incompatibility alleles are generally recessive in hybrids, and the “faster-male theory,” which posits that genes causing hybrid male sterility diverge more rapidly than those causing hybrid female sterility (Muller 1942; Wu and Davis 1993; Turelli and Orr 1995; reviewed in Coyne and Orr 2004). In some cases, however, additional factors might contribute to Haldane''s rule, including meiotic drive, a faster-evolving X chromosome, dosage compensation, and Y chromosome incompatibilities (reviewed in Laurie 1997; Turelli and Orr 2000; Coyne and Orr 2004).The second broad pattern affecting the evolution of postzygotic isolation is the disproportionately large effect of the X chromosome on heterogametic F₁ hybrid sterility (Coyne 1992). This “large X effect” has been documented in genetic analyses of backcross hybrid sterility (e.g., Dobzhansky 1936; Grula and Taylor 1980; Orr 1987; Masly and Presgraves 2007) and inferred from patterns of introgression across natural hybrid zones (e.g., Machado et al. 2002; Saetre et al. 2003; Payseur et al. 2004). However, in only one case has the cause of the large X effect been unambiguously determined: incompatibilities causing hybrid male sterility between Drosophila mauritiana and D. sechellia occur at a higher density on the X than on the autosomes (Masly and Presgraves 2007). Testing the generality of this pattern will require additional high-resolution genetic analyses in diverse taxa (Presgraves 2008). But whatever its causes, there is now general consensus that the X chromosome often plays a special role in the evolution of postzygotic isolation (Coyne and Orr 2004).The contribution of the Y chromosome to animal speciation is less clear. Y chromosomes have far fewer genes than the X or autosomes, and most of these genes are male specific (Lahn and Page 1997; Carvalho et al. 2009). In Drosophila species, the Y chromosome is typically required for male fertility, but not for viability (Voelker and Kojima 1971). How often, then, does the Y chromosome play a role in reproductive isolation? In crosses between Drosophila species, hybrid male sterility is frequently caused by incompatibilities between the X and Y chromosomes (Schafer 1978; Heikkinen and Lumme 1998; Mishra and Singh 2007) or between the Y and heterospecific autosomal alleles (Patterson and Stone 1952; Vigneault and Zouros 1986; Lamnissou et al. 1996). In crosses between D. yakuba and D. santomea, the Y chromosome causes F₁ hybrid male sterility, and accordingly, shows no evidence for recent introgression across a species hybrid zone (Coyne et al. 2004; Llopart et al. 2005). In mammals, reduced introgression of Y-linked loci (relative to autosomal loci) has been shown across natural hybrid zones of mice (Tucker et al. 1992) and rabbits (Geraldes et al. 2008), suggesting that the Y chromosome contributes to reproductive barriers.Here I examine the genetic basis of hybrid male sterility between two species of Drosophila, D. virilis and D. americana. These species show considerable genetic divergence (K_s ∼0.11, Morales-Hojas et al. 2008) and are currently allopatric: D. virilis is a human commensal worldwide with natural populations in Asia, and D. americana is found in riparian habitats throughout much of North America (Throckmorton 1982; McAllister 2002). Nearly 70 years ago, Patterson et al. (1942) showed that incompatibilities between the D. americana Y chromosome and the second and fifth chromosomes from D. virilis cause hybrid male sterility, a result that was confirmed in a more recent study (Lamnissou et al. 1996). Another study suggested that the X chromosome might play the predominant role in causing hybrid male sterility between D. virilis and D. americana (Orr and Coyne 1989). But because previous genetic analyses had to rely on only a few visible markers to map hybrid male sterility, they lacked the resolution to examine the genomic distribution of incompatibility loci.Using the D. virilis genome sequence, I have developed a dense set of molecular markers to investigate the genetic architecture of hybrid male sterility between D. virilis and D. americana. In this study, I perform a comprehensive set of crosses to address several key questions: What is the effect of the X chromosome on hybrid male sterility between D. virilis and D. americana? What is the effect of the Y chromosome? Approximately how many loci contribute to hybrid male sterility between these Drosophila species? Perhaps surprisingly, the answers to these questions differ dramatically from what has been found for other Drosophila species, including the well-studied D. melanogaster group.     

Virulence Gene Regulation by l-Arabinose in Salmonella enterica

Invasion of the intestinal epithelium is a critical step in Salmonella enterica infection and requires functions encoded in the gene cluster known as Salmonella Pathogenicity Island 1 (SPI-1). Expression of SPI-1 genes is repressed by l-arabinose, and not by other pentoses. Transport of l-arabinose is necessary to repress SPI-1; however, repression is independent of l-arabinose metabolism and of the l-arabinose-responsive regulator AraC. SPI-1 repression by l-arabinose is exerted at a single target, HilD, and the mechanism appears to be post-translational. As a consequence of SPI-1 repression, l-arabinose reduces translocation of SPI-1 effectors to epithelial cells and decreases Salmonella invasion in vitro. These observations reveal a hitherto unknown role of l-arabinose in gene expression control and raise the possibility that Salmonella may use L-arabinose as an environmental signal.     

A New Resource for Characterizing X-Linked Genes in Drosophila melanogaster: Systematic Coverage and Subdivision of the X Chromosome With Nested,Y-Linked Duplications

Interchromosomal duplications are especially important for the study of X-linked genes. Males inheriting a mutation in a vital X-linked gene cannot survive unless there is a wild-type copy of the gene duplicated elsewhere in the genome. Rescuing the lethality of an X-linked mutation with a duplication allows the mutation to be used experimentally in complementation tests and other genetic crosses and it maps the mutated gene to a defined chromosomal region. Duplications can also be used to screen for dosage-dependent enhancers and suppressors of mutant phenotypes as a way to identify genes involved in the same biological process. We describe an ongoing project in Drosophila melanogaster to generate comprehensive coverage and extensive breakpoint subdivision of the X chromosome with megabase-scale X segments borne on Y chromosomes. The in vivo method involves the creation of X inversions on attached-XY chromosomes by FLP-FRT site-specific recombination technology followed by irradiation to induce large internal X deletions. The resulting chromosomes consist of the X tip, a medial X segment placed near the tip by an inversion, and a full Y. A nested set of medial duplicated segments is derived from each inversion precursor. We have constructed a set of inversions on attached-XY chromosomes that enable us to isolate nested duplicated segments from all X regions. To date, our screens have provided a minimum of 78% X coverage with duplication breakpoints spaced a median of nine genes apart. These duplication chromosomes will be valuable resources for rescuing and mapping X-linked mutations and identifying dosage-dependent modifiers of mutant phenotypes.MANY eukaryotes of biomedical and agricultural importance—including humans, other mammals, birds, and Drosophila—are heterogametic. Their sex chromosomes differ drastically in size and genetic composition. In species with X and Y chromosomes, males carry only one copy of each X-linked gene. This poses a serious challenge for experimental geneticists, because males inheriting a mutation in a vital X-linked gene die before they can be used in genetic crosses. In fact, the hemizygosity of X-linked genes in males has been a significant barrier to the functional analysis of many X-linked genes and is largely responsible for the poor genetic characterization of X chromosomes relative to autosomes in most organisms.The lethality of X-linked mutations can be rescued by providing a wild-type copy of the mutated gene elsewhere in the genome. This can be accomplished with a transgenic construct if the molecular identity of the mutated gene is known. In many cases, however, the mutated gene has not been identified and it is necessary to provide wild-type function with a multigene interchromosomal duplication, i.e., a segment of the X inserted in another chromosome. If the proximal and distal extents of the duplicated segment are known, phenotypic rescue maps the mutated gene to the defined X chromosome region.Multigene deletions can also be used to map X-linked mutations by complementation, but crosses between individuals carrying deletions and X-linked lethal mutations are impossible without rescuing the lethality of either the deletion or the lethal mutation in males. Projects at the Bloomington Drosophila Stock Center and elsewhere (Parks et al. 2004; Ryder et al. 2007) have generated large collections of deletions with molecularly defined breakpoints in Drosophila melanogaster, but the utility of the X deletions is limited without duplications of the corresponding chromosomal regions.Duplications are potentially important for gene discovery. Identifying sets of genes involved in the same cellular process is a major focus of functional genomics research and this can be accomplished genetically by identifying dosage-sensitive modifiers of mutant phenotypes. Often, increasing or decreasing the copy number of a gene will enhance or suppress the phenotype associated with mutating another gene involved in the same process. Screening collections of deletions is a popular way to identify interacting genes in Drosophila (for examples, see Seher et al. 2007; Zhao et al. 2008; Aerts et al. 2009; Salzer et al. 2010) and was a major impetus for the assembly of the Bloomington Stock Center “Deficiency Kit,” which provides maximal coverage of the genome with the fewest deletions. Though dosage-sensitive modifiers could also be identified using increased gene dosage, the use of duplications in enhancer and suppressor screens remains largely unexplored. Assembling sets of duplications providing efficient genomic coverage would likely popularize this experimental approach.The size of duplicated segments determines how duplication chromosomes are used experimentally. Small duplicated segments allow high resolution gene mapping, but they are not suitable for other purposes. Only large duplicated segments are capable of rescuing the lethality of sizable multigene X deletions. Likewise, large duplicated segments provide efficiency in initially localizing mutations and identifying dosage-dependent modifiers. Despite their usefulness, interchromosomal duplications of large segments are among the hardest chromosomal rearrangements to isolate. In Drosophila, many existing duplications were recovered fortuitously as three-breakpoint aberrations following irradiation, but such rearrangements are rare and difficult to identify in screens. Other duplications were methodically constructed from preexisting rearranged chromosomes. This approach works well when it is possible, but it can be used only when progenitor aberrations with appropriate breakpoints are available. Because of these difficulties, the selection of duplication strains generated by Drosophila workers over the past several decades is not satisfactory for many purposes. The duplications are often difficult to use experimentally, their breakpoints are sparsely distributed along the X chromosome and only roughly mapped, and substantial gaps in coverage exist. Obviously, improved duplication resources are needed.Here we present the methodology and progress of a project at the Bloomington Drosophila Stock Center to construct interchromosomal duplications of large, megabase-scale X segments. Our approach builds on the long history of manipulating Drosophila chromosomes in vivo (Novitski and Childress 1976; Ashburner et al. 2005), but we have eliminated the need for preexisting aberrations by generating progenitor chromosomes using the FLP-FRT system. Indeed, this site-specific recombination system has had an enormous impact on the ability of fly geneticists to engineer many kinds of novel chromosomes (Golic and Golic 1996; Parks et al. 2004; Ryder et al. 2007). We will demonstrate how we have combined FLP-mediated recombination and other chromosome manipulation techniques to produce Y-linked duplications of large X segments. As we will show, appending X segments to Y chromosomes rather than autosomes has advantages both for the synthesis and experimental use of X duplications.To date, we have generated a minimum of 78% X coverage with duplication breakpoints spaced a median of nine genes apart. We anticipate completion of the project within the coming year. Using these duplications, mutations and genetic modifiers can be mapped first to large X intervals using a tiling set of the largest duplicated segments and then to small chromosome intervals using subsets of the duplications. These duplications will also facilitate deletion mapping. The creation of a set of stocks providing complete duplication coverage and extensive breakpoint subdivision of the X chromosome in a consistent genetic background will remove an impediment to investigating the functions of X-linked genes that has frustrated generations of Drosophila geneticists.     

Map-Based Cloning of the Gene Associated With the Soybean Maturity Locus E3

Photosensitivity plays an essential role in the response of plants to their changing environments throughout their life cycle. In soybean [Glycine max (L.) Merrill], several associations between photosensitivity and maturity loci are known, but only limited information at the molecular level is available. The FT3 locus is one of the quantitative trait loci (QTL) for flowering time that corresponds to the maturity locus E3. To identify the gene responsible for this QTL, a map-based cloning strategy was undertaken. One phytochrome A gene (GmPhyA3) was considered a strong candidate for the FT3 locus. Allelism tests and gene sequence comparisons showed that alleles of Misuzudaizu (FT3/FT3; JP28856) and Harosoy (E3/E3; PI548573) were identical. The GmPhyA3 alleles of Moshidou Gong 503 (ft3/ft3; JP27603) and L62-667 (e3/e3; PI547716) showed weak or complete loss of function, respectively. High red/far-red (R/FR) long-day conditions enhanced the effects of the E3/FT3 alleles in various genetic backgrounds. Moreover, a mutant line harboring the nonfunctional GmPhyA3 flowered earlier than the original Bay (E3/E3; PI553043) under similar conditions. These results suggest that the variation in phytochrome A may contribute to the complex systems of soybean flowering response and geographic adaptation.FLOWERING represents the transition from the vegetative to the reproductive phase in plants. Various external cues, such as photoperiod and temperature, are known to initiate plant flowering under the appropriate seasonal conditions. Of these cues, light is the most important, being received by several photoreceptors, including the red light (R) and the far-red light (FR)-absorbing phytochromes and the blue/UV-A absorbing cryptochromes and phototorpins (Chen et al. 2004).Phytochrome is the best characterized of these photoreceptors. All higher plant phytochromes are thought to exist as specific dimer combinations (Sharrock and Clack 2004), with each monomer being attached to a light-absorbing linear tetrapyrrole, phytochromobilin. The phytochrome apoproteins are synthesized within the cytosol and assemble autocatalytically with a chromophore to form the phytochrome holoproteins. The R-absorbing form (Pr) is thought to be inactive but is then converted to the active FR-absorbing form (Pfr) by R absorption. The absorption of light triggers the transfer of the phytochrome to the nucleus, where it regulates gene expression. In most plant species, the phytochrome apoproteins are encoded by a small gene family. Type I phytochrome is degraded in the light and is abundant in dark-grown seedlings, whereas type II phytochrome is relatively stable in the light (reviewed by Bae and Choi 2008). In Arabidopsis, five phytochromes (PhyA–E) have been characterized (Clack et al. 1994; Quail et al. 1995). PhyA is type I and is responsible for the very low fluence response and high irradiance response, whereas the other phytochromes are type II and are responsible for red-far/red reversible low fluence response (reviewed by Whitelam et al. 1998).It is well known that mutations in the phytochrome A gene affect the photoperiodic control of flowering. In Arabidopsis, a phyA mutant flowered later in either long-day or short-day conditions with a night break (Johnson et al. 1994; Reed et al. 1994). In rice, combinations of mutant alleles of phytochrome genes conferred various effects on the flowering phenotype. For example, the phyA phyB and phyA phyC double mutants grown under natural-day-length conditions showed earlier flowering phenotypes than wild-type plants (Takano et al. 2005). In pea, a long-day plant, loss- or gain-of-function phyA mutants displayed late or early flowering phenotypes, respectively (Weller et al. 1997, 2001). It is likely that photoperiodic response via phyA signaling is important for crop adaptation to a wide range of growing conditions.In soybean [Glycine max (L.) Merrill], several maturity loci, designated as E loci (Cober et al. 1996a), have been characterized by classical methods. These are E1 and E2 (Bernard 1971), E3 (Buzzell 1971), E4 (Buzzell and Voldeng 1980), E5 (McBlain and Bernard 1987), E6 (Bonato and Vello 1999), and E7 (Cober and Voldeng 2001). Of these, the E1, E3, and E4 loci have been suggested to be related to photoperiod sensitivity under various light conditions (Saidon et al. 1989; Cober et al. 1996b; Abe et al. 2003). In previous studies, using the same populations as in this study, three flowering-time quantitative trait loci (QTL)—FT1, FT2, and FT3 loci—were identified and considered to be identical with the maturity loci E1, E2, and E3, respectively (Yamanaka et al. 2001; Watanabe et al. 2004). Although many loci related to soybean flowering and maturity have been identified, and some candidate genes were recognized using near isogenic lines (NILs) (Tasma and Shoemaker 2003), most of the genes responsible for these loci have not yet been isolated except for the E4 gene. Liu et al. (2008) reported an association between phytochrome A and photoperiod sensitivity. A retrotransposon sequence inserted into the exon of the e4 allele conferred an early flowering phenotype under long-day conditions extended by incandescent lighting.A relationship between the E3 gene and some photoreceptor genes was suggested from different photosensitivity responses of various soybean NILs (Cober et al. 1996a). Cober and Voldeng (1996) also reported a linkage relationship between the E3 and Dt1 loci, which is related to a determinate or indeterminate growth habit phenotype. Additionally, Molnar et al. (2003) reported that Satt229, on linkage group (LG) L, was a proximal simple sequence repeat (SSR) marker to the E3 loci. According to the Soybean Genome Database (Shultz et al. 2006a,b, 2007; http://soybeangenome.siu.edu/) and the Legume Information System (LIS; http://www.comparative-legumes.org/), there are numerous QTL and >60 loci associated with various agronomic traits in the region between Dt1 and Satt373 (∼30–40 cM). This extremely large number of QTL may be the result of linkage between the Dt1 and E3 loci because both loci can affect many aspects of plant morphology. Among these QTL, several associations with the E3 gene have been reported (Mansur et al. 1996; Orf et al. 1999; Funatsuki et al. 2005; Kahn et al. 2008).To identify the genes responsible for the target QTL, fine mapping and map-based cloning strategies are necessary (Salvi and Tuberosa 2005). QTL analysis using intercross-derived populations, such as F₂ and recombinant inbred lines (RILs), have some limitations in genome resolution (10–30 cM) because of the simultaneous segregation of several loci affecting the same trait (Kearsey and Farquhar 1998). Additional strategies are therefore required to locate QTL more precisely. The use of NILs that differ at a single QTL is an effective approach for fine mapping and characterization of an individual locus (Salvi and Tuberosa 2005). However, the development of NILs through repeated backcrossing is time-consuming and laborious (Tuinstra et al. 1997). The use of a residual heterozygous line (RHL), as proposed by Yamanaka et al. (2004), and which is derived from RIL, is a powerful tool for precisely evaluating QTL (Haley et al. 1994). An RHL harbors a heterozygous region where the target QTL is located and a homozygous background in most other regions of the genome. Tuinstra et al. (1997) used a similar term, heterogeneous inbred family, for a selfed RHL population to identify the QTL associated with seed weight in sorghum.This RHL strategy has already been used to identify loci underlying resistance to pathogens in soybean (Njiti et al. 1998; Meksem et al. 1999; Triwitayakorn et al. 2005). After identification of the target loci, novel DNA markers tightly linked to the loci were developed using the amplified fragment length polymorphism (AFLP) method (Meksem et al. 2001a,b). Physical contigs, screened by sequence-characterized amplified region (SCAR) markers converted from these AFLP fragments, are ideal sources for identifying candidate genes for the target traits (Ruben et al. 2006).The aim of this study is to characterize the FT3 locus using a map-based cloning strategy and to confirm the gene responsible for the E3/FT3 locus by allelism tests through comparisons of gene sequences and photosensitivity of several alleles.     

High-Level Heterologous Production of d-Cycloserine by Escherichia coli

Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 μM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 μM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H₂S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 μM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 μM).     

Prediction of Genetic Values of Quantitative Traits in Plant Breeding Using Pedigree and Molecular Markers

The availability of dense molecular markers has made possible the use of genomic selection (GS) for plant breeding. However, the evaluation of models for GS in real plant populations is very limited. This article evaluates the performance of parametric and semiparametric models for GS using wheat (Triticum aestivum L.) and maize (Zea mays) data in which different traits were measured in several environmental conditions. The findings, based on extensive cross-validations, indicate that models including marker information had higher predictive ability than pedigree-based models. In the wheat data set, and relative to a pedigree model, gains in predictive ability due to inclusion of markers ranged from 7.7 to 35.7%. Correlation between observed and predictive values in the maize data set achieved values up to 0.79. Estimates of marker effects were different across environmental conditions, indicating that genotype × environment interaction is an important component of genetic variability. These results indicate that GS in plant breeding can be an effective strategy for selecting among lines whose phenotypes have yet to be observed.PEDIGREE-BASED prediction of genetic values based on the additive infinitesimal model (Fisher 1918) has played a central role in genetic improvement of complex traits in plants and animals. Animal breeders have used this model for predicting breeding values either in a mixed model (best linear unbiased prediction, BLUP) (Henderson 1984) or in a Bayesian framework (Gianola and Fernando 1986). More recently, plant breeders have incorporated pedigree information into linear mixed models for predicting breeding values (Crossa et al. 2006, 2007; Oakey et al. 2006; Burgueño et al. 2007; Piepho et al. 2007).The availability of thousands of genome-wide molecular markers has made possible the use of genomic selection (GS) for prediction of genetic values (Meuwissen et al. 2001) in plants (e.g., Bernardo and Yu 2007; Piepho 2009; Jannink et al. 2010) and animals (Gonzalez-Recio et al. 2008; VanRaden et al. 2008; Hayes et al. 2009; de los Campos et al. 2009a). Implementing GS poses several statistical and computational challenges, such as how models can cope with the curse of dimensionality, colinearity between markers, or the complexity of quantitative traits. Parametric (e.g., Meuwissen et al. 2001) and semiparametric (e.g., Gianola et al. 2006; Gianola and van Kaam 2008) methods address these problems differently.In standard genetic models, phenotypic outcomes, , are viewed as the sum of a genetic value, , and a model residual, ; that is, . In parametric models for GS, is described as a regression on marker covariates (j = 1,  …  , p molecular markers) of the form , such that(or , in matrix notation), where is the regression of on the jth marker covariate .Estimation of via multiple regression by ordinary least squares (OLS) is not feasible when p > n. A commonly used alternative is to estimate marker effects jointly using penalized methods such as ridge regression (Hoerl and Kennard 1970) or the Least Absolute Shrinkage and Selection Operator (LASSO) (Tibshirani 1996) or their Bayesian counterpart. This approach yields greater accuracy of estimated genetic values and can be coupled with geostatistical techniques commonly used in plant breeding to model multienvironments trials (Piepho 2009).In ridge regression (or its Bayesian counterpart) the extent of shrinkage is homogeneous across markers, which may not be appropriate if some markers are located in regions that are not associated with genetic variance, while markers in other regions may be linked to QTL (Goddard and Hayes 2007). To overcome this limitation, many authors have proposed methods that use marker-specific shrinkage. In a Bayesian setting, this can be implemented using priors of marker effects that are mixtures of scaled-normal densities. Examples of this are methods Bayes A and Bayes B of Meuwissen et al. (2001) and the Bayesian LASSO of Park and Casella (2008).An alternative to parametric regressions is to use semiparametric methods such as reproducing kernel Hilbert spaces (RKHS) regression (Gianola and van Kaam 2008). The Bayesian RKHS regression regards genetic values as random variables coming from a Gaussian process centered at zero and with a (co)variance structure that is proportional to a kernel matrix K (de los Campos et al. 2009b); that is, , where , are vectors of marker genotypes for the ith and jth individuals, respectively, and is a positive definite function evaluated in marker genotypes. In a finite-dimensional setting this amounts to modeling the vector of genetic values, , as multivariate normal; that is, where is a variance parameter. One of the most attractive features of RKHS regression is that the methodology can be used with almost any information set (e.g., covariates, strings, images, graphs). A second advantage is that with RKHS the model is represented in terms of n unknowns, which gives RKHS a great computational advantage relative to some parametric methods, especially when p ≫ n.This study presents an evaluation of several methods for GS, using two extensive data sets. One contains phenotypic records of a series of wheat trials and recently generated genomic data. The other data set pertains to international maize trials in which different traits were measured in maize lines evaluated under severe drought and well-watered conditions.     

Identification of two AFLP markers linked to bacterial wilt resistance in tomato and conversion to SCAR markers

Tomato bacterial wilt (BW) incited by Ralstonia solanacearum is a constraint on tomato production in tropical, subtropical and humid regions of the world. In this paper, we present the results of a research aimed at the identification of PCR-based markers amplified fragment length polymorphism (AFLP) linked to the genes that confer resistance to tomato BW. To this purpose, bulked segregant analysis was applied to an F₂ population segregating for the BW resistant gene and derived from the pair-cross between a BW resistant cultivar T51A and the susceptible cultivar T9230. Genetic analysis indicated that tomato BW was conferred by two incomplete dominant genes. A CTAB method for total DNA extraction, developed by Murray and Thompson with some modifications was used to isolation the infected tomato leaves. Thirteen differential fragments were detected using 256 primer combinations, and two AFLP markers were linked to the BW resistance. Subsequently, the AFLP markers were converted to co-dominant SCAR markers, named TSCAR_AAT/CGA and TSCAR_AAG/CAT. Linkage analysis showed that the two markers are on the contralateral side of TRSR-1. Genetic distance between TSCAR_AAT/CGA and TRS-1 was estimated to 4.6 cM, while 8.4 cM between TSCAR_AAG/CAT and TRS-1.     

Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b₅ were found to support a high NOD activity. Cygb-NOD activity shows respective K_m values for ascorbate, cytochrome b₅, NO, and O₂ of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a k_cat of 0.5 s⁻¹. Ascorbate and cytochrome b₅ reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m⁻¹ s⁻¹ and 3 × 10⁶ m⁻¹ s⁻¹, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k_cat of 1.2 s⁻¹, a K_m(NO) of 40 nm, and a k_cat/K_m(NO) (k′_NOD) value of 3 × 10⁷ m⁻¹ s⁻¹, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O₂] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b₅ as reductants.     

DdlN from Vancomycin-Producing Amycolatopsis orientalis C329.2 Is a VanA Homologue with d-Alanyl-d-Lactate Ligase Activity

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in d-alanyl-d-lactate. A key enzyme in this process is a d-alanyl-d-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide d-alanyl-d-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for d-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.The origin of antibiotic resistance determinants is of significant interest for several reasons, including the prediction of the emergence and spread of resistance patterns, the design of new antimicrobial agents, and the identification of potential reservoirs for resistance elements. Antibiotic resistance can occur either through spontaneous mutation in the target or by the acquisition of external genetic elements such as plasmids or transposons which carry resistance genes (7). The origins of these acquired genes are varied, but it has long been recognized that potential reservoirs are antibiotic-producing organisms which naturally harbor antibiotic resistance genes to protect themselves from the actions of toxic compounds (6).High-level resistance to glycopeptide antibiotics such as vancomycin and teicoplanin in vancomycin-resistant enterococci (VRE) is conferred by the presence of three genes, vanH, vanA (or vanB), and vanX, which, along with auxiliary genes necessary for inducible gene expression, are found on transposons integrated into plasmids or the bacterial genome (1, 20). These three genes are essential to resistance and serve to change the C-terminal peptide portion of the peptidoglycan layer from d-alanyl-d-alanine (d-Ala-d-Ala) to d-alanyl-d-lactate (d-Ala-d-Lac). This change results in the loss of a critical hydrogen bond between vancomycin and the d-Ala-d-Ala terminus and in a 1,000-fold decrease in binding affinity between the antibiotic and the peptidoglycan layer, which is the basis for the bactericidal action of this class of compounds (5). The vanH gene encodes a d-lactate dehydrogenase which provides the requisite d-Lac (3, 5), while the vanX gene encodes a highly specific dd-peptidase which cleaves only d-Ala-d-Ala produced endogenously while leaving d-Ala-d-Lac intact (19, 21). The final gene, vanA or vanB, encodes an ATP-dependent d-Ala-d-Lac ligase (4, 8, 10). This enzyme has sequence homology with the chromosomal d-Ala-d-Ala ligases, which are essential for peptidoglycan synthesis but which generally lack the ability to synthesize d-Ala-d-Lac (9).We have recently cloned vanH, vanA, and vanX homologues from two glycopeptide antibiotic-synthesizing organisms: Amycolatopsis orientalis C329.2, which produces vancomycin, and Streptomyces toyocaensis NRRL 15009, which produces {"type":"entrez-nucleotide","attrs":{"text":"A47934","term_id":"2301796","term_text":"A47934"}}A47934 (14). In addition, the vanH-vanA-vanX gene cluster was identified in several other glycopeptide producers. We have also demonstrated that the VanA homologue from S. toyocaensis NRRL 15009 can synthesize d-Ala-d-Lac in vitro and in the glycopeptide-sensitive host Streptomyces lividans (15, 16). We now report the expression of the A. orientalis C329.2 VanA homologue DdlN in Escherichia coli, its purification, and its enzymatic characterization. These data reinforce the striking similarity between vancomycin resistance elements in VRE and glycopeptide-producing organisms and support the possibility of a common origin for these enzymes.

Expression, purification, and specificity of DdlN.

DdlN was overexpressed in E. coli under the control of the bacteriophage T7 promoter. The construct gave good yields of highly purified enzyme following a four-step purification procedure (Table ​(Table1;1; Fig. ​Fig.1).1). Like other dd-ligases, DdlN behaved like a dimer in solution (not shown).

TABLE 1

Purification of DdlN from E. coli BL21 (DE3)/pETDdlN

Linking ascorbic acid production in Ribes nigrum with fruit development and changes in sources and sinks

Background and Aims

Understanding the synthesis of ascorbic acid (l-AsA) in green tissues in model species has advanced considerably; here we focus on its production and accumulation in fruit. In particular, our aim is to understand the links between organs which may be sources of l-AsA (leaves) and those which accumulate it (fruits). The work presented here tests the idea that changes in leaf and fruit number influence the accumulation of l-AsA. The aim was to understand the importance of leaf tissue in the production of l-AsA and to determine how this might provide routes for the manipulation of fruit tissue l-AsA.

Methods

The experiments used Ribes nigrum (blackcurrant), predominantly in field experiments, where the source–sink relationship was manipulated to alter potential leaf l-AsA production and fruit growth and accumulation of l-AsA. These manipulations included reductions in reproductive capacity, by raceme removal, and the availability of assimilates by leaf removal and branch phloem girdling. Natural variation in fruit growth and fruit abscission is also described as this influences subsequent experimental design and the interpretation of l-AsA data.

Key Results

Results show that fruit l-AsA concentration is conserved but total yield of l-AsA per plant is dependent on a number of innate factors many of which relate to raceme attributes. Leaf removal and phloem girdling reduced fruit weight, and a combination of both reduced fruit yields further. It appears that around 50 % of assimilates utilized for fruit growth came from apical leaves, while between 20 and 30 % came from raceme leaves, with the remainder from ‘storage’.

Conclusions

Despite being able to manipulate leaf area and therefore assimilate availability and stored carbohydrates, along with fruit yields, rarely were effects on fruit l-AsA concentration seen, indicating fruit l-AsA production in Ribes was not directly coupled to assimilate supply. There was no supporting evidence that l-AsA production occurred predominantly in green leaf tissue followed by its transfer to developing fruits. It is concluded that l-AsA production occurs predominantly in the fruit of Ribes nigrum.     

Conversion of l-Sorbosone to l-Ascorbic Acid by a NADP-Dependent Dehydrogenase in Bean and Spinach Leaf

An NADP-dependent dehydrogenase catalyzing the conversion of l-sorbosone to l-ascorbic acid has been isolated from Phaseolus vulgaris L. and Spinacia oleracea L. and partially purified. It is stable at −20°C for up to 8 months. Molecular masses, as determined by gel filtration, were 21 and 29 kilodaltons for bean and spinach enzymes, respectively. K_m for sorbosone were 12 ± 2 and 18 ± 2 millimolar and for NADP⁺, 0.14 ± 0.05 and 1.2 ± 0.5 millimolar, for bean and spinach, respectively. Lycorine, a purported inhibitor of l-ascorbic acid biosynthesis, had no effect on the reaction.     

The Functional Transfer of Genes From the Mitochondria to the Nucleus: The Effects of Selection,Mutation, Population Size and Rate of Self-Fertilization

The transfer of mitochondrial genes to the nucleus is a recurrent and consistent feature of eukaryotic genome evolution. Although many theories have been proposed to explain such transfers, little relevant data exist. The observation that clonal and self-fertilizing plants transfer more mitochondrial genes to their nuclei than do outcrossing plants contradicts predictions of major theories based on nuclear recombination and leaves a gap in our conceptual understanding how the observed pattern of gene transfer could arise. Here, with a series of deterministic and stochastic simulations, we show how epistatic selection and relative mutation rates of mitochondrial and nuclear genes influence mitochondrial-to-nuclear gene transfer. Specifically, we show that when there is a benefit to having a mitochondrial gene present in the nucleus, but absent in the mitochondria, self-fertilization dramatically increases both the rate and the probability of gene transfer. However, absent such a benefit, when mitochondrial mutation rates exceed those of the nucleus, self-fertilization decreases the rate and probability of transfer. This latter effect, however, is much weaker than the former. Our results are relevant to understanding the probabilities of fixation when loci in different genomes interact.GENOMIC investigations of obligate intracellular endosymbionts (Moran and Wernegreen 2000; Akman et al. 2002; Tamas et al. 2002; Wernegreen et al. 2002; Klasson and Andersson 2004; Foster et al. 2005) reveal a reduction in genome size and number of protein-coding genes compared to their free-living relatives (Charles et al. 1999; Gil et al. 2002; Wernegreen et al. 2002; Moran 2003; Van Ham et al. 2003; Klasson and Andersson 2004; Khachane et al. 2007). Similarly, mitochondria—ancient obligate intracellular symbionts of eukaryotes—have retained very few protein-coding genes (Boore 1999; Adams et al. 2002) [Reclinomonas americanas is at the extreme of retention of mitochondrial genes (Lang et al. 1997)]. Understanding the process of gene loss in mitochondria and other endosymbionts is a major research focus of mitochondrial and endosymbiont genomics (Moran 2003; Timmis et al. 2004; Khachane et al. 2007; Bock and Timmis 2008).The loss of endosymbiont genes can be complete, in which lost genes are absent from the host–endosymbiont complex, a substitution, in which a nuclear allele functions in place of the lost symbiont gene, or a functional transfer of an endosymbiont gene to the nucleus, followed by its loss (Adams and Palmer 2003). Such “functional transfer” involves the relocation of a mitochondrial gene to the nucleus, its acquisition of a promoter, successful targeting to the mitochondria for proper function, and the eventual loss of the gene from the mitochondrial genome altogether. Although this process is probably quite complex and requires numerous evolutionary modifications (Murcha et al. 2005), there is evidence that some mitochondrial genes are preadapted to functional transfer as they contain signals that target them to the mitochondria before functional transfer to the nucleus (Ueda et al. 2008a). The complex evolution of rps16 is an illuminating case of both functional gene transfer and substitution. In some lineages, the mitochondrial rps16 is functionally expressed in the nucleus but absent from the mitochondria (functional transfer) while in a subset of taxa, the chloroplast copy is also absent and the nuclear gene is also targeted to the chloroplast [substitution (Ueda et al. 2008b)].A number of evolutionary scenarios have been proposed to account for the massive loss of genes from endosymbionts. A subset of models argues that endosymbiont gene loss is a neutral or nearly neutral process. Since endosymbiosis reduces the strength of selection on genes that are unnecessary or redundant in an obligate intracellular environment, these genes may be quickly lost by the neutral fixation of a deletion or other loss-of-function mutations. Moreover, even when selection favors the retention of genes in endosymbionts, such selection may be ineffective because of reduction in effective population size due to recurrent bottlenecking (Rispe and Moran 2000). Additionally, frequent input of functional endosymbiont genes into the nucleus makes symbiont genes redundant, exacerbating gene loss via functional transfer (Berg and Kurland 2000).An alternative class of explanations views the loss of mitochondrial genes (be it complete loss, substitution, or functional transfer) as an adaptive process. The “mitochondrial competition theory” argues that mitochondrial genomes that either do not contain or do not express a given allele have a replicative advantage over other mitochondria, providing a within-host selective advantage to mitochondrial gene loss (Albert et al. 1996; Selosse et al. 2001; Yamauchi 2005). The “benefits of sex” model posits that the genomic diploid nuclear environment (diploid, sexual) is in some way preferable (e.g., as an escape from Muller''s ratchet or Hill–Robertson interference) to a haploid asexual mitochondrial environment (Blanchard and Lynch 2000). The epistatic model (Wade and Goodnight 2006) does not advance a specific or consistent benefit to transfer, but posits that transfer is explicitly a process of coevolution between mitochondrial and nuclear genomes, where fitness is a function of the gene combination rather than of either gene separately.Because few species are currently undergoing mitochondrial to nuclear gene transfer, these alternative hypotheses are difficult to distinguish with direct experimentation. However, the distribution of transferred genes across lineages allows for evaluation of the alternative hypotheses. For example, self-pollination reduces the rate of heteroplasmy and consequently the opportunity for competition among genetically distinct mitochondria. Thus, the mitochondrial competition theory predicts an excess of transfer events in sexual, outcrossing lineages, with high degrees of “paternal leakage.” Similarly, frequent self-fertilization diminishes the benefits of sex, and thus the benefits of sex hypothesis predicts fewer transfers in selfing and clonally reproducing plants than in outcrossing taxa. The epistatic model makes the opposite prediction. Selfing and clonal reproduction maintain cyto-nuclear gene combinations and increase the response to selection on epistatic combinations, potentially encouraging transfer. On the other hand, outcrossing tends to break apart adaptive cyto-nuclear gene combinations, potentially decreasing the amount of adaptive transfer in outcrossing lineages.Plant lineages with high levels of self-fertilization or asexual reproduction transfer more mitochondrial genes to their nuclei than predominantly sexual and outcrossing lineages (Brandvain et al. 2007). This result is consistent with predictions of the epistatic model and is contrary to predictions of the mitochondrial competition or benefits of sex models. More specific predictions allowing further empirical tests require more detailed theoretical investigations of the gene transfer process. Here, we investigate the roles of mutation, selection, and random drift in gene transfer using both deterministic models and stochastic simulations to refine and extend predictions of patterns of functional mitochondrial to nuclear gene transfer.