Genomewide single nucleotide polymorphism discovery in Atlantic salmon (Salmo salar): validation in wild and farmed American and European populations.

A considerable number of single nucleotide polymorphisms (SNPs) are required to elucidate genotype-phenotype associations and determine the molecular basis of important traits. In this work, we carried out de novo SNP discovery accounting for both genome duplication and genetic variation from American and European salmon populations. A total of 9 736 473 nonredundant SNPs were identified across a set of 20 fish by whole-genome sequencing. After applying six bioinformatic filtering steps, 200 K SNPs were selected to develop an Affymetrix Axiom(®) myDesign Custom Array. This array was used to genotype 480 fish representing wild and farmed salmon from Europe, North America and Chile. A total of 159 099 (79.6%) SNPs were validated as high quality based on clustering properties. A total of 151 509 validated SNPs showed a unique position in the genome. When comparing these SNPs against 238 572 markers currently available in two other Atlantic salmon arrays, only 4.6% of the SNP overlapped with the panel developed in this study. This novel high-density SNP panel will be very useful for the dissection of economically and ecologically relevant traits, enhancing breeding programmes through genomic selection as well as supporting genetic studies in both wild and farmed populations of Atlantic salmon using high-resolution genomewide information.

Two-phase analysis in consensus genetic mapping.

Numerous mapping projects conducted on different species have generated an abundance of mapping data. Consequently, many multilocus maps have been constructed using diverse mapping populations and marker sets for the same organism. The quality of maps varies broadly among populations, marker sets, and software used, necessitating efforts to integrate the mapping information and generate consensus maps. The problem of consensus genetic mapping (MCGM) is by far more challenging compared with genetic mapping based on a single dataset, which by itself is also cumbersome. The additional complications introduced by consensus analysis include inter-population differences in recombination rate and exchange distribution along chromosomes; variations in dominance of the employed markers; and use of different subsets of markers in different labs. Hence, it is necessary to handle arbitrary patterns of shared sets of markers and different level of mapping data quality. In this article, we introduce a two-phase approach for solving MCGM. In phase 1, for each dataset, multilocus ordering is performed combined with iterative jackknife resampling to evaluate the stability of marker orders. In this phase, the ordering problem is reduced to the well-known traveling salesperson problem (TSP). Namely, for each dataset, we look for order that gives minimum sum of recombination distances between adjacent markers. In phase 2, the optimal consensus order of shared markers is selected from the set of allowed orders and gives the minimal sum of total lengths of nonconflicting maps of the chromosome. This criterion may be used in different modifications to take into account the variation in quality of the original data (population size, marker quality, etc.). In the foregoing formulation, consensus mapping is considered as a specific version of TSP that can be referred to as "synchronized TSP." The conflicts detected after phase 1 are resolved using either a heuristic algorithm over the entire chromosome or an exact/heuristic algorithm applied subsequently to the revealed small non-overlapping regions with conflicts separated by non-conflicting regions. The proposed approach was tested on a wide range of simulated data and real datasets from maize.

On the origins of species: does evolution repeat itself in polyploid populations of independent origin?

Multiple origins of the same polyploid species pose the question: Does evolution repeat itself in these independently formed lineages? Tragopogon is a unique evolutionary model for the study of recent and recurrent allopolyploidy. The allotetraploids T. mirus (T. dubius x T. porrifolius) and T. miscellus (T. dubius x T. pratensis) formed repeatedly following the introduction of three diploids to the United States. Concerted evolution has consistently occurred in the same direction (resulting in loss of T. dubius rDNA copies). Both allotetraploids exhibit homeolog loss, with the same genes consistently showing loss, and homeologs of T. dubius preferentially lost in both allotetraploids. We have also documented repeated patterns of tissue-specific silencing in multiple populations of T. miscellus. Hence, some aspects of genome evolution may be "hardwired," although the general pattern of loss is stochastic within any given population. On the basis of the study of F(1) hybrids and synthetics, duplicate gene loss and silencing do not occur immediately following hybridization or polyploidization, but gradually and haphazardly. Genomic approaches permit analysis of hundreds of loci to assess the frequency of homeolog loss and changes in gene expression. This methodology is particularly promising for groups such as Tragopogon for which limited genetic and genomic resources are available.

Turning repeats to advantage: scaffolding genomic contigs using LTR retrotransposons.

The abundance of repeat elements in the maize genome complicates its assembly. Retrotransposons alone are estimated to constitute at least 50% of the genome. In this paper, we introduce a problem called retroscaffolding, which is a new variant of the well known problem of scaffolding that orders and orients a set of assembled contigs in a genome assembly project. The key feature of this new formulation is that it takes advantage of the structural characteristics and abundance of a particular type of retrotransposons called the Long Terminal Repeat (LTR) retrotransposons. This approach is not meant to supplant but rather to complement other scaffolding approaches. The advantages of retroscaffolding are twofold: (i) it allows detection of regions containing LTR retrotransposons within the unfinished portions of a genome and can therefore guide the process of finishing, and (ii) it provides a mechanism to lower sequencing coverage without impacting the quality of the final assembled genic portions. Sequencing and finishing costs dominate the expenditures in whole genome projects, and it is often desired in the interest of saving cost to reduce such efforts spent on repetitive regions of a genome. The retroscaffolding technique provides a viable mechanism to this effect. Results of preliminary studies on maize genomic data validate the utility of our approach. We also report on the on-going development of an algorithmic framework to perform retroscaffolding.

Using the biological taxonomy to access biological literature with PathBinderH.

UNLABELLED: PathBinderH allows users to make queries that retrieve sentences and the abstracts containing them from PubMed. Another aspect of PathBinderH is that users can specify biological taxa in order to limit searches by mentioning either the specified taxa, or their subordinate taxa, in the biological taxonomy. Although the current project requires this function only for plant taxa, the principle is extensible to the entire taxonomy. AVAILABILITY: www.plantgenomics.iastate.edu/PathBinderH. Source code and databases on request.

Mitochondrial aldehyde dehydrogenase activity is required for male fertility in maize.

Some plant cytoplasms express novel mitochondrial genes that cause male sterility. Nuclear genes that disrupt the accumulation of the corresponding mitochondrial gene products can restore fertility to such plants. The Texas (T) cytoplasm mitochondrial genome of maize expresses a novel protein, URF13, which is necessary for T cytoplasm-induced male sterility. Working in concert, functional alleles of two nuclear genes, rf1 and rf2, can restore fertility to T cytoplasm plants. Rf1 alleles, but not Rf2 alleles, reduce the accumulation of URF13. Hence, Rf2 differs from typical nuclear restorers in that it does not alter the accumulation of the mitochondrial protein necessary for T cytoplasm-induced male sterility. This study established that the rf2 gene encodes a soluble protein that accumulates in the mitochondrial matrix. Three independent lines of evidence establish that the RF2 protein is an aldehyde dehydrogenase (ALDH). The finding that T cytoplasm plants that are homozygous for the rf2-R213 allele are male sterile but accumulate normal amounts of RF2 protein that lacks normal mitochondrial (mt) ALDH activity provides strong evidence that rf2-encoded mtALDH activity is required to restore male fertility to T cytoplasm maize. Detailed genetic analyses have established that the rf2 gene also is required for anther development in normal cytoplasm maize. Hence, it appears that the rf2 gene was recruited recently to function as a nuclear restorer. ALDHs typically have very broad substrate specificities. Indeed, the RF2 protein is capable of oxidizing at least three aldehydes. Hence, the specific metabolic pathway(s) within which the rf2-encoded mtALDH acts remains to be discovered.

Molecular biology of acetyl-CoA metabolism.

We have characterized the expression of potential acetyl-CoA-generating genes (acetyl-CoA synthetase, pyruvate decarboxylase, acetaldehyde dehydrogenase, plastidic pyruvate dehydrogenase complex and ATP-citrate lyase), and compared these with the expression of acetyl-CoA-metabolizing genes (heteromeric and homomeric acetyl-CoA carboxylase). These comparisons have led to the development of testable hypotheses as to how distinct pools of acetyl-CoA are generated and metabolized. These hypotheses are being tested by combined biochemical, genetic and molecular biological experiments, which is providing insights into how acetyl-CoA metabolism is regulated.

Mitochondrial transcript processing and restoration of male fertility in T-cytoplasm maize.

Cytoplasmic male sterility (CMS) systems have been useful in the production of hybrid seed in a number of crops. The Texas or T-cytoplasmic male-sterile (cms-T) system was used extensively in the 1960s to eliminate the need for hand detasseling in hybrid maize production. As a consequence of the 1970 epidemic of southern corn leaf blight, cms-T is no longer widely used commercially. However, it has been developed as a model system to study the genetic and molecular mechanisms underlying male sterility and fertility restoration. Male sterility in T-cytoplasm maize results from the action of a T-cytoplasm-specific mitochondrial gene, T-urf13. Full (or partial) fertility restoration of T-cytoplasm maize is mediated by the Rf2 nuclear restorer in combination with one of three other restorers: Rf1, Rf8, or Rf*. Rf2 encodes a protein highly similar to mitochondrial aldehyde dehydrogenases; Rf1, Rf8, and Rf* each mediate discrete T-urf13 mitochondrial transcript processing events. To test the functionality of Rf1, Rf8, or Rf*, a T-cytoplasm transformation system is under development. AFLP bulk-segregant analysis has been used to identify DNA markers closely linked to the Rf8 locus. These tools will provide a foundation for determining mechanisms of nuclear-directed mitochondrial RNA processing and fertility restoration.

Genetic recombination in plants.

Meiotic recombination generates novel allelic arrays on chromosomes. Recent experiments have revealed an extraordinarily nonrandom distribution of recombination breakpoints along the lengths of plant chromosomes; for example, recombination breakpoints often resolve within genic sequences, and thereby generate novel alleles. The mechanism by which recombination breakpoints are determined is an area of active investigation. In addition, recent developments are providing recombination-based technologies for creating targeted alterations in the architecture of plant genomes.

Developmental and hormonal regulation of the arabidopsis CER2 gene that codes for a nuclear-localized protein required for the normal accumulation of cuticular waxes.

The previously cloned CER2 gene is required for the normal accumulation of cuticular waxes and encodes a novel protein. Earlier reports suggested that the CER2 protein is either a membrane-bound component of the fatty acid elongase complex or a regulatory protein. Cell fractionation and immunoblot analyses using polyclonal antibodies raised against a chemically synthesized peptide with a sequence based on the predicted CER2 protein sequence have demonstrated that the 47-kD CER2 protein is soluble and nuclear localized. These results are consistent with CER2 being a regulatory protein. Detailed studies of plants harboring a CER2 promoter/GUS transgene (CER2-GUS), in combination with immunoblot analyses, revealed that CER2 is expressed and the CER2 protein accumulates in a variety of organs and cell types. Expression is highest early in the development of these organs and is epidermis specific in most tissues. In agreement with the activity of the CER2 promoter in hypocotyls, cuticular wax accumulates on this organ in a CER2-dependent fashion. In leaves CER2 expression is confined to the guard cells, trichomes, and petioles. However, application of the cytokinin 6-benzylaminopurine induces ectopic expression of CER2-GUS in all cell types of leaves that emerge following treatment.

Sequence analysis of the cloned glossy8 gene of maize suggests that it may code for a beta-ketoacyl reductase required for the biosynthesis of cuticular waxes.

The gl8 locus of maize (Zea mays L.) was previously defined by a mutation that reduces the amount and alters the composition of seedling cuticular waxes. Sixty independently derived gl8 mutant alleles were isolated from stocks that carried the Mutator transposon system. A DNA fragment that contains a Mu8 transposon and that co-segregates with one of these alleles, gl8-Mu3142, was identified and cloned. DNA flanking the Mu8 transposon was shown via allelic cross-referencing experiments to represent the gl8 locus. The gl8 probe revealed a 1.4-kb transcript present in wild-type seedling leaves and, in lesser amounts, in other organs and at other developmental stages. The amino acid sequence deduced from an apparently full-length gl8 cDNA exhibits highly significant sequence similarity to a group of enzymes from plants, eubacteria, and mammals that catalyzes the reduction of ketones. This finding suggests that the GL8 protein probably functions as a reductase during fatty acid elongation in the cuticular wax biosynthetic pathway.

The glossy1 locus of maize and an epidermis-specific cDNA from Kleinia odora define a class of receptor-like proteins required for the normal accumulation of cuticular waxes.

Mutations at the glossy1 (gl1) locus of maize (Zea mays L.) quantitatively and qualitatively affect the deposition of cuticular waxes on the surface of seedling leaves. The gl1 locus has been molecularly cloned by transposon tagging with the Mutator transposon system. The epi23 cDNA was isolated by subtractive hybridization as an epidermis-specific mRNA from Senecio odora (Kleinia odora). The deduced amino acid sequence of the GL1 and EPI23 proteins are very similar to each other and to two other plant proteins in which the sequences were deduced from their respective mRNAs. These are the Arabidopsis CER1 protein, which is involved in cuticular wax deposition on siliques, stems, and leaves of that plant, and the protein coded by the rice expressed sequence tag RICS2751A. All four proteins are predicted to be localized in a membrane via a common NH2-terminal domain, which consists of either five or seven membrane-spanning helices. The COOH-terminal portion of each of these proteins, although less conserved, is predicted to be a water-soluble, globular domain. These sequence similarities indicate that these plant orthologs may belong to a superfamily of membrane-bound receptors that have been extensively characterized from animals, including the HIV co-receptor fusin (also termed CXCR4).

Rf8 and Rf* mediate unique T-urf13-transcript accumulation, revealing a conserved motif associated with RNA processing and restoration of pollen fertility in T-cytoplasm maize.

Rf8 is a newly described nuclear gene that can substitute for Rf1 to partially restore pollen fertility to male-sterile, T-cytoplasm maize. Families segregating for Rf8 were used to investigate the mechanism of this fertility restoration and to compare it to the restoration conditioned by Rf1. Although Rf8 is unlinked to the rf1 locus, it also alters T-urf13 mitochondrial transcript accumulation and reduces the accumulation of the URF13 protein. Like the 1.6- and 0.6-kilobase (kb) T-urf13 transcripts that accumulate in T-cytoplasm plants carrying Rf1, 1.42- and 0.42-kb transcripts accumulate in plants that are partially restored by Rf8. A survey of T-cytoplasm maize lines, inbreds, and F1 hybrids by mitochondrial RNA gel blot analyses revealed that Rf8 is rare in maize germplasm. These surveys revealed the presence of another rare, weak restorer factor, Rf*, which is uniquely associated with the accumulation of 1.4- and 0.4-kb T-urf13 transcripts. Primer extension analyses position the 5' termini of the 1.42/0.42-kb and 1.4/0.4-kb transcripts at +137 and +159 nucleotides, respectively, 3' of the AUG initiation codon of the T-urf13 reading frame. The conserved motif, 5'-CNACNNU-3', overlaps the 5' termini of the Rf1-, Rf8-, and Rf*-associated transcripts and the 380 nucleotide, Rf3-associated orf107 transcript from cytoplasmic male sterility sorghum. These results demonstrate that multiple unlinked, nuclear genes can have similar but distinct effects on the expression of the unique T-urf13 mitochondrial coding sequence to restore pollen fertility to T-cytoplasm maize.

Cloning and characterization of CER2, an Arabidopsis gene that affects cuticular wax accumulation.

Cuticular waxes are complex mixtures of very long chain fatty acids and their derivatives that cover plant surfaces. Mutants of the ECERIFERUM2 (cer2) gene of Arabidopsis condition bright green stems and siliques, indicative of the relatively low abundance of the cuticular wax crystals that comprise the wax bloom on wild-type plants. We cloned the CER2 gene via chromosome walking. Three lines of evidence establish that the cloned sequence represents the CER2 gene: (1) this sequence is capable of complementing the cer2 mutant phenotype in transgenic plants; (2) the corresponding DNA sequence isolated from plants homozygous for the cer2-2 mutant allele contains a sequence polymorphism that generates a premature stop codon; and (3) the deduced CER2 protein sequence exhibits sequence similarity to that of a maize gene (glossy2) that also is involved in cuticular wax accumulation. The CER2 gene encodes a novel protein with a predicted mass of 47 kD. We studied the expression pattern of the CER2 gene by in situ hybridization and analysis of transgenic Arabidopsis plants carrying a CER2-beta-glucuronidase gene fusion that includes 1.0 kb immediately upstream of CER2 and 0.2 kb of CER2 coding sequences. These studies demonstrate that the CER2 gene is expressed in an organ- and tissue-specific manner; CER2 is expressed at high levels only in the epidermis of young siliques and stems. This finding is consistent with the visible phenotype associated with mutants of the CER2 gene. Hence, the 1.2-kb fragment of the CER2 gene used to construct the CER2-beta-glucuronidase gene fusion includes all of the genetic information required for the epidermis-specific accumulation of CER2 mRNA.

Mutator-induced mutations of the rf1 nuclear fertility restorer of T-cytoplasm maize alter the accumulation of T-urf13 mitochondrial transcripts.

Dominant alleles of the rf1 and rf2 nuclear-encoded fertility restorer genes are necessary for restoration of pollen fertility in T-cytoplasm maize. To further characterize fertility restoration mediated by the Rf1 allele, 123,500 gametes derived from plants carrying the Mutator transposable element family were screened for rf1-mutant alleles (rf1-m) Four heritable rf1-m alleles were recovered from these populations. Three rf1-m alleles were derived from the progenitor allele Rf1-IA153 and one was derived from Rf1-Ky21. Cosegregation analysis revealed 5.5- and 2.4-kb Mu1-hybridizing EcoRI restriction fragments in all of the male-sterile and none of the male-fertile plants in families segregating for rf1-m3207 and rf1-m3310, respectively. Mitochondrial RNA gel blot analyses indicated that all four rf1-m alleles in male-sterile plants cosegregated with the altered steady-state accumulation of 1.6- and 0.6-kb T-urf13 transcripts, demonstrating that these transcripts are Rf1 dependent. Plants carrying a leaky mutant, rf1-m7323, revealed variable levels of Rf1-associated, T-urf13 transcripts and the degree of pollen fertility. The ability to obtain rf1-m derivatives from Rf1 indicates that Rf1 alleles produce a functional gene product necessary for the accumulation of specific T-urf13 transcripts in T-cytoplasm maize.

The rf2 nuclear restorer gene of male-sterile T-cytoplasm maize.

The T cytoplasm of maize serves as a model for the nuclear restoration of cytoplasmic male sterility. The rf2 gene, one of two nuclear genes required for fertility restoration in male-sterile T-cytoplasm (cmsT) maize, was cloned. The protein predicted by the rf2 sequence is a putative aldehyde dehydrogenase, which suggests several mechanisms that might explain Rf2-mediated fertility restoration in cmsT maize. Aldehyde dehydrogenase may be involved in the detoxification of acetaldehyde produced by ethanolic fermentation during pollen development, may play a role in energy metabolism, or may interact with URF13, the mitochondrial protein associated with male sterility in cmsT maize.

DNA sequence analyses support the role of interrupted gap repair in the origin of internal deletions of the maize transposon, MuDR.

Previous research has demonstrated that the autonomous Cy transposon can activate the excision of Mu transposons. To determine the relationship between Cy and the more recently described autonomous Mu transposon, MuDR, a Cy transposon inserted at the mutable a1 allele, a1-m5216, was isolated and cloned. DNA sequence analyses established that this Cy insertion is identical to MuDR (Mu9, GenBank accession No.: m76978.gb-pl). Therefore, Cy will henceforth be termed MuDR:Cy. Defective derivatives of MuDR:Cy were isolated that had lost their capacity to activate their own excision or the excision of a Mu7 transposon. Most of these derivatives are nonautonomous transposons because they can excise, but only in the presence of unlinked MuDR:Cy transposons. Physical mapping and DNA sequence analyses have established that six of these defective derivatives carry internal deletions. It has been proposed previously that such deletions arise via interrupted gap repair. The DNA sequences of the break points associated with all four sequenced deletions are consistent with this model. The finding that three of the excision-defective derivatives carry deletions that disrupt the coding region of the mudrA (but not the mudrB) transcript supports the view that mudrA plays a role in the excision of Mu transposons.

Meiotic recombination break points resolve at high rates at the 5' end of a maize coding sequence.

Sequence analysis of recombination break points has defined a 377-bp recombination hot spot within the anthocyanin 1 (a1) gene. One-fifth of all recombination events that occurred within the 140-kb a1-shrunken 2 interval resolved within this 377-bp hot spot. In yeast, meiotic double-strand breaks in chromosomal DNA are thought to initiate recombination and are generally located 5' of coding regions, near transcription promoter sequences. Because the a1 recombination hot spot is located within the 5' transcribed region of the a1 gene, the sites at which recombination events initiate and resolve appear to be different, but both appear to be regulated in relation to transcribed sequences. Although transposon insertions are known to suppress recombination and alter the ratio of crossovers to apparent gene conversions, the Mutator 1 transposon insertion in the a1-mum2 allele does not alter the sites at which recombination events resolve.

Cloning and characterization of the maize An1 gene.

The Anther ear1 (An1) gene product is involved in the synthesis of ent-kaurene, the first tetracyclic intermediate in the gibberellin (GA) biosynthetic pathway. Mutations causing the loss of An1 function result in a GA-responsive phenotype that includes reduced plant height, delayed maturity, and development of perfect flowers on normally pistillate ears. The an1::Mu2-891339 allele was recovered from a Mutator (Mu) F2 family. Using Mu elements as molecular probes, an An1-containing restriction fragment was identified and cloned. The identity of the cloned gene as An1 was confirmed by using a reverse genetics screen for maize families that contain a Mu element inserted into the cloned gene and then by demonstrating that the insertion causes an an1 phenotype. The predicted amino acid sequence of the An1 cDNA shares homology with plant cyclases and contains a basic N-terminal sequence that may target the An1 gene product to the chloroplast. The sequence is consistent with the predicted subcellular localization of AN1 in the chloroplast and with its biochemical role in the cyclization of geranylgeranyl pyrophosphate, a 20-carbon isoprenoid, to ent-kaurene. The semidwarfed stature of an1 mutants is in contrast with the more severely dwarfed stature of GA-responsive mutants at other loci in maize and may be caused by redundancy in this step of the GA biosynthetic pathway. DNA gel blot analysis indicated that An1 is a single-copy gene that lies entirely within the deletion of the an1-bz2-6923 mutant. However, homozygous deletion mutants accumulated ent-kaurene to 20% of the wild-type level, suggesting that the function of An1 is supplemented by an additional activity.

The relationship between genetic and physical distances in the cloned a1-sh2 interval of the Zea mays L. genome.

A 470-kb segment from the long arm of chromosome 3 of Zea mays (inbred LH82), encompassing the a1-sh2 interval, was cloned as a yeast artificial chromosome. Comparison of the sizes of the restriction fragments generated from the cloned DNA fragment and from the DNA isolated from the maize inbred line LH82 established the colinearity of the a1-sh2 interval in these DNAs. By utilizing a chromosome fragmentation technique, a yeast artificial chromosome encompassing the a1-sh2 interval was separately fragmented at the a1 and sh2 loci. Comparison of the sizes of these fragmentation products established the physical distance between the a1 and sh2 loci to be 140 kb. Furthermore, these fragmentation experiments established the physical orientation of the a1 and sh2 genes relative to the maize centromere. The molecular cloning of the contiguous region between the a1 and sh2 loci made it possible to define the relationship between physical and genetic distances over a relatively large segment of the maize genome. In this interval, the relationship between physical and genetic distances is 1560 kb/centimorgan, which compares with 1460 kb/centimorgan for the entire maize genome, and 217 kb/centimorgan for a 1-kb segment within the a1 locus. Therefore, these findings are consistent with the hypothesis that genes per se are preferred sites for meiotic recombination rather than the hypothesis that genes reside in large recombinationally active segments of the genome.