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The widely recounted story of the origin of cultivated strawberry (Fragaria × ananassa) oversimplifies the complex interspecific hybrid ancestry of the highly admixed populations from which heirloom and modern cultivars have emerged. To develop deeper insights into the three-century-long domestication history of strawberry, we reconstructed the genealogy as deeply as possible-pedigree records were assembled for 8,851 individuals, including 2,656 cultivars developed since 1775. The parents of individuals with unverified or missing pedigree records were accurately identified by applying an exclusion analysis to array-genotyped single-nucleotide polymorphisms. We identified 187 wild octoploid and 1,171 F. × ananassa founders in the genealogy, from the earliest hybrids to modern cultivars. The pedigree networks for cultivated strawberry are exceedingly complex labyrinths of ancestral interconnections formed by diverse hybrid ancestry, directional selection, migration, admixture, bottlenecks, overlapping generations, and recurrent hybridization with common ancestors that have unequally contributed allelic diversity to heirloom and modern cultivars. Fifteen to 333 ancestors were predicted to have transmitted 90% of the alleles found in country-, region-, and continent-specific populations. Using parent-offspring edges in the global pedigree network, we found that selection cycle lengths over the past 200 years of breeding have been extraordinarily long (16.0-16.9 years/generation), but decreased to a present-day range of 6.0-10.0 years/generation. Our analyses uncovered conspicuous differences in the ancestry and structure of North American and European populations, and shed light on forces that have shaped phenotypic diversity in F. × ananassa., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Background: The ability to accurately and comprehensively identify genomic variations is critical for plant studies utilizing high-throughput sequencing. Most bioinformatics tools for processing next-generation sequencing data were originally developed and tested in human studies, raising questions as to their efficacy for plant research. A detailed evaluation of the entire variant calling pipeline, including alignment, variant calling, variant filtering, and imputation was performed on different programs using both simulated and real plant genomic datasets., Results: A comparison of SOAP2, Bowtie2, and BWA-MEM found that BWA-MEM was consistently able to align the most reads with high accuracy, whereas Bowtie2 had the highest overall accuracy. Comparative results of GATK HaplotypCaller versus SAMtools mpileup indicated that the choice of variant caller affected precision and recall differentially depending on the levels of diversity, sequence coverage and genome complexity. A cross-reference experiment of S. lycopersicum and S. pennellii reference genomes revealed the inadequacy of single reference genome for variant discovery that includes distantly-related plant individuals. Machine-learning-based variant filtering strategy outperformed the traditional hard-cutoff strategy resulting in higher number of true positive variants and fewer false positive variants. A 2-step imputation method, which utilized a set of high-confidence SNPs as the reference panel, showed up to 60% higher accuracy than direct LD-based imputation., Conclusions: Programs in the variant discovery pipeline have different performance on plant genomic dataset. Choice of the programs is subjected to the goal of the study and available resources. This study serves as an important guiding information for plant biologists utilizing next-generation sequencing data for diversity characterization and crop improvement.

Describing the genetic diversity in the gene pool of crops will provide breeders with novel resources for varietal improvement. Nested Association Mapping (NAM) populations are uniquely suited for characterizing parental diversity through the shuffling and fixation of parental haplotypes. Here, we describe a set of 1879 rice NAM lines created through the selfing and single-seed descent of F 1 hybrids derived from elite IR64 indica crossed with 10 diverse tropical japonica lines. Genotyping data indicated tropical japonica alleles were captured at every queried locus despite the presence of segregation distortion factors. Several distortion loci were mapped, both shared and unique, among the 10 populations. Using two-point and multi-point genetic map calculations, our datasets achieved the ∼1500 cM expected map size in rice. Finally, we highlighted the utility of the NAM lines for QTL mapping, including joint analysis across the 10 populations, by confirming known QTL locations for the trait days to heading., (Copyright © 2017 Fragoso et al.)

Wide crosses have been used for decades as a method for transferring novel genetic material and traits in plant breeding. Historically, many products of wide crosses require tedious and inefficient surgical embryo rescue prior to embryo abortion to recover single plantlets. We have utilized transgenic switchgrass (Panicum virgatum L. cv Alamo) as a pollen donor in conjunction with antibiotic or herbicide selection for recovery of intra-and interspecific F 1 crosses by using developing ovules from the female parent and selecting for embryogenic cultures derived from the in situ immature embryo. Using this approach, several intravarietial crosses were generated between transgenic Alamo and the switchgrass varieties Kanlow, Blackwell and Cave-in-Rock as well as an interspecific cross with Atlantic coastal panicgrass. This procedure selected F 1 embryogenic callus produced from the developing embryo contained within isolated immature ovules. Several clonal plants were successfully regenerated from each cross. Southern blot, PCR, phenotypic analyses and genomic analysis confirmed F 1 hybrids. Using genotyping-by-sequencing shows the hybridization of the recovered plants by determining the ratio of transgressive markers to total compared markers between parents and their potential offspring. The ratio of transgressive markers to total compared markers was significantly lower between parents and their predicted offspring than between parents and offspring unrelated to them. This approach provides the possibility to move useful transgenes into varieties that are recalcitrant to direct transformation which can be optionally segregated thus useful to create new hybrids, as well as recovery of wide crosses that are either difficult or impossible using traditional techniques., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Sex determination in maize involves the production of staminate and pistillate florets from an initially bisexual floral meristem. Pistil elimination in staminate florets requires jasmonic acid signaling, and functional pistils are protected by the action of the silkless 1 ( sk1 ) gene. The sk1 gene was identified and found to encode a previously uncharacterized family 1 uridine diphosphate glycosyltransferase that localized to the plant peroxisomes. Constitutive expression of an sk1 transgene protected all pistils in the plant, causing complete feminization, a gain-of-function phenotype that operates by blocking the accumulation of jasmonates. The segregation of an sk1 transgene was used to effectively control the production of pistillate and staminate inflorescences in maize plants.

Low-coverage next-generation sequencing methodologies are routinely employed to genotype large populations. Missing data in these populations manifest both as missing markers and markers with incomplete allele recovery. False homozygous calls at heterozygous sites resulting from incomplete allele recovery confound many existing imputation algorithms. These types of systematic errors can be minimized by incorporating depth-of-sequencing read coverage into the imputation algorithm. Accordingly, we developed Low-Coverage Biallelic Impute (LB-Impute) to resolve missing data issues. LB-Impute uses a hidden Markov model that incorporates marker read coverage to determine variable emission probabilities. Robust, highly accurate imputation results were reliably obtained with LB-Impute, even at extremely low (<1×) average per-marker coverage. This finding will have implications for the design of genotype imputation algorithms in the future. LB-Impute is publicly available on GitHub at https://github.com/dellaporta-laboratory/LB-Impute., (Copyright © 2016 by the Genetics Society of America.)

Switchgrass (Panicum virgatum L.) and its relatives are regarded as top bioenergy crop candidates; however, one critical barrier is the introduction of useful genetic diversity and the development of new cultivars and hybrids. Combining genomes from related cultivars and species provides an opportunity to introduce new traits. In switchgrass, a breeding advantage would be achieved by combining the genomes of intervarietal ecotypes or interspecific hybrids. The recovery of wide crosses, however, is often tedious and may involve complicated embryo rescue and numerous backcrosses. Here, we demonstrate a straightforward approach to wide crosses involving the use of a selectable transgene for recovery of interspecific [P. virgatum cv. Alamo × Panicum amarum Ell. var amarulum or Atlantic Coastal Panicgrass (ACP)] F 1 hybrids followed by backcrossing to generate a nontransgenic admixture population. A nontransgenic herbicide-sensitive (HbS) admixture population of 83 F 1 BC 1 progeny was analyzed by genotyping-by-sequencing (GBS) to characterize local ancestry, parental contribution, and patterns of recombination. These results demonstrate a widely applicable breeding strategy that makes use of transgenic selectable resistance to identify and recover true hybrids., (© 2015 The Authors.)

Background: Many areas critical to agricultural production and research, such as the breeding and trait mapping in plants and livestock, require robust and scalable genotyping platforms. Genotyping-by-sequencing (GBS) is a one such method highly suited to non-human organisms. In the GBS protocol, genomic DNA is fractionated via restriction digest, then reduced representation is achieved through size selection. Since many restriction sites are conserved across a species, the sequenced portion of the genome is highly consistent within a population. This makes the GBS protocol highly suited for experiments that require surveying large numbers of markers within a population, such as those involving genetic mapping, breeding, and population genomics. We have modified the GBS technology in a number of ways. Custom, enzyme specific adaptors have been replaced with standard Illumina adaptors compatible with blunt-end restriction enzymes. Multiplexing is achieved through a dual barcoding system, and bead-based library preparation protocols allows for in-solution size selection and eliminates the need for columns and gels., Results: A panel of eight restriction enzymes was selected for testing on B73 maize and Nipponbare rice genomic DNA. Quality of the data was demonstrated by identifying that the vast majority of reads from each enzyme aligned to restriction sites predicted in silico. The link between enzyme parameters and experimental outcome was demonstrated by showing that the sequenced portion of the genome was adaptable by selecting enzymes based on motif length, complexity, and methylation sensitivity. The utility of the new GBS protocol was demonstrated by correctly mapping several in a maize F2 population resulting from a B73×Country Gentleman test cross., Conclusions: This technology is readily adaptable to different genomes, highly amenable to multiplexing and compatible with over forty commercially available restriction enzymes. These advancements represent a major improvement in genotyping technology by providing a highly flexible and scalable GBS that is readily implemented for studies on genome-wide variation.

Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.

We surveyed genetic variation in alr2, an allodeterminant of the colonial hydroid Hydractinia symbiolongicarpus. We generated cDNA from a sample of 239 Hydractinia colonies collected at Lighthouse Point, Connecticut, and identified 473 alr2 alleles, 198 of which were unique. Rarefaction analysis suggested that the sample was near saturation. Most alleles were rare, with 86% occurring at frequencies of 1% or less. Alleles were highly variable, diverging on average by 18% of the amino acids in a predicted extracellular domain of the molecule. Analysis of 152 full-length alleles confirmed the existence of two structural types, defined by exons 4-8 of the gene. Several residues of the predicted immunoglobulin superfamily-like domains display signatures of positive selection. We also identified 77 unique alr2 pseudogene sequences from 85 colonies. Twenty-seven of these sequences matched expressed alr2 sequences from other colonies. This observation is consistent with pseudogenes contributing to alr2 diversification through sequence donation. A more limited collection of animals was made from a distant, relict population of H. symbiolongicarpus. Sixty percent of the unique sequences identified in this sample were found to match sequences from the Lighthouse Point population. The large number of alr2 alleles, their degree of divergence, the predominance of rare alleles in the population, their persistence over broad spatial and temporal scales, and the signatures of positive selection in multiple residues of the putative recognition domain paint a consistent picture of negative-frequency-dependent selection operating in this system. The genetic diversity observed at alr2 is comparable to that of the most highly polymorphic genetic systems known to date.

The Hydractinia allorecognition complex (ARC) was initially identified as a single chromosomal interval using inbred and congenic lines. The production of defined lines necessarily homogenizes genetic background and thus may be expected to obscure the effects of unlinked allorecognition loci should they exist. Here, we report the results of crosses in which inbred lines were out-crossed to wild-type animals in an attempt to identify dominant, codominant, or incompletely dominant modifiers of allorecognition. A claim for the existence of modifiers unlinked to ARC was rejected for three different genetic backgrounds. Estimates of the genetic map distance of ARC in two wild-type haplotypes differed markedly from one another and from that measured in congenic lines. These results suggest that additional allodeterminants exist in the Hydractinia ARC.

Hydractinia symbiolongicarpus, a colonial cnidarian (class Hydrozoa) epibiont on hermit crab shells, is well established as a model for genetic studies of allorecognition. Recently, two linked loci, allorecognition (alr) 1 and alr2, were identified by positional cloning and shown to be major determinants of histocompatibility. Both genes encode putative transmembrane proteins with hypervariable extracellular domains similar to immunoglobulin (Ig)-like domains. We sought to characterize the naturally occurring variation at the alr2 locus and to understand the origins of this molecular diversity. We examined full-length cDNA coding sequences derived from a sample of 21 field-collected colonies, including 18 chosen haphazardly and two laboratory reference strains. Of the 35 alleles recovered from the 18 unbiased samples, 34 encoded unique gene products. We identified two distinct structural classes of alleles that varied over a large central region of the gene but both possessed highly polymorphic extracellular domains I, similar to an Ig-like V-set domain. The discovery of structurally chimeric alleles provided evidence that interallelic recombination may contribute to alr2 variation. Comparisons of the genomic region encompassing alr2 from two field-derived haplotypes and one laboratory reference sequence revealed a history of structural variation at the haplotype level as well. Maintenance of large numbers of equally rare alleles in a natural population is a hallmark of negative frequency-dependent selection and is expected to produce high levels of heterozygosity. The observed alr2 allelic diversity is comparable with that found in immune recognition molecules such as human leukocyte antigens, B cell Igs, or natural killer cell Ig-like receptors.

Allorecognition, the ability to discriminate between self and nonself, is ubiquitous among colonial metazoans and widespread among aclonal taxa. Genetic models for the study of allorecognition have been developed in the jawed vertebrates, invertebrate chordate Botryllus, and cnidarian Hydractinia. In Botryllus, two genes contribute to the histocompatibility response, FuHC and fester. In the cnidarian Hydractinia, one of the two known allorecognition loci, alr2, has been isolated, and a second linked locus, alr1, has been mapped to the same chromosomal region, called the allorecognition complex (ARC). Here we isolate alr1 by positional cloning and report it to encode a transmembrane receptor protein with two hypervariable extracellular regions similar to immunoglobulin (Ig)-like domains. Variation in the extracellular domain largely predicts fusibility within and between laboratory strains and wild-type isolates. alr1 was found embedded in a family of immunoglobulin superfamily (IgSF)-like genes, thus establishing that the ARC histocompatibility complex is an invertebrate IgSF-like gene complex., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Colonial marine invertebrates, such as sponges, corals, bryozoans, and ascidians, often live in densely populated communities where they encounter other members of their species as they grow over their substratum. Such encounters typically lead to a natural histocompatibility response in which colonies either fuse to become a single, chimeric colony or reject and aggressively compete for space. These allorecognition phenomena mediate intraspecific competition, support allotypic diversity, control the level at which selection acts, and resemble allogeneic interactions in pregnancy and transplantation. Despite the ubiquity of allorecognition in colonial phyla, however, its molecular basis has not been identified beyond what is currently known about histocompatibility in vertebrates and protochordates. We positionally cloned an allorecognition gene by using inbred strains of the cnidarian, Hydractinia symbiolongicarpus, which is a model system for the study of invertebrate allorecognition. The gene identified encodes a putative transmembrane receptor expressed in all tissues capable of allorecognition that is highly polymorphic and predicts allorecognition responses in laboratory and field-derived strains. This study reveals that a previously undescribed hypervariable molecule bearing three extracellular domains with greatest sequence similarity to the immunoglobulin superfamily is an allodeterminant in a lower metazoan.

Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with presently over 46,000 members. In phylogenetic comparisons, members of this superfamily show early divergence where the majority have only low pairwise sequence identity, although sharing common structural properties. The SDR enzymes are present in virtually all genomes investigated, and in humans over 70 SDR genes have been identified. In humans, these enzymes are involved in the metabolism of a large variety of compounds, including steroid hormones, prostaglandins, retinoids, lipids and xenobiotics. It is now clear that SDRs represent one of the oldest protein families and contribute to essential functions and interactions of all forms of life. As this field continues to grow rapidly, a systematic nomenclature is essential for future annotation and reference purposes. A functional subdivision of the SDR superfamily into at least 200 SDR families based upon hidden Markov models forms a suitable foundation for such a nomenclature system, which we present in this paper using human SDRs as examples.

For more than a century, the origin of metazoan animals has been debated. One aspect of this debate has been centered on what the hypothetical "urmetazoon" bauplan might have been. The morphologically most simply organized metazoan animal, the placozoan Trichoplax adhaerens, resembles an intriguing model for one of several "urmetazoon" hypotheses: the placula hypothesis. Clear support for a basal position of Placozoa would aid in resolving several key issues of metazoan-specific inventions (including, for example, head-foot axis, symmetry, and coelom) and would determine a root for unraveling their evolution. Unfortunately, the phylogenetic relationships at the base of Metazoa have been controversial because of conflicting phylogenetic scenarios generated while addressing the question. Here, we analyze the sum of morphological evidence, the secondary structure of mitochondrial ribosomal genes, and molecular sequence data from mitochondrial and nuclear genes that amass over 9,400 phylogenetically informative characters from 24 to 73 taxa. Together with mitochondrial DNA genome structure and sequence analyses and Hox-like gene expression patterns, these data (1) provide evidence that Placozoa are basal relative to all other diploblast phyla and (2) spark a modernized "urmetazoon" hypothesis., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Sex determination in maize is controlled by a developmental cascade leading to the formation of unisexual florets derived from an initially bisexual floral meristem. Abortion of pistil primordia in staminate florets is controlled by a tasselseed-mediated cell death process. We positionally cloned and characterized the function of the sex determination gene tasselseed1 (ts1). The TS1 protein encodes a plastid-targeted lipoxygenase with predicted 13-lipoxygenase specificity, which suggests that TS1 may be involved in the biosynthesis of the plant hormone jasmonic acid. In the absence of a functional ts1 gene, lipoxygenase activity was missing and endogenous jasmonic acid concentrations were reduced in developing inflorescences. Application of jasmonic acid to developing inflorescences rescued stamen development in mutant ts1 and ts2 inflorescences, revealing a role for jasmonic acid in male flower development in maize.

The presence of highly specific histocompatibility reactions in colonial marine invertebrates that lack adaptive immune systems (such as the sponges, cnidarians, bryozoans and ascidians) provides a unique opportunity to investigate the evolutionary roots of allorecognition and to explore whether homologous innate recognition systems exist in vertebrates. Conspecific interactions among adult animals in these groups are regulated by highly specific allorecognition systems that restrict somatic fusion to self or close kin. In Hydractinia (Cnidaria:Hydrozoa), fusion/rejection responses are controlled by two linked genetic loci. Alleles at each locus are co-dominantly inherited. Colonies fuse if they share at least one haplotype, reject if they share no haplotypes, and display transitory fusion if they share only one allele in a haplotype-a pattern that echoes natural killer cell responses in mice and humans. Allorecognition in Hydractinia and other marine invertebrates serves as a safeguard against stem cell or germline parasitism thus, limiting chimerism to closely related individuals. These animals fail to become tolerant even if exposed during early development to cells from a histoincompatible individual. Detailed analysis of the structure and function of molecules responsible for allorecognition in basal marine invertebrates could provide clues to the innate mechanisms by which higher animals respond to organ and cell allografts, including embryonic tissues.

The evolution of ANTP genes in the Metazoa has been the subject of conflicting hypotheses derived from full or partial gene sequences and genomic organization in higher animals. Whole genome sequences have recently filled in some crucial gaps for the basal metazoan phyla Cnidaria and Porifera. Here we analyze the complete genome of Trichoplax adhaerens, representing the basal metazoan phylum Placozoa, for its set of ANTP class genes. The Trichoplax genome encodes representatives of Hox/ParaHox-like, NKL, and extended Hox genes. This repertoire possibly mirrors the condition of a hypothetical cnidarian-bilaterian ancestor. The evolution of the cnidarian and bilaterian ANTP gene repertoires can be deduced by a limited number of cis-duplications of NKL and "extended Hox" genes and the presence of a single ancestral "ProtoHox" gene.

As arguably the simplest free-living animals, placozoans may represent a primitive metazoan form, yet their biology is poorly understood. Here we report the sequencing and analysis of the approximately 98 million base pair nuclear genome of the placozoan Trichoplax adhaerens. Whole-genome phylogenetic analysis suggests that placozoans belong to a 'eumetazoan' clade that includes cnidarians and bilaterians, with sponges as the earliest diverging animals. The compact genome shows conserved gene content, gene structure and synteny in relation to the human and other complex eumetazoan genomes. Despite the apparent cellular and organismal simplicity of Trichoplax, its genome encodes a rich array of transcription factor and signalling pathway genes that are typically associated with diverse cell types and developmental processes in eumetazoans, motivating further searches for cryptic cellular complexity and/or as yet unobserved life history stages.

Background: Mitochondrial genomes (mtDNA) of numerous sponges have been sequenced as part of an ongoing effort to resolve the class-level phylogeny of the Porifera, as well as to place the various lower metazoan groups on the animal-kingdom tree. Most recently, the partial mtDNA of two glass sponges, class Hexactinellida, were reported. While previous phylogenetic estimations based on these data remain uncertain due to insufficient taxon sampling and accelerated rates of evolution, the mtDNA molecules themselves reveal interesting traits that may be unique to hexactinellids. Here we determined the first complete mitochondrial genome of a hexactinellid sponge, Aphrocallistes vastus, and compared it to published poriferan mtDNAs to further describe characteristics specific to hexactinellid and other sponge mitochondrial genomes., Results: The A. vastus mtDNA consisted of a 17,427 base pair circular molecule containing thirteen protein-coding genes, divergent large and small subunit ribosomal RNAs, and a reduced set of 18 tRNAs. The A. vastus mtDNA showed a typical hexactinellid nucleotide composition and shared a large synteny with the other sequenced glass sponge mtDNAs. It also contained an unidentified open reading frame and large intergenic space region. Two frameshifts, in the cox3 and nad6 genes, were not corrected by RNA editing, but rather possessed identical shift sites marked by the extremely rare tryptophan codon (UGG) followed by the common glycine codon (GGA) in the +1 frame., Conclusion: Hexactinellid mtDNAs have shown similar trends in gene content, nucleotide composition, and codon usage, and have retained a large gene syntenty. Analysis of the mtDNA of A. vastus has provided evidence diagnostic for +1 programmed translational frameshifting, a phenomenon disparately reported throughout the animal kingdom, but present in the hexactinellid mtDNAs that have been sequenced to date.

The allorecognition complex of Hydractinia symbiolongicarpus is a chromosomal interval containing two loci, alr1 and alr2, that controls fusion between genetically distinct colonies. Recombination between these two loci has been associated with a heterogeneous class of phenotypes called transitory fusion. A large-scale backcross was performed to generate a population of colonies (N = 106) with recombination breakpoints within the allorecognition complex. Two distinct forms of transitory fusion were correlated with reciprocal recombination products, suggesting that alr1 and alr2 contributed differentially to the allorecognition response. Specifically, type I transitory fusion is associated with rapid and persistent separation of allogeneic tissues, whereas type II transitory fusion generates a patchwork of continuously fusing and separating tissues.

The maize sex determination pathway results in the arrest of stamen in ear spikelets and the abortion of pistils in both the tassel spikelets and in the secondary florets of ear spikelets. Arrested stamen cells showed no signs of DNA fragmentation, an absence of CYCLIN B expression, and an accumulation of the negative cell cycle regulator WEE1 RNA.

In colonial marine invertebrates, allorecognition restricts somatic fusion and thus, chimerism, to histocompatible individuals. Little is understood, however, about how invertebrates respond to chimerism formed across histocompatibility barriers or whether embryonic exposure to histoincompatible cells induces allotolerance. We here evaded natural allorecognition barriers by generating well mixed embryonic chimeras of Hydractinia symbiolongicarpus (Cnidaria:Hydrozoa) and developed molecular markers to detect chimerism in both histocompatible and histoincompatible settings. Histocompatible chimeras exhibited markedly higher growth rates and survivorship than histoincompatible pairings. Histoincompatible chimeras were unstable, with chimerism being undetectable by 4 wk of age. In contrast, colonies generated from histocompatible pairings remained chimeric at markedly higher frequencies and longer durations. Histoincompatible chimeras that lost detectable chimerism retained the fusibility/rejection characteristics of the remaining component of the chimera but not that of the lost component. Chimerism across histocompatibility barriers in an invertebrate model organism was unstable and did not induce tolerance.

The development of unisexual flowers in maize and other plants proceeds through selective elimination of floral organs in an initially bisexual floral meristem. The essential character of the tasselseed 2 gene (TS2) in this cell-death pathway has been established previously. Molecular cloning of TS2 reveals membership to the evolutionarily conserved superfamily of short-chain dehydrogenases/reductases, but its substrate specificity remained unknown. Recombinant TS2 protein was produced in Escherichia coli, and purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration experiments show that TS2 is a tetrameric enzyme. Thermal denaturation followed by circular dichroism spectroscopy reveals that TS2 binds NAD(H) and NAD(P)(H). Substrate screening demonstrates that TS2 converts steroids with specificities found at positions 3 and 17, and several dicarbonyl and quinone compounds, thus establishing TS2 as a plant 3beta/17beta-hydroxysteroid dehydrogenase and carbonyl/quinone reductase. Taken together, the genetic data and the substrate specificities determined suggest that TS2 converts specific plant compounds and acts as a prereceptor control mechanism, in a manner similar to that of mammalian hydroxysteroid dehydrogenases.

The first sequenced mitochondrial genome of a placozoan, Trichoplax adhaerens, challenged the conventional wisdom that a compact mitochondrial genome is a common feature among all animals. Three additional placozoan mitochondrial genomes representing highly divergent clades have been sequenced to determine whether the large Trichoplax mtDNA is a shared feature among members of the phylum Placozoa or a uniquely derived condition. All three mitochondrial genomes were found to be very large, 32- to 37-kb, circular molecules, having the typical 12 respiratory chain genes, 24 tRNAs, rnS, and rnL. They share with the Trichoplax mitochondrial genome the absence of atp8, atp9, and all ribosomal protein genes, the presence of several cox1 introns, and a large open reading frame containing an intron group I LAGLIDADG endonuclease domain. The differences in mtDNA size within Placozoa are due to variation in intergenic spacer regions and the presence or absence of long open reading frames of unknown function. Phylogenetic analyses of the 12 respiratory chain genes support the monophyly of Placozoa. The similarities in composition and structure between the three mitochondrial genomes reported here and that of Trichoplax's mtDNA suggest that their uncompacted state is a shared ancestral feature to other nonmetazoans while their gene content is a derived feature shared only among the Metazoa., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

We here address placozoan distribution and phylogeography in five locations in the Caribbean Sea. We performed a coarse-resolution presence/absence survey of placozoans in Belize, Bermuda, Grenada, Jamaica, and Panama and a fine-resolution study of the distribution of placozoans in Twin Cays, Belize. Placozoans were recovered in every country sampled. Animals were sequenced at the mitochondrial 16S rDNA locus, and our analysis identified four of the five previously identified clades present in the Caribbean. In addition, we discovered two new haplotypes within one of these clades, and we found sympatric clades in Belize, Bermuda, Jamaica, and Panama. These studies provide further molecular evidence for species diversity within the Phylum Placozoa.

Mitochondrial genomes of multicellular animals are typically 15- to 24-kb circular molecules that encode a nearly identical set of 12-14 proteins for oxidative phosphorylation and 24-25 structural RNAs (16S rRNA, 12S rRNA, and tRNAs). These genomes lack significant intragenic spacers and are generally without introns. Here, we report the complete mitochondrial genome sequence of the placozoan Trichoplax adhaerens, a metazoan with the simplest known body plan of any animal, possessing no organs, no basal membrane, and only four different somatic cell types. Our analysis shows that the Trichoplax mitochondrion contains the largest known metazoan mtDNA genome at 43,079 bp, more than twice the size of the typical metazoan mtDNA. The mitochondrion's size is due to numerous intragenic spacers, several introns and ORFs of unknown function, and protein-coding regions that are generally larger than those found in other animals. Not only does the Trichoplax mtDNA have characteristics of the mitochondrial genomes of known metazoan outgroups, such as chytrid fungi and choanoflagellates, but, more importantly, it shares derived features unique to the Metazoa. Phylogenetic analyses of mitochondrial proteins provide strong support for the placement of the phylum Placozoa at the root of the Metazoa.

Across the animal kingdom, Hox genes are organized in clusters whose genomic organization reflects their central roles in patterning along the anterior/posterior (A/P) axis . While a cluster of Hox genes was present in the bilaterian common ancestor, the origins of this system remain unclear (cf. ). With new data for two representatives of the closest extant phylum to the Bilateria, the sea anemone Nematostella and the hydromedusa Eleutheria, we argue here that the Cnidaria predate the evolution of the Hox system. Although Hox-like genes are present in a range of cnidarians, many of these are paralogs and in neither Nematostella nor Eleutheria is an equivalent of the Hox cluster present. With the exception of independently duplicated genes, the cnidarian genes are unlinked and in several cases are flanked by non-Hox genes. Furthermore, the cnidarian genes are expressed in patterns that are inconsistent with the Hox paradigm. We conclude that the Cnidaria/Bilateria split occurred before a definitive Hox system developed. The spectacular variety in morphological and developmental characteristics shown by extant cnidarians demonstrates that there is no obligate link between the Hox system and morphological diversity in the animal kingdom and that a canonical Hox system is not mandatory for axial patterning.

Homeobox genes of the ANTP and PRD classes play important roles in body patterning of metazoans, and a large diversity of these genes have been described in bilaterian animals and cnidarians. Trichoplax adhaerens (Phylum Placozoa) is a small multicellular marine animal with one of the simplest body organizations of all metazoans, showing no symmetry and a small number of distinct cell types. Only two ANTP class genes have been described from Trichoplax: the Hox/ParaHox gene Trox-2 and a gene related to the Not family. Here we report an extensive screen for ANTP class genes in Trichoplax, leading to isolation of three additional ANTP class genes. These can be assigned to the Dlx, Mnx and Hmx gene families. Sequencing approximately 12-20 kb around each gene indicates that none are part of tight gene clusters, and in situ hybridization reveals that at least two have spatially restricted expression around the periphery of the animal. The low diversity of ANTP class genes isolated in Trichoplax can be reconciled with the low anatomical complexity of this animal, although the finding that these genes are assignable to recognized gene families is intriguing.

Placozoans, the simplest free-living animals, have never been observed to reproduce sexually. Here, we describe molecular evidence for sexual reproduction within one clade of the Placozoa. In a population sample of 10 individuals, within-individual and overall nucleotide diversity were similar to each other and consistent with levels observed in sexually reproducing species. Intergenic recombination as well as the sharing of alleles between heterozygous and homozygous individuals was also observed. These hallmarks of sexual reproduction establish that sex is indeed present in this phylum.

Stomatal pores located on the plant epidermis regulate CO(2) uptake for photosynthesis and the loss of water by transpiration. The opening and closing of the pore is mediated by turgor-driven volume changes of two surrounding guard cells. These highly specialized cells integrate internal signals and environmental stimuli to modulate stomatal aperture for plant survival under diverse conditions. Modulation of transcription and mRNA processing play important roles in controlling guard-cell activity, although the details of these levels of regulation remain mostly unknown. Here we report the characterization of AtMYB60, a R2R3-MYB gene of Arabidopsis, as the first transcription factor involved in the regulation of stomatal movements. AtMYB60 is specifically expressed in guard cells, and its expression is negatively modulated during drought. A null mutation in AtMYB60 results in the constitutive reduction of stomatal opening and in decreased wilting under water stress conditions. Transcript levels of a limited number of genes are altered in the mutant, and many of these genes are involved in the plant response to stress. Our data indicate that AtMYB60 is a transcriptional modulator of physiological responses in guard cells and open new possibilities to engineering stomatal activity to help plants survive desiccation.

Frequently, crop plants do not take up adequate amounts of iron from the soil, leading to chlorosis, poor yield and decreased nutritional quality. Extremely limited soil bioavailability of iron has led plants to evolve two distinct uptake strategies: chelation, which is used by the world's principal grain crops; and reduction, which is used by other plant groups. The chelation strategy involves extrusion of low-molecular-mass secondary amino acids (mugineic acids) known as 'phytosiderophores' which chelate sparingly soluble iron. The Fe(III)-phytosiderophore complex is then taken up by an unknown transporter at the root surface. The maize yellow stripe1 (ys1) mutant is deficient in Fe(III)-phytosiderophore uptake, therefore YS1 has been suggested to be the Fe(III)-phytosiderophore transporter. Here we show that ys1 is a membrane protein that mediates iron uptake. Expression of YS1 in a yeast iron uptake mutant restores growth specifically on Fe(III)-phytosiderophore media. Under iron-deficient conditions, ys1 messenger RNA levels increase in both roots and shoots. Cloning of ys1 is an important step in understanding iron uptake in grasses, and has implications for mechanisms controlling iron homeostasis in all plants.

In planta Agrobacterium-mediated transformation combined with a soil-based herbicide selection for transgenic plants was used to recover large numbers of transgenic Arabidopsis plants for functional genomic studies. A tissue-culture-free system for generating transgenic plants was achieved by infiltrating Arabidopsis plants with Agrobacterium tumefaciens harboring a binary T-DNA vector containing the phosphinothricin acetyltransferase gene from Streptomyces hygroscopicus, and by selecting transgenic Arabidopsis growing in soil by foliar application of the herbicide Finale (phosphinothricin). Analysis of herbicide-resistant plants indicated that all were transgenic and that the T-DNA transformation process occurred late during flower development, resulting in a preponderance of independently derived T-DNA insertions. T-DNA insertions were usually integrated in a concatenated, rearranged form, and using linkage analysis, we estimated that T1 plants carried between one and five T-DNA loci. Using pooling strategies, both DNA and seed pools were generated from about 38,000 Arabidopsis plants representing over 115,000 independent T-DNA insertions. We show the utility of these transgenic lines for identifying insertion mutations using gene sequence and PCR-based screening.

The formation of unisexual flowers in maize requires the selective elimination and sexual maturation of floral organs in an initially bisexual floral meristem. Elimination of pistil primordia occurs in the primary and secondary florets of the tassel spikelets, and in the secondary florets of ear spikelets. Ill-fated pistil cells undergo a cell death process associated with nuclear degeneration in a specific spatial-temporal pattern that begins in the subepidermis, eventually aborting the entire organ. The sex determination genes tasselseed1 and tasselseed2 are required for death of pistil cells. tasselseed1 is required for the accumulation of TASSELSEED2 mRNA in pistil cells. All pistil primordia express TASSELSEED2 RNA but functional pistils found in ear spikelets are protected from cell death by the action of the silkless1 gene. silkless1 blocks tasselseed-induced cell death in the pistil primordia of primary ear florets. A model is proposed for the control of pistil fate by the action of the ts1-ts2-sk1 pathway.

A novel derivative of the maize transposable element Ac, termed Ac-st2, that displays a positive dosage effect in maize has been identified. Although identical in sequence to other Ac elements, increasing the copy number of the element in the endosperm results in earlier and more frequent Ds excision. Ac-st2 autonomously transposes and catalyzes somatic excision of Ds elements. Germinal transpositions of either Ac-st2 or Ds, however, were not observed. The Ac-st2 phenotype includes a reduction in Ac transcript accumulation that is associated with increased methylation at specific sites in the promoter region of the major transcriptional start site within Ac (ORFa). This element differs from metastable (cycling) Ac derivatives in that Ac-st2 conditions a uniform transposition pattern throughout endosperm and plant development. Ac-st2 undergoes frequent increases in activity after its association with an active Ac element. This change in activity correlates with reduced levels of methylation in the ORFa promoter region. Using a competitive PCR assay, Ac transcript accumulation was followed through endosperm development. From these data, a model is proposed to explain the patterns of variegation associated with both "wild type" active Ac and Ac-st2 elements.

The duplicated R and Sn genes are involved in the regulation of the maize anthocyanin biosynthetic pathway, encoding tissue-specific products that are homologous to the helix-loop-helix transcriptional activators. Sn determines the pigmentation of the mesocotyl, leaf basis and pericarp, while R determines pigmentation in various tissues, but not in the mesocotyl. In the progeny derived from test-crosses of R/Sn heterozygous plants, a high frequency of R plants exhibiting mesocotyl pigmentation was observed; these derivatives were defined as R*. In R* plants, the presence of this novel trait was not accompanied by the acquisition of Sn or by gross DNA rearrangements in the R profile. Accordingly, RT-PCR analysis showed that mesocotyl pigmentation in R* was attributable to the resident R gene. The occurrence of R* was observed with all R alleles tested, and was enhanced when a P component was present. The heritability of R* was shown only in the case of the standard R-r allele, which carries a functional P component. In addition, we observed that R* can influence other R alleles, transferring the ability to pigment the mesocotyl. R* is unstable, showing a tendency to return to its original state after a few generations. In R* plants there was a correlation between observed ectopic pigmentation and an increase in the level of A1 transcript but, surprisingly, not in the accumulation of R transcript. The results obtained from the analysis of test crosses of rSn/r delta plants suggest that an unlinked genetic factor accounts for the ectopic pigmentation. Concomitant occurrence of epigenetic events might explain the observed instability and reversibility noted above. Further study of this phenomenon might help to elucidate the basis of the interaction between homologous and non-homologous regulators.

The r locus of maize regulates anthocyanin synthesis in various tissues of maize through the production of helix-loop-helix DNA binding proteins capable of inducing expression of structural genes in the anthocyanin biosynthetic pathway. The complex r variant, R-r: standard (R.r), undergoes frequent mutation through a variety of mechanisms including displaced synapsis and crossing over, and intrachromosomal recombination. Here we report a new mechanism for mutation at the R-r complex: insertion of a novel family of transposable elements. Because the elements were first identified in the R-p gene of the R-r complex, they have been named P instability Factor (PIF). Two different PIF elements were cloned and found to have identical sequences at their termini but divergent internal sequences. In addition, the PIF elements showed a marked specificity of insertion sites. Six out of seven PIF-containing derivatives examined had an element inserted at an identical location. Two different members of the PIF element family were identified at this position. The seventh PIF-containing derivative examined had the element inserted at a distinct position within r. Even at this location, however, the element inserted into a conserved target sequence. The timing of PIF excision is unusual. Germinal excision rates can range up to several percent of progeny. Yet somatic sectors are rare, even in lines exhibiting high germinal reversion rates.

Cultivated maize (Zea mays) and several other members of the Tribe Andropogoneae produce unisexual florets. In maize, the formation of two staminate florets in each spikelet on the tassel and a single pistillate floret in each spikelet on the ear includes a pistil abortion process that requires the action of the TASSELSEED2 gene. In Eastern gamagrass (Tripsacum dactyloides) the GYNOMONOECIOUS SEX FORM1 gene appears to perform a similar role in pistil abortion. These genes were shown to be homeologs by restriction fragment length polymorphism mapping and by the failure of the gsf1 and ts2 alleles to complement one another in intergeneric hybrids. Molecular analysis of the gsf1 allele shows that it is caused by a 1.4-kb deletion mutation. Both TASSELSEED2 and GYNOMONOECIOUS SEX FORM1 show similar expression patterns in subepidermal cells of pistils just before abortion. These results suggest that the formation of staminate florets in the Andropogoneae represents a monophyletic trait.

The maize ligule and auricle are structures on the maize leaf that develop at the boundary of the sheath and blade. In the absence of liguleless1 (Ig1) gene expression, ligule and auricle are not formed, and the blade-sheath boundary does not develop as an exact line between sheath and blade. By using the Activator (Ac) transposable element as a molecular tag, a novel Ig1 allele, Ig1-m1, was isolated and cloned. Analysis of somatic revertant sectors confirmed that the LG1 gene product functions in a cell-autonomous fashion. cDNA cloning as well as RT-PCR analysis of the LG1 mRNA indicate that the Ig1 gene is expressed at very low levels in the ligular region of developing maize leaf primordia, perhaps as early as plastochron 6 or earlier. Cellular localization studies in a heterologous system indicate that the LG1 product localizes exclusively to the nucleus. The predicted amino acid sequence of the LG1 protein is largely novel but contains an internal domain of 77 amino acids with significant similarity to a domain present in two recently identified SQUAMOSA PROMOTER-BINDING proteins 1 and 2 (SBP1 and SBP2) in Antirhinum majus.

The function of DNA methylation in higher plants was investigated by expression of a complementary DNA encoding a cytosine methyltransferase (MET1) from Arabidopsis thaliana as an antisense RNA in transgenic plants. This expression resulted in a 34 to 71 percent reduction in total genomic cytosine methylation. Loss of methylation was observed in both repetitive DNA and single-copy gene sequences. Developmental effects included altered heterochrony, changes in meristem identity and organ number, and female sterility. Cytosine demethylation prolonged both vegetative and reproductive phases of development. These findings implicate DNA methylation in establishing or maintaining epigenetic developmental states in the meristem.

Adjacent bundle sheath and mesophyll cells cooperate for carbon fixation in the leaves of C4 plants. Mutants with compromised plastid development should reveal the degree to which this cooperation is obligatory, because one can assay whether mesophyll cells with defective bundle sheath neighbors retain C4 characteristics or revert to C3 photosynthesis. The leaf permease1-mutable1 (lpe1-m1) mutant of maize exhibits disrupted chloroplast ultrastructure, preferentially affecting bundle sheath choroplasts under lower light. Despite the disrupted ultrastructure, the metabolic cooperation of bundle sheath and mesophyll cells for C4 photosynthesis remains intact. To investigate this novel mutation, the Activator transposon-tagged allele and cDNAs corresponding to the Lpe1 mRNA from wild-type plants were cloned. The Lpe1 gene encodes a polypeptide with significant similarity to microbial pyrimidine and purine transport proteins. An analysis of revertant sectors generated by Activator excision suggests that the Lpe1 gene product is cell autonomous and can be absent up to the last cell divisions in the leaf primordium without blocking bundle sheath chloroplast development.

R-r controls the production of anthocyanin pigment in plant parts and the aleurone layer of seeds through the production of a family of related transcriptional activating proteins of the helix-loop-helix type. The R-r complex comprises a series of repeated, homologous components arranged in both direct and inverted orientations. These include the P component, a simple R gene that confers pigmentation of plant parts, and the S subcomplex that consists of a truncated inactive R gene called q, and two functional R genes, S1 and S2, that pigment the aleurone. The S genes are arranged in an unusual inverted head-to-head orientation. The identity of each functional component was confirmed by microprojectile bombardment of intact maize tissues with cloned genomic DNA and by analysis of in vivo mRNA populations. Sequence analysis suggests that the S subcomplex was derived through the rearrangement of a simple P-like progenitor element. At the rearrangement breakpoints, features typical of the CACTA family of transposable elements were found. The location and arrangement of these CACTA element sequences implies that this element may have mediated the chromosomal rearrangements that led to the formation of the R-r complex. The unusual structure of R-r explains much of the meiotic instability of the complex.

Maize partitions the sexes into different flowers on the plant, a condition called monoecy, which facilitates outcrossing. Sex determination in maize is a complex process involving an interplay between genetic determinants, the environment, and hormones. Unisexuality of flowers is achieved by the process of selective arrest and abortion of the inappropriate organ primordia within a bisexual floral meristem. Floral organ abortion is associated with the degeneration of cells within an immature primordia. Masculinizing genes are required for gynoecial abortion, feminizing genes arrest stamen development, and both types also control secondary sexual traits involving morphological characteristics of floral tissues. Gibberellins, steroid-like plant hormones, appear to play a pivotal role in the stamen abortion process and the feminization of floral tissues.

Several metastable Ac alleles at the maize p locus were identified that produced novel pericarp variegation patterns. From the transmission analysis of pericarp sectors, we show that Ac inactivation is a somatic process. Ac excision from P and transactivation of an unlinked Ds was delayed or absent in plants with metastable Ac alleles. These decreases in Ac activity were correlated to increases in cytosine methylation at specific sites near the start of Ac transcription (open reading frame a; ORFa) and at sites in some flanking P sequence. Reactivation of inactive alleles was accompanied by decreased methylation of Ac and P sequences. Using a competitive polymerase chain reaction assay, steady state levels of ORFa transcript were quantitatively compared among the various metastable alleles. We propose that changes in the methylation profile of Ac correspond to changes in Ac activity through the differential accumulation of Ac transcript.

In many ways, plants offer unique systems through which to study sex determination. Because the production of unisexual flowers has evolved independently in many plant species, different and novel mechanisms may be operational. Hence, there is probably not one unifying mechanism that explains sex determination in plants. Advances in our understanding of sex determination will come from the analysis of the genetics, molecular biology, and biochemistry of genes controlling sexual determination in plants. Several excellent model systems for bisexual floral development (Arabidopsis and Antirrhinum), monoecy (maize), and dioecy (Silene, asparagus, and mercury) are available for such analyses. The important questions that remain concern the mechanism of action of sex determination genes and their interrelationship, if any, with homeotic genes that determine the sexual identity of floral organ primordia. At the physiological level, the connection between hormone signaling and sexuality is not well understood, although significant correlations have been discovered. Finally, once the genes that regulate these processes are identified, cloned, and studied, new strategies for the manipulation of sexuality in plants should be forthcoming.

Maize produces separate unisexual flowers through programmed abortion of preformed organ primordia. In the male inflorescence (tassel), stamen primordia develop to sexual maturity, while gynoecia (pistil primordia) are aborted. In tasselseed2 (ts2) mutant plants, floral structures in the tassel adopt a female developmental program. Here we report the transposon tagging and cloning of the TS2 gene, which plays a late but pivotal role in determining the sexual fate of floral meristems. Shortly before abortion of the gynoecium, Ts2 mRNA is expressed subepidermally in that primordium. Phenotypic instability of the Activator (Ac)-induced allele ts2-m1 indicates that late restoration of TS2 action in somatic tissues, which is correlated with Ac excision, reactivates the male developmental program. The predicted amino acid sequence of the Ts2 protein shows significant similarity to short-chain alcohol dehydrogenases, particularly hydroxysteroid dehydrogenases.

We used Random Amplified Polymorphic DNA (RAPD) fingerprinting to address issues of paternity in two odonate species. Amplification artifacts of RAPD markers were controlled by assessing paternity patterns relative to the banding patterns generated by quantitative mixtures of DNA from putative parents ('synthetic offspring'). In the aeshnid dragonfly Anax parthenope, for which the mating histories of both males and females were unknown, we found strong evidence for complete paternity success for the contact guarding male. In the highly polygamous libellulid dragonfly Orthetrum coerulescens, we detected and quantified mixed paternity in sequentially produced offspring clutches and demonstrated that fertilization success is correlated with the duration of copulation. Our results suggest that RAPD fingerprinting is suitable to address issues of paternity in systems which are genetically uncharacterized and produce large offspring clutches.