Alan E. Pepper

CMD: a Cotton Microsatellite Database resource for Gossypium genomics

BackgroundThe Cotton Microsatellite Database (CMD) http://www.cottonssr.org is a curated and integrated web-based relational database providing centralized access to publicly available cotton microsatellites, an invaluable resource for basic and applied research in cotton breeding.DescriptionAt present CMD contains publication, sequence, primer, mapping and homology data for nine major cotton microsatellite projects, collectively representing 5,484 microsatellites. In addition, CMD displays data for three of the microsatellite projects that have been screened against a panel of core germplasm. The standardized panel consists of 12 diverse genotypes including genetic standards, mapping parents, BAC donors, subgenome representatives, unique breeding lines, exotic introgression sources, and contemporary Upland cottons with significant acreage. A suite of online microsatellite data mining tools are accessible at CMD. These include an SSR server which identifies microsatellites, primers, open reading frames, and GC-content of uploaded sequences; BLAST and FASTA servers providing sequence similarity searches against the existing cotton SSR sequences and primers, a CAP3 server to assemble EST sequences into longer transcripts prior to mining for SSRs, and CMap, a viewer for comparing cotton SSR maps.ConclusionThe collection of publicly available cotton SSR markers in a centralized, readily accessible and curated web-enabled database provides a more efficient utilization of microsatellite resources and will help accelerate basic and applied research in molecular breeding and genetic mapping in Gossypium spp.

Geographic patterns of microsatellite variation in Boechera stricta, a close relative of Arabidopsis

The genus Boechera is a widespread North American group with great potential for studies of ecology and evolution: Boechera is closely related to Arabidopsis and exhibits different ecological and reproductive strategies. Boechera stricta (previously Arabis drummondii) is a morphologically and genetically well‐defined, perennial crucifer species. Fifteen natural populations of diploid individuals from the Rocky Mountains were analysed using 21 microsatellite loci. In accordance with our expectation for this predominately inbreeding species, a high FIS value (0.89) was observed. Furthermore, populations of B. stricta were highly differentiated, as indicated by FST = 0.56. Three clusters were identified using structure— the majority of populations belonged to either the Northern or Southern cluster. Together, the north–south partitioning and evenness of genetic variation across the two clusters suggested multiple refugia for this perennial herb in the Rocky Mountains. Pleistocene glaciation, together with the topographically and climatologically heterogeneous cordillera, has profoundly influenced the genetic architecture of B. stricta. Genetic population structure was also influenced by relatively recent genome admixture at two levels: within species (involving individuals from the Northern and Southern clusters) and between species (with the hybridization of B. stricta and Boechera holboellii). This complexity of population structure at presumably neutral microsatellite loci located throughout the genome in B. stricta provides a baseline against which to test whether functional genetic variation is undergoing local adaptive evolution throughout the natural species range.

A High-Density Simple Sequence Repeat and Single Nucleotide Polymorphism Genetic Map of the Tetraploid Cotton Genome

Genetic linkage maps play fundamental roles in understanding genome structure, explaining genome formation events during evolution, and discovering the genetic bases of important traits. A high-density cotton (Gossypium spp.) genetic map was developed using representative sets of simple sequence repeat (SSR) and the first public set of single nucleotide polymorphism (SNP) markers to genotype 186 recombinant inbred lines (RILs) derived from an interspecific cross between Gossypium hirsutum L. (TM-1) and G. barbadense L. (3-79). The genetic map comprised 2072 loci (1825 SSRs and 247 SNPs) and covered 3380 centiMorgan (cM) of the cotton genome (AD) with an average marker interval of 1.63 cM. The allotetraploid cotton genome produced equivalent recombination frequencies in its two subgenomes (At and Dt). Of the 2072 loci, 1138 (54.9%) were mapped to 13 At-subgenome chromosomes, covering 1726.8 cM (51.1%), and 934 (45.1%) mapped to 13 Dt-subgenome chromosomes, covering 1653.1 cM (48.9%). The genetically smallest homeologous chromosome pair was Chr. 04 (A04) and 22 (D04), and the largest was Chr. 05 (A05) and 19 (D05). Duplicate loci between and within homeologous chromosomes were identified that facilitate investigations of chromosome translocations. The map augments evidence of reciprocal rearrangement between ancestral forms of Chr. 02 and 03 versus segmental homeologs 14 and 17 as centromeric regions show homeologous between Chr. 02 (A02) and 17 (D02), as well as between Chr. 03 (A03) and 14 (D03). This research represents an important foundation for studies on polyploid cottons, including germplasm characterization, gene discovery, and genome sequence assembly.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

Latitudinal variation in cold hardiness in introduced Tamarix and native Populus

To investigate the evolution of clinal variation in an invasive plant, we compared cold hardiness in the introduced saltcedar (Tamarix ramosissima, Tamarix chinensis, and hybrids) and the native plains cottonwood (Populus deltoides subsp. monilifera). In a shadehouse in Colorado (41°N), we grew plants collected along a latitudinal gradient in the central United States (29–48°N). On 17 occasions between September 2005 and June 2006, we determined killing temperatures using freeze‐induced electrolyte leakage and direct observation. In midwinter, cottonwood survived cooling to −70°C, while saltcedar was killed at −33 to −47°C. Frost sensitivity, therefore, may limit northward expansion of saltcedar in North America. Both species demonstrated inherited latitudinal variation in cold hardiness. For example, from September through January killing temperatures for saltcedar from 29.18°N were 5–21°C higher than those for saltcedar from 47.60°N, and on September 26 and October 11, killing temperatures for cottonwood from 33.06°N were >43°C higher than those for cottonwood from 47.60°N. Analysis of nine microsatellite loci showed that southern saltcedars are more closely related to T. chinensis while northern plants are more closely related to T. ramosissima. Hybridization may have introduced the genetic variability necessary for rapid evolution of the cline in saltcedar cold hardiness.

The Arabidopsis thaliana carboxyl-terminal domain phosphatase-like 2 regulates plant growth, stress and auxin responses

More than 20 genes in the Arabidopsis genome encode proteins similar to phosphatases that act on the carboxyl-terminal domain (CTD) of RNA polymerase II. One of these CTD-phosphatase-like (CPL) proteins, CPL2, dephosphorylates CTD-Ser5-PO4 in an intact RNA polymerase II complex and contains a double-stranded (ds)-RNA-binding motif (DRM). Although the dsRNA-binding activity of CPL2 DRM has not been shown to date, T-DNA insertion mutants that express CPL2 variants lacking either a part of DRM (cpl2-1) or the entire DRM (cpl2-2) exhibited leaf expansion defects, early flowering, low fertility, and increased salt sensitivity. cpl2 mutant plants produced shorter hypocotyls than wild-type plants in the light, but were indistinguishable from wild type in the dark. CPL2 was expressed in shoot and root meristems and vasculatures, expanding rosette leaves, and floral organs suggesting a focal role for growth. Microarray and RT-PCR analyses revealed that basal levels of several auxin-responsive transcripts were reduced in cpl2. On the other hand, the levels of endogenous auxin and its conjugates were similar in wild type and cpl2. Overexpression of ARF5 but not all activator ARF transcription factors restored the auxin-responsive DR5-GUS reporter gene expression and the leaf expansion of cpl2 mutant plants but not early flowering phenotype. These results establish CPL2 as a multifunctional regulator that modulates plant growth, stress, and auxin responses.

Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L

Simultaneous improvement of fibre quality, early-flowering, early-maturity and productivity in Upland cotton (G. hirsutum) is a challenging task for conventional breeding. The influence of red/far-red light ratio on the fibre length prompted us to examine the phenotypic effects of RNA interference (RNAi) of the cotton PHYA1 gene. Here we show a suppression of up to ~70% for the PHYA1 transcript, and compensatory overexpression of up to ~20-fold in the remaining phytochromes in somatically regenerated PHYA1 RNAi cotton plants. Two independent transformants of three generations exhibited vigorous root and vegetative growth, early-flowering, significantly improved upper half mean fibre length and an improvement in other major fibre characteristics. Small decreases in lint traits were observed but seed cotton yield was increased an average 10-17% compared with controls. RNAi-associated phenotypes were heritable and transferable via sexual hybridization. These results should aid in the development of early-maturing and productive Upland cultivars with superior fibre quality.

Microsatellites Uncover Multiple Introductions of Clonal Giant Reed (Arundo donax)

Abstract Giant reed (Arundo donax) is an invasive weed that is native to the Old World. Tens of thousands of hectares of riparian habitat in the Rio Grande Basin (RGB) in Texas and Mexico have been heavily affected by invasions of Arundo. Additionally, many other watersheds across the southwestern United States have also been affected. Giant reed is being targeted for biological control because it displaces native vegetation and consumes water that could potentially be used for agricultural and municipal purposes, especially in areas with limited access to water. Finding the best-adapted insects for biological control involves locating the origin(s) of this plant. To narrow down the proximal source(s) of invasion of giant reed in the RGB, 10 microsatellite markers were developed. An analysis of 203 Old World and 159 North American plants, with an emphasis on the RGB, indicated a reduction in the allelic diversity in the introduced range compared with the Old World. Clonal assignment, neighbor joining, principal coordinates analyses, and STRUCTURE analyses were consistent and implied multiple introductions in North America, with one (likely clonal) lineage responsible for the invasion of the RGB, northern Mexico, and other parts of the southwestern United States. Although no identical matches with the RGB lineage were found in the Old World, several close matches were found on the Mediterranean coast of Spain. Nomenclature: Giant reed, Arundo donax L Management Implications: Giant reed is a clonal, rhizomatous grass that has invaded tens of thousands of hectares of riparian habitat throughout the Rio Grande Basin (RGB) and other parts of the southwestern United States. In this paper, we used microsatellites to determine the original population source(s) of the invasive Arundo donax in the RGB to locate biocontrol agents from the Old World. Biological control is deemed the best long-term option for control of giant reed. Chemical and mechanical control of A. donax is expensive, especially in heavily affected areas. Although A. donax is clonal, some genetic variation was found throughout the RGB. We also discovered multiple introductions in the United States, but only one lineage is responsible for the invasion in the RGB. This indicates that a limited sampling of biocontrol insects might be effective in controlling A. donax along the Rio Grande. Additionally, these biocontrol agents might also be effective in controlling giant reed in others areas where this lineage has been introduced, such as California and Mexico.

De novo assembly and characterization of the transcriptome of the toxic dinoflagellate Karenia brevis

BackgroundKarenia brevis is a harmful algal species that blooms in the Gulf of Mexico and produces brevetoxins that cause neurotoxic shellfish poisoning. Elevated brevetoxin levels in K. brevis cells have been measured during laboratory hypo-osmotic stress treatments. To investigate mechanisms underlying K. brevis osmoacclimation and osmoregulation and establish a valuable resource for gene discovery, we assembled reference transcriptomes for three clones: Wilson-CCFWC268, SP3, and SP1 (a low-toxin producing variant). K. brevis transcriptomes were annotated with gene ontology terms and searched for putative transmembrane proteins that may elucidate cellular responses to hypo-osmotic stress. An analysis of single nucleotide polymorphisms among clones was used to characterize genetic divergence.ResultsK. brevis reference transcriptomes were assembled with 58.5 (Wilson), 78.0 (SP1), and 51.4 million (SP3) paired reads. Transcriptomes contained 86,580 (Wilson), 93,668 (SP1), and 84,309 (SP3) predicted transcripts. Approximately 40% of the transcripts were homologous to proteins in the BLAST nr database with an E value ≤ 1.00E-6. Greater than 80% of the highly conserved CEGMA core eukaryotic genes were identified in each transcriptome, which supports assembly completeness. Seven putative voltage-gated Na+ or Ca2+ channels, two aquaporin-like proteins, and twelve putative VATPase subunits were discovered in all clones using multiple bioinformatics approaches. Furthermore, 45% (Wilson) and 43% (SP1 and SP3) of the K. brevis putative peptides > 100 amino acids long produced significant hits to a sequence in the NCBI nr protein database. Of these, 77% (Wilson and SP1) and 73% (SP3) were successfully assigned gene ontology functional terms. The predicted single nucleotide polymorphism (SNP) frequencies between clones were 0.0028 (Wilson to SP1), 0.0030 (Wilson to SP3), and 0.0028 (SP1 to SP3).ConclusionsThe K. brevis transcriptomes assembled here provide a foundational resource for gene discovery and future RNA-seq experiments. The identification of ion channels, VATPases, and aquaporins in all three transcriptomes indicates that K. brevis regulates cellular ion and water concentrations via transmembrane proteins. Additionally, > 40,000 unannotated loci may include potentially novel K. brevis genes. Ultimately, the SNPs identified among the three ecologically diverse clones with different toxin profiles may help to elucidate variations in K. brevis brevetoxin production.

Genetic analyses of nickel tolerance in a North American serpentine endemic plant, Caulanthus amplexicaulis var. barbarae (Brassicaceae)

PREMISE OF THE STUDY The evolution of metal tolerance in plants is an important model for studies of adaptation to environment, population genetics, and speciation. Here, we investigated nickel tolerance in the North American serpentine endemic Caulanthus amplexicaulis var. barbarae in comparison with its nonserpentine sister taxon C. amplexicaulis var. amplexicaulis. We hypothesized that the serpentine endemic would have a heritable growth advantage on nickel-containing substrates. METHODS We employed an artificial growth assay to quantify biomass accumulation. Study plants were crossed to create an F(2:3) population that was used to determine the heritability of nickel tolerance and to map quantitative trait loci (QTL). Nickel accumulation in both laboratory populations and native specimens was examined using energy-dispersive x-ray fluorescence (EDXRF). KEY RESULTS The serpentine endemic had a dramatic growth advantage at concentrations of nickel >30 µmol/L. Caulanthus amplexicaulis var. barbarae and its nonserpentine sister taxon both accumulated nickel to substantial levels. Nickel tolerance was highly heritable (h(2) = 0.59) and not associated with accumulation. The QTL analyses identified two major loci for nickel tolerance, on linkage group 2 (LG2) and linkage group 9 (LG9). CONCLUSIONS In our study, nickel tolerance was determined by two major loci with large effects. At both loci, alleles from the serpentine parent conferred positive effects on nickel tolerance, suggesting that they are adaptive in the natural serpentine environment. The mechanism of nickel tolerance in the serpentine plant was not exclusion of nickel. Nickel tolerance may have an inducible component in C. amplexicaulis var. barbarae.