Improving phylogenetic resolution of the Lamiales using the complete plastome sequences of six Penstemon species.

The North American endemic genus Penstemon (Mitchell) has a recent geologic origin of ca. 3.6 million years ago (MYA) during the Pliocene/Pleistocene transition and has undergone a rapid adaptive evolutionary radiation with ca. 285 species of perennial forbs and sub-shrubs. Penstemon is divided into six subgenera occupying all North American habitats including the Arctic tundra, Central American tropical forests, alpine meadows, arid deserts, and temperate grasslands. Due to the rapid rate of diversification and speciation, previous phylogenetic studies using individual and concatenated chloroplast sequences have failed to resolve many polytomic clades. We investigated the efficacy of utilizing the plastid genomes (plastomes) of 29 species in the Lamiales order, including five newly sequenced Penstemon plastomes, for analyzing phylogenetic relationships and resolving problematic clades. We compared whole-plastome based phylogenies to phylogenies based on individual gene sequences (matK, ndhF, psaA, psbA, rbcL, rpoC2, and rps2) and concatenated sequences. We also We found that our whole-plastome based phylogeny had higher nodal support than all other phylogenies, which suggests that it provides greater accuracy in describing the hierarchal relationships among taxa as compared to other methods. We found that the genus Penstemon forms a monophyletic clade sister to, but separate from, the Old World taxa of the Plantaginaceae family included in our study. Our whole-plastome based phylogeny also supports the rearrangement of the Scrophulariaceae family and improves resolution of major clades and genera of the Lamiales.

The Complete Plastome Sequence Of Penstemon fruticosus (Pursh) Greene (Plantaginaceae).

The genus Penstemon is an emerging model for the study of continental adaptive radiation. We report here the first complete plastome sequence for this genus. The P. fruticosus (shrubby, or brush penstemon) plastome is 152,704 bp in length with a quadripartite structure consisting of a large single-copy region (83,693 bp) and a small single-copy region (17,820 bp) that are separated by two inverted repeats (25,594 bp). The plastome contained 24 tRNA genes, 8 rRNA genes, and 83 protein-coding genes for a total of 115 unique genes. Phylogenetic analysis of whole chloroplast sequences shows that the nearest relatives of P. fruticosus are the Plantago and Veronica genera in the Plantaginaceae family.

Molecular and morphological evidence for Penstemon luculentus (Plantaginaceae): a replacement name for Penstemon fremontii var. glabrescens.

Penstemon luculentus R.L.Johnson & M.R.Stevens, nom. nov. replaces Penstemon fremontii var. glabrescens Dorn & Lichvar. The varietal name glabrescens was not elevated because it was already occupied by Penstemon glabrescens Pennell, a different species. This new arrangement is supported by molecular and morphological evidence. An analysis of genetic diversity in populations of both varieties of Penstemon fremontii Torr. & A. Gray (glabrescens and fremontii) from the Piceance Basin, Colorado, using SSR (simple sequences repeats) or microsatellites markers, revealed significant genetic differentiation between the two. Penstemon fremontii var. glabrescens was also genetically different from Penstemon gibbensii Dorn and Penstemon scariosus var. garrettii (Pennell) N.H. Holmgren. The combination of hirtellous stems, glabrous leaves, non-glandular inflorescence, and long anther hairs distinguish Penstemon luculentus from other morphologically similar species.

Identification and characterization of microsatellite markers in Penstemon scariosus (Plantaginaceae).

PREMISE OF THE STUDY: Penstemon scariosus var. albifluvis (Plantaginaceae) has been proposed to be federally listed as threatened due to its unique, geologically oil-rich habitat. Developing simple sequence repeat (SSR) markers to study its genetic diversity would be most useful. METHODS AND RESULTS: Using genomic reduction in combination with next-generation sequencing, we identified SSR motifs with five to 15 perfect repeats in 1067 P. scariosus contigs. After multiple qualifying tests, 16 SSRs were selected for their robust polymorphic reliability across 12 taxa with as high as 21 alleles in a given taxon. With the exception of two monomorphic loci, the observed and expected heterozygosity values ranged from 0.083 to 1.000 and 0.398 to 0.920, respectively. CONCLUSIONS: These microsatellite markers will directly aid in studies of the genetic diversity and relatedness of P. scariosus, P. comarrhenus, P. compactus, P. cyananthus var. cyananthus, P. fremontii var. fremontii, P. fremontii var. glabrescens, P. gibbensii, P. strictus, and P. subglaber.

Developing molecular tools and insights into the Penstemon genome using genomic reduction and next-generation sequencing.

BACKGROUND: Penstemon's unique phenotypic diversity, hardiness, and drought-tolerance give it great potential for the xeric landscaping industry. Molecular markers will accelerate the breeding and domestication of drought tolerant Penstemon cultivars by, creating genetic maps, and clarifying of phylogenetic relationships. Our objectives were to identify and validate interspecific molecular markers from four diverse Penstemon species in order to gain specific insights into the Penstemon genome. RESULTS: We used a 454 pyrosequencing and GR-RSC (genome reduction using restriction site conservation) to identify homologous loci across four Penstemon species (P. cyananthus, P. davidsonii, P. dissectus, and P. fruticosus) representing three diverse subgenera with considerable genome size variation. From these genomic data, we identified 133 unique interspecific markers containing SSRs and INDELs of which 51 produced viable PCR-based markers. These markers produced simple banding patterns in 90% of the species × marker interactions (~84% were polymorphic). Twelve of the markers were tested across 93, mostly xeric, Penstemon taxa (72 species), of which ~98% produced reproducible marker data. Additionally, we identified an average of one SNP per 2,890 bp per species and one per 97 bp between any two apparent homologous sequences from the four source species. We selected 192 homologous sequences, meeting stringent parameters, to create SNP markers. Of these, 75 demonstrated repeatable polymorphic marker functionality across the four sequence source species. Finally, sequence analysis indicated that repetitive elements were approximately 70% more prevalent in the P. cyananthus genome, the largest genome in the study, than in the smallest genome surveyed (P. dissectus). CONCLUSIONS: We demonstrated the utility of GR-RSC to identify homologous loci across related Penstemon taxa. Though PCR primer regions were conserved across a broadly sampled survey of Penstemon species (93 taxa), DNA sequence within these amplicons (12 SSR/INDEL markers) was highly diverse. With the continued decline in next-generation sequencing costs, it will soon be feasible to use genomic reduction techniques to simultaneously sequence thousands of homologous loci across dozens of Penstemon species. Such efforts will greatly facilitate our understanding of the phylogenetic structure within this important drought tolerant genus. In the interim, this study identified thousands of SNPs and over 50 SSRs/INDELs which should provide a foundation for future Penstemon phylogenetic studies and breeding efforts.

Developmental contributions to phenotypic variation in functional leaf traits within quaking aspen clones.

Phenotypic variation in plant traits is strongly influenced by genetic and environmental factors. Over the life span of trees, developmental factors may also strongly influence leaf phenotypes. The objective of this study was to fill gaps in our understanding of developmental influences on patterns of phenotypic trait variation among different-aged ramets within quaking aspen (Populus tremuloides Michx.) clones. We hypothesized that phenotypic variation in leaf functional traits is strongly influenced by developmental cues as trees age. We surveyed eight aspen clones, each with eight distinct age classes ranging from 1 to 160 years in age, and selected three ramets per age class for sample collection. Leaf traits measured included photosynthesis, stomatal conductance, water use efficiency, specific leaf area, and concentrations of N, phosphorus, sucrose, starch, condensed tannins and phenolic glycosides. Using regression analysis, we examined the relationships between ramet age and expression of leaf functional traits. The data showed significant correlations between ramet age and 10 of the 12 phenotypic traits measured. Eight of the phenotypic traits demonstrated a non-linear relationship in which large changes in phenotype occurred in the early stages of ramet development and stabilized thereafter. Water relations, nutrient concentration, leaf gas exchange and phenolic glycosides tended to decrease from early to late development, whereas sucrose, condensed tannin concentrations and water use efficiency increased with ramet age. We hypothesize that ontogenetically derived phenotypic variation leads to fitness differentials among different-aged ramets, which may have important implications for clone fitness. Age-related increases in phenotypic diversity may partially underlie aspen's ability to tolerate the large environmental gradients that span its broad geographical range.

A survey of Penstemon's genome size.

Penstemon is the largest genus in North America with more than 270 reported species. However, little is known about its genome size. This information may be useful in developing hybrids for landscape use and for gaining insight into its current taxonomy. Using flow cytometry, we estimated the genome size of approximately 40% of the genus (115 accessions from 105 different species). Genome sizes for both reported and probable diploids range from P. dissectus 2C = 0.94 pg (1C = 462 Mbp) to P. pachyphyllus var. mucronatus 2C = 1.88 pg (1C = 919 Mbp), and the polyploids range from P. attenuatus var. attenuatus 2C = 2.35 pg (1C = 1148 Mbp) to P. digitalis 2C = 6.45 pg (1C = 3152 Mbp). Chromosome counts were done for ten previously published and four previously unreported Penstemon species (P. dissectus, P. navajoa, P. caespitosus var. desertipicti, and P. ramaleyi). These counts were compiled with all previously published chromosome data and compared with the flow cytometry results. Ploidy within this study ranged from diploid to dodecaploid. These data were compared and contrasted with the current taxonomy of Penstemon and previously published internal transcribed spacer and chloroplast DNA phylogenetic work. Based on genome size and previous studies, reassigning P. montanus to the subgenus Penstemon and P. personatus to the subgenus Dasanthera, would better reflect the phylogeny of the genus. Furthermore, our data concur with previous studies suggesting that the subgenus Habroanthus be included in the subgenus Penstemon. The DNA content of subgenus Penstemon exhibits high plasticity and spans a sixfold increase from the smallest to the largest genome (P. linarioides subsp. sileri and P. digitalis, respectively). Our study found flow cytometry to be useful in species identification and verification.

Screening Two Lycopersicon peruvianum Collections for Resistance to Tomato spotted wilt virus.

The United States Department of Agriculture (USDA) Research Service and the Tomato Genetics Resource Center (TGRC) Lycopersicon peruvianum germplasm collections (16,335 plants from 285 accessions) were screened with the Tomato spotted wilt virus (TSWV) isolates TSWV6 from Hawaii, and Anwa-1 from Western Australia. Using TSWV6 to screen for resistance, 10,634 L. peruvianum plants from 280 accessions were screened for resistance, resulting in 168 (60%) accessions with 1,437 (14%) plants indicating resistance, with all 1,404 89S (Sw-5+/Sw-5+) and 1,456 89R (Sw-5/Sw-5) controls infected. When using Anwa-1 for screening, 864 (15%) of 5,701 L. peruvianum plants were uninfected from 106 of the 181 accessions tested, and 472 (95%) of the 495 89S and 421 (73%) of the 574 89R controls were infected. Of the 172 accessions tested with both isolates, 54 were resistant to one isolate but not the other. Additionally, more accessions from the USDA than from the TGRC collection indicated resistance. TSWV-resistant accessions were somewhat equally distributed throughout the L. peruvianum geographic range, with an observation that northern Chile and southern Peru seemed to have an unusually high portion of accession indicating resistance. The value of Sw-5 is discussed in relationship to potential additional sources of TSWV resistance.

Correlation between molecular markers and adaptively significant genetic variation in Bromus tectorum (Poaceae), an inbreedingannual grass.

Single sequence repeat (SSR) and amplified fragment length polymorphic (AFLP) molecular marker genotypes in cheatgrass (Bromus tectorum) were compared to published data on phenotypic variation in seed dormancy, vernalization requirement, and resistance to the pathogen Ustilago bullata. Several features of cheatgrass facilitated this study: it is a recent invader in the western United States, has considerable phenotypic polymorphism, and is an obligate self-pollinator. Forty self-pollinating lines from four populations common to the three phenotypic data sets were analyzed for molecular genetic variation using seven SSR loci and 31 AFLP loci. We examined correlations between distance matrices using the Mantel test for each pair of studies. The two molecular data sets were significantly correlated (r = 0.636). The AFLP markers often distinguished among several lines with identical SSR genotypes. The AFLP data were also significantly correlated with the phenotypic data (r values from 0.4640 to 0.5658), but the SSR data were much more highly correlated (r values from 0.677 to 0.844). The difference between molecular marker systems was especially notable when an outlier population from Potosi Pass, Nevada, was excluded from the analysis. These results suggest that SSR markers may be good surrogates for phenotypic traits in population genetic studies of strongly inbreeding species such as cheatgrass.