Phenotypic differences among and within extant populations of Chrysanthemum arcticum L. and C. a. subsp. arcticum.

BACKGROUND: Chrysanthemum arcticum, arctic daisy and its two subspecies (Chrysanthemum arcticum subsp. arcticum, Chrysanthemum arcticum subsp. polaré) are the only chrysanthemum species native to North America. A study on species' variation in morphological and diagnostic traits is important to link morphological traits with previously described single nucleotide polymorphism (SNP) markers, particularly when the genomes are sequenced. The purpose of this study was to establish phenotypic differences and soil conditions among wild C. arcticum and C. a. subsp. arcticum populations, when grown in a uniform environment for two years, for potential linkages with our SNP library. Sixteen quantitative morphological traits and five qualitative morphological traits were investigated for 255 individuals from nine C. arcticum populations and 326 individuals from 21 C. a. subsp. arcticum populations. RESULTS: In long-day controlled environment, C. arcticum flowering rate was 0% in Year 1, increased to 2.7% in Year 2, while C. a. subsp. arcticum flowering rate was 98.5% in Year 2. Two distinct clusters, distributed by taxonomic classification, were detected by Principal component analysis (PCoA) for 551 individuals from C. arcticum and C. a. subsp. arcticum. Pearson's correlation coefficient analysis indicated a positive and significant correlation between plant height, flower fresh and dry weights. Flower fresh weights were correlated with Δflower weight, while inflorescence length had showed a negative correlation with leaf number. Soil samples had high Na levels along with heavy metals. Thus, the species are salt-tolerant. CONCLUSION: A high level of salt tolerance (Na) is tolerated by these maritime species which is a unique trait in Chrysanthemum. A new diagnostic trait of inflorescence length was discovered to distinguish among C. arcticum and C. a. subsp. arcticum. Significant flowering differences occurred among the species C. arcticum and C. a. subsp. arcticum under same photoperiodic environment, including flowering rates and visible bud date. This study on the species' variation in morphological and diagnostic traits is of importance to link morphological traits with single nucleotide polymorphism (SNP) markers.

Variability in ITS1 and ITS2 sequences of historic herbaria and extant (fresh) Phalaris species (Poaceae).

BACKGROUND: Phalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica. Phalaris arundinacea is an important forage, ornamental, wetland restoration and biofuel crop grown globally as well as being a wetland invasive. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for Phalaris barcoding as a DNA region with high nucleotide diversity for Phalaris species identification. Recent findings that P. arundinacea populations in Minnesota USA are most likely native and not European prompted this analysis to determine whether Eurasian vs. native North American P. arundinacea differed in ITS regions. Our objectives were to amplify and compare ITS regions (ITS1 and ITS2) of historic herbaria (1882-2001) and extant (fresh) Phalaris specimens; analyze ITS regions for species-specific polymorphisms (diagnostic SNPs) and compare ITS regions of historic Phalaris specimens with known, extant Phalaris species. RESULTS: We obtained complete ITS1 and ITS2 sequences from 31 Phalaris historic (herbaria samples, 1908 to 2001) and five extant (fresh) specimens. Herbaria Phalaris specimens did not produce new SNPs (single nucleotide polymorphisms) not present in extant specimens. Diagnostic SNPs were identified in 8/12 (66.6%) Phalaris species. This study demonstrates the use of herbaria tissue for barcoding as a means for improved species identification of Phalaris herbaria specimens. No significant correlation between specimen age and genomic DNA concentration was found. Phalaris arundinacea showed high SNP variation within its clade, with the North American being distinctly different than other USA and most Eurasian types, potentially allowing for future identification of specific SNPs to geographic origin. CONCLUSIONS: While not as efficient as extant specimens to obtain DNA, Phalaris herbaria specimens can produce high quality ITS sequences to evaluate historic genetic resources and facilitate identification of new species-specific barcodes. No correlation between DNA concentration and age of historic samples (119 year range) occurred. Considerable polymorphism was exhibited in the P. arundinacea clade with several N. American accessions being distinct from Eurasian types. Further development of within species- and genus-specific barcodes could contribute to designing PCR primers for efficient and accurate identification of N. American P. arundinacea. Our finding of misidentified Phalaris species indicates the need to exercise stringent quality control measures on newly generated sequence data and to approach public sequence databases in a critical way.

PELPIII: the class III pistil-specific extensin-like Nicotiana tabacum proteins are essential for interspecific incompatibility.

Pre-zygotic interspecific incompatibility (II) involves an active inhibition mechanism between the pollen of one species and the pistil of another. As a barrier to fertilization, II effectively prevents hybridization and maintains species identity. Transgenic ablation of the mature transmitting tract (TT) in Nicotiana tabacum resulted in the loss of inhibition of pollen tube growth in Nicotiana obtusifolia (synonym Nicotiana trigonophylla) and Nicotiana repanda. The role of the TT in the II interaction between N. tabacum and N. obtusifolia was characterized by evaluating N. obtusifolia pollen tube growth in normal and TT-ablated N. tabacum styles at various post-pollination times and developmental stages. The II activity of the TT slowed and then arrested N. obtusifolia pollen tube growth, and was developmentally synchronized. We hypothesize that proteins produced by the mature TT and secreted into the extracellular matrix inhibit interspecific pollen tubes. When extracts from the mature TT of N. tabacum were injected into the TT-ablated style prior to pollination, the growth of incompatible pollen tubes of N. obtusifolia and N. repanda was inhibited. The class III pistil-specific extensin-like protein (PELPIII) was consistently associated with specific inhibition of pollen tubes, and its requirement for II was confirmed through use of plants with antisense suppression of PELPIII. Inhibition of N. obtusifolia and N. repanda pollen tube growth required accumulation of PELPIII in the TT of N. tabacum, supporting PELPIII function in pre-zygotic II.

Genetic Differentiation of Reed Canarygrass (Phalaris arundinacea L.) within Eastern Europe and Eurasia.

Worldwide molecular research of economically important Phalaris arundinacea (Poaceae) is mainly focused on the invasions of this species from Europe to North America. Until the present study, the genetic diversity of the P. arundinacea had not been studied across the Baltic countries. The objective of this research is to evaluate the diversity of Lithuanian populations of P. arundinacea at simple sequence repeat (SSR) loci comparatively among populations of the Baltic countries, Luxembourg, and the Russian Far East (Eurasian), evaluating differentiation between Lithuanian populations and ornamental accessions, and relating these with environmental features. For six selected Lithuanian river basin populations, GBS low density SNPs were used to determine genetic diversity. Bayesian analysis showed that Eurasian populations of Phalaris arundinacea consist of two gene clusters. Statistically significant genetic differentiation among European and Eurasian populations was documented. Lithuanian genotypes growing naturally along rivers are genetically distinct from cultivated ornamentals. GBS-SNPs divided the six selected Nemunas river basins into three distinct groups with one, two, or three rivers in separate groupings for genetic diversity. Genetic diversity is primarily within, rather than among, Lithuanian, eastern European, and Eurasian populations of P. arundinacea across the continent. Thus, restoration efforts would benefit from local population seed origination.

A novel pollen tube growth assay utilizing a transmitting tract-ablated Nicotiana tabacum style.

Sexual plant reproduction requires multiple pollen-pistil interactions from the stigma (pollen adhesion, hydration, and germination) to the ovary (fertilization). Understanding the factors that regulate pollen tube growth is critical to understanding the processes essential to sexual reproduction. Many pollen tube growth assays (PTGAs) have shorter and slower pollen tube growth when compared to pollen tube growth through the style. The identification and study of factors that regulate pollen tube growth have been impeded by a lack of an efficient and reproducible PTGA. The objective of this research is to develop a robust assay for Nicotiana tabacum pollen tube growth in an environment that supports sustained and normal growth yet is amenable to testing the effects of specific factors. In this paper, we introduce a novel PTGA, which uses pistils from N. tabacum that lack a mature transmitting tract (TT) due to tissue-specific ablation. The TT-ablated style supports normal pollen tube growth and the hollow structure of the style allows modification of the growth environment by direct injection of test material. This PTGA is robust and allows for rapid and accurate measurement of pollen tube length and pollen tube morphology, supporting pollen tube growth from 20 to 35°C and at pH ranging from 4.8 to 7.6. Use of the ablated style for a PTGA is a novel method for the culture of pollen tubes with sustained growth in vivo while permitting the application of treatments to the growing pollen tubes.

How many marker loci are necessary? Analysis of dominant marker data sets using two popular population genetic algorithms.

The number of marker loci required to answer a given research question satisfactorily is especially important for dominant markers since they have a lower information content than co-dominant marker systems. In this study, we used simulated dominant marker data sets to determine the number of dominant marker loci needed to obtain satisfactory results from two popular population genetic analyses: STRUCTURE and AMOVA (analysis of molecular variance). Factors such as migration, level of population differentiation, and unequal sampling were varied in the data sets to mirror a range of realistic research scenarios. AMOVA performed well under all scenarios with a modest quantity of markers while STRUCTURE required a greater number, especially when populations were closely related. The popular ΔK method of determining the number of genetically distinct groups worked well when sampling was balanced, but underestimated the true number of groups with unbalanced sampling. These results provide a window through which to interpret previous work with dominant markers and we provide a protocol for determining the number of markers needed for future dominant marker studies.

The transmitting tissue of Nicotiana tabacum is not essential to pollen tube growth, and its ablation can reverse prezygotic interspecific barriers.

The Nicotiana tabacum transmitting tissue is a highly specialized file of metabolically active cells that is the pathway for pollen tubes from the stigma to the ovules where fertilization occurs. It is thought to be essential to pollen tube growth because of the nutrients and guidance it provides to the pollen tubes. It also regulates gametophytic self-incompatibility in the style. To test the function of the transmitting tissue in pollen tube growth and to determine its role in regulating prezygotic interspecific incompatibility, genetic ablation was used to eliminate the mature transmitting tissue, producing a hollow style. Despite the absence of the mature transmitting tissue and greatly reduced transmitting-tissue-specific gene expression, self-pollen tubes had growth to the end of the style. Pollen tubes grew at a slower rate in the transmitting-tissue-ablated line during the first 24 h post-pollination. However, pollen tubes grew to a similar length 40 h post-pollination with and without a transmitting tissue. Ablation of the N. tabacum transmitting tissue significantly altered interspecific pollen tube growth. These results implicate the N. tabacum transmitting tissue in facilitating or inhibiting interspecific pollen tube growth in a species-dependent manner and in controlling prezygotic reproductive barriers.