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.

Structure and function of class III pistil-specific extensin-like protein in interspecific reproductive barriers.

BACKGROUND: The transmitting tissue of the style is the pathway for pollen tube growth to the ovules and has components that function in recognizing and discriminating appropriate pollen genotypes. In Nicotiana tabacum, the class III pistil extensin-like (PELPIII) arabinogalactan protein is essential for the inhibition of N. obtusifolia pollen tube growth. The transmitting tissue-specific (TTS) arabinogalactan protein amino acid sequence and expression pattern is similar to PELPIII, but it facilitates self-pollinated N. tabacum. The TTS and PELPIII arabinogalactan protein can be divided into the less conserved N-terminal (NTD) and the more conserved C-terminal (CTD) domains. This research tested whether the NTD is the key domain in determining PELPIII function in the inhibition of interspecific pollen tube growth. Three variant PELPIII gene constructs were produced where the PELPIII NTD was exchanged with the TTS NTD and a single amino acid change (cysteine to alanine) was introduced into the PELPIII NTD. The PELPIII variants of N. tabacum were tested for activity by measuring the inhibition N. obtusifolia pollen tube growth by using them to complement a 3'UTR RNAi transgenic line with reduced PELPIII mRNA. RESULTS: The RNAi N. tabacum line had reduced PELPIII mRNA accumulation and reduced inhibition of N. obtusifolia pollen tube growth, but had no effect on self-pollen tube growth or pollen tube growth of 12 other Nicotiana species. The NTD of PELPIII with either the PELPIII or TTS CTDs complemented the loss PELPIII activity in the RNAi transgenic line as measured by inhibition of N. obtusifolia pollen tube growth. The TTS NTD with the PELPIII CTD and a variant PELPIII with a cysteine to alanine mutation in its NTD failed to complement the loss of PELPIII activity and did not inhibit N. obtusifolia pollen tube growth. CONCLUSION: The NTD is a key determinant in PELPIII's function in regulating interspecific pollen tube growth and is a first step toward understanding the mechanism of how PELPIII NTD regulates pollen tube growth.

Polymorphism and structure of style-specific arabinogalactan proteins as determinants of pollen tube growth in Nicotiana.

BACKGROUND: Pollen tube growth and fertilization are key processes in angiosperm sexual reproduction. The transmitting tract (TT) of Nicotiana tabacum controls pollen tube growth in part by secreting pistil extensin-like protein III (PELPIII), transmitting-tract-specific (TTS) protein and 120 kDa glycoprotein (120 K) into the stylar extracellular matrix. The three arabinogalactan proteins (AGP) are referred to as stylar AGPs and are the focus of this research. The transmitting tract regulates pollen tube growth, promoting fertilization or rejecting pollen tubes. RESULTS: The N-terminal domain (NTD) of the stylar AGPs is proline rich and polymorphic among Nicotiana spp. The NTD was predicted to be mainly an intrinsically disordered region (IDR), making it a candidate for protein-protein interactions. The NTD is also the location for the majority of the predicted O-glycosylation sites that were variable among Nicotiana spp. The C-terminal domain (CTD) contains an Ole e 1-like domain, that was predicted to form beta-sheets that are similar in position and length among Nicotiana spp. and among stylar AGPs. The TTS protein had the greatest amino acid and predicted O-glycosylation conservation among Nicotiana spp. relative to the PELPIII and 120 K. The PELPIII, TTS and 120 K genes undergo negative selection, with dn/ds ratios of 0.59, 0.29 and 0.38 respectively. The dn/ds ratio for individual species ranged from 0.4 to 0.9 and from 0.1 to 0.8, for PELPIII and TTS genes, respectively. These data indicate that PELPIII and TTS genes are under different selective pressures. A newly discovered AGP gene, Nicotiana tabacum Proline Rich Protein (NtPRP), was found with a similar intron-exon configuration and protein structure resembling other stylar AGPs, particularly TTS. CONCLUSIONS: Further studies of the NtPRP gene are necessary to elucidate its biological role. Due to its high similarity to the TTS gene, NtPRP may be involved in pollen tube guidance and growth. In contrast to TTS, both PELPIII and 120 K genes are more diverse indicating a possible role in speciation or mating preference of Nicotiana spp. We hypothesize that the stylar AGPs and NtPRP share a common origin from a single gene that duplicated and diversified into four distinct genes involved in pollen-style interactions.

Accelerated evolution of the mitochondrial genome in an alloplasmic line of durum wheat.

BACKGROUND: Wheat is an excellent plant species for nuclear mitochondrial interaction studies due to availability of large collection of alloplasmic lines. These lines exhibit different vegetative and physiological properties than their parents. To investigate the level of sequence changes introduced into the mitochondrial genome under the alloplasmic condition, three mitochondrial genomes of the Triticum-Aegilops species were sequenced: 1) durum alloplasmic line with the Ae. longissima cytoplasm that carries the T. turgidum nucleus designated as (lo) durum, 2) the cytoplasmic donor line, and 3) the nuclear donor line. RESULTS: The mitochondrial genome of the T. turgidum was 451,678 bp in length with high structural and nucleotide identity to the previously characterized T. aestivum genome. The assembled mitochondrial genome of the (lo) durum and the Ae. longissima were 431,959 bp and 399,005 bp in size, respectively. The high sequence coverage for all three genomes allowed analysis of heteroplasmy within each genome. The mitochondrial genome structure in the alloplasmic line was genetically distant from both maternal and paternal genomes. The alloplasmic durum and the Ae. longissima carry the same versions of atp6, nad6, rps19-p, cob and cox2 exon 2 which are different from the T. turgidum parent. Evidence of paternal leakage was also observed by analyzing nad9 and orf359 among all three lines. Nucleotide search identified a number of open reading frames, of which 27 were specific to the (lo) durum line. CONCLUSIONS: Several heteroplasmic regions were observed within genes and intergenic regions of the mitochondrial genomes of all three lines. The number of rearrangements and nucleotide changes in the mitochondrial genome of the alloplasmic line that have occurred in less than half a century was significant considering the high sequence conservation between the T. turgidum and the T. aestivum that diverged from each other 10,000 years ago. We showed that the changes in genes were not limited to paternal leakage but were sufficiently significant to suggest that other mechanisms, such as recombination and mutation, were responsible. The newly formed ORFs, differences in gene sequences and copy numbers, heteroplasmy, and substoichiometric changes show the potential of the alloplasmic condition to accelerate evolution towards forming new mitochondrial genomes.

Nicotiana tabacum pollen-pistil interactions show unexpected spatial and temporal differences in pollen tube growth among genotypes.

KEY MESSAGE: This research revealed diverse PTG rates among intraspecific pollen-pistil interactions that showed variable dependency on the stigma and mature TT. Pollen-pistil interactions regulate pollen tube growth (PTG) rates and are determinants of fertilization and seed set. This research focuses on the diversity of intraspecific PTG rates and the spatial and temporal regulation of PTG among Nicotiana tabacum genotypes. Nonrandom mating within self-compatible species has been noted, but little is known on the mechanisms involved. To begin research on nonrandom mating, we took advantage of the model reproductive system of N. tabacum and used seventeen diverse N. tabacum genotypes in a complete pollination diallel to measure the diversity of intraspecific pollen-pistil interactions. The 289 intraspecific interactions showed surprisingly large differences in PTG rates. The interaction between specific males and females resulted in 18 specific combining abilities that were significantly different, indicating the importance of the specific genotype interaction in regulating intraspecific PTG. No single female or male genotype exerted overall control of PTG rates, as determined by a general combining ability analysis. Slow and fast pollen-pistil interactions showed spatial differences in growth rates along the style. Slower interactions had a slower initial PTG rate while fast interactions had faster consistent rates of growth indicating spatial regulation of PTG in the pistil. Removal of the stigma or the mature transmitting tissue (TT) showed the tissue-specific component of PTG regulation. Stigma removal resulted in slower or no change in PTG rate depending on the pollen and pistil genotypes. Removal of the TT, which necessitated removal of the stigma, showed no change, slower or unexpectedly, increased growth rates relative to growth rates without a stigma. These data show the diverse nature of pollen-pistil interactions in N. tabacum genotypes providing a system to further investigate the regulation of PTG.