Sample sequence analysis was employed to investigate the repetitive DNAs that
Sample sequence analysis was employed to investigate the repetitive DNAs that were most responsible for the evolved variation in genome content across seven panicoid grasses with >5-fold variation in genome size and different histories of polyploidy. One of the LTR retrotransposon amplification bursts in may have been initiated buy AMD 3465 Hexahydrobromide by polyploidy, but the great majority of transposable element activations are not. Instead, the results suggest random activation of a few or many LTR retrotransposons families in particular lineages over evolutionary time, with some families especially prone to future activation and hyper-amplification. (Piegu spp.) and pearl millet (designated TAMU) and John Doebley of the University Wisconsin-Madison for the same teosinte lines used to estimate nuclear DNA content for (Iltis G-5 and G-42) and (Iltis 1190) (Laurie and Bennett, 1985). Nuclear DNA from sugarcane (species share an allotetraploidy that occurred <12 mya (Figure 1) (Swigonova analysis. For the other panicoid grasses, two 384-well plates were chosen and clones were sequenced from both directions, except for sugarcane where four plates were sequenced because of small insert sizes for many clones. The electropherograms obtained from the ABI3730 sequencing machine (Applied Biosystems) were analyzed with Phred (Ewing (inbred B73) by downloading data from the 3C4?kb unfiltered genomic shotgun data set (NCBI accession # 33825241C34849215) from GenBank (Schnable comparisons, where quantitation was important, the annotation information was transformed into a repeat percentage for each sequence by dividing the repeat length in that sequence by the total sequence read length. The transformed data were then bootstrapped using SAS with 1000 permutations. The values produced in the bootstrap statistic were multiplied by genome size for each library. The 1C/1N genome size values utilized were all from the Kew C value database (http://data.kew.org/cvalues): (2620?Mb), (730?Mb), (740?Mb), (3960?Mb for the octoploid 1C/4N genome), (2590?Mb); (2365?Mb) and (4470?Mb). The mean and a 95% confidence interval for repeat quantities in each species or genotype were then graphed to display the genome comparisons and test the null hypothesis that the two samples being compared have equal amounts (Mb) of the TE buy AMD 3465 Hexahydrobromide family. If the 95% confidence interval in any pairwise comparison did not overlap, we rejected the null hypothesis and argue that the samples are significantly different in the amount of the TE family being compared. Because sorghum and maize have excellent repeat databases, masking of SSA data was employed to find and quantify buy AMD 3465 Hexahydrobromide repeats using the prototypic repeat representatives from the Repbase Update data (AFA Smit, R Hubley and P Green RepeatMasker at http://repeatmasker.org). A custom PERL script (R Hubley, pers. comm.) returned the percent sample masked by each repeat. Retroelement phylogenetic analysis Annotated sequences of retroelements were translated into all six reading frames and then searched by BLASTp with a translated copy of the reverse transcriptase or the integrase genes to find the sequences in the data set that could be used to reconstruct a phylogeny for each high copy retroelement. The BLAST results with the highest number of sequence hits were aligned in clustalX and trimmed to incorporate the largest number of taxa with the longest alignment. Neighbor joining trees were constructed in PAUP using the default settings (using an uncorrected (the closest related species with sequence data) were used as the out group for the resulting trees. In the same manner, a nucleotide alignment and NJ tree were also produced for the 180-bp knob repeat. For comparison with and repeats assembled with AAARF, SSA data were produced for and Ten thousand random unfiltered shotgun sequence reads from maize (average read Rabbit Polyclonal to ASC length 782?bp, accession numbers EI697885.1EI684889.2) were downloaded from TIGR. For sorghum, 10?000 random sequences (average read length 975?bp) were downloaded from the NCBI GSS database (http://www.ncbi.nlm.nih.gov/projects/dbGSS/). A custom database was used to identify LTR retrotransposon sequences from the five panicoid grasses’ SSA data. The database was assembled from Panicoid-specific LTR retrotransposons from Repbase Update (Jarka and retrotransposons from the TIGR Plant Repeat Databases (Ouyang and Buell, 2004) (from http://plantrepeats.plantbiology.msu.edu/), and the full MIPS-REdat database (v. 4.3) (http://mips.helmholtz-muenchen.de/plant). Results The four most abundant repeats in five panicoid grass species AAARF assemblies (for and and species, several repeats were found to account for >1% each of the buy AMD 3465 Hexahydrobromide total genome. In each case, the largest contribution was from an LTR retrotransposon family, although this was a different family in each genus. Table buy AMD 3465 Hexahydrobromide 1 The four most abundant LTR retrotransposon families in five panicoid genomes The family was found to be an abundant element in most of the panicoids investigated, including among the top four in maize and pearl millet, and the sixth most abundant LTR retrotransposon in element is only a middle-repetitive DNA in (Peterson.