Towards Improved Molecular Identification Tools in Fine Fescue (Festuca L., Poaceae) Turfgrasses: Nuclear Genome Size, Ploidy, and Chloroplast Genome Sequencing

Abstract

Fine fescues (Festuca L., Poaceae) are turfgrass species that perform well in low-input environments. Based on morphological characteristics, the most commonly-utilized fine fescues are divided into five taxa: three are subspecies within F. rubra L. and the remaining two are treated as species within the F. ovina L. complex. Morphologically, these five taxa are very similar; both identification and classification of fine fescues remain challenging. In an effort to develop identification methods for fescues, we used flow cytometry to estimate genome size and ploidy level and sequenced the chloroplast genome of all five taxa. Fine fescue chloroplast genome sizes ranged from 133,331 to 133,841 bp and contained 113-114 genes. Phylogenetic relationship reconstruction using whole chloroplast genome sequences agreed with previous work based on morphology. Comparative genomics suggested unique repeat signatures for each fine fescue taxon that could potentially be used for marker development for taxon identification.

Keywords: chloroplast genome; comparative genomics; fine fescue; low input turfgrass; phylogeny.

Figure 1

Flow cytometry results for the five fine fescue taxa. Lolium perenne (2n = 2x = 14) was used as the reference. Flow cytometry was able to separate F. rubra ssp. rubra from the other two taxa in the F. rubra complex. The mean PI-A values of F. rubra ssp. fallax and F. rubra ssp. litoralis were similar (145.86 to 137.85).

Figure 2

Whole chloroplast genome structure of F. ovina complex (A) and F. rubra complex (B). Genes inside the circle are transcribed clockwise, and genes outside are transcribed counter-clockwise. Genes belong to different functional groups are color coded. GC content is represented by the dark gray inner circle, and the light gray corresponded to the AT content. IRA(B), inverted repeat A(B); LSC, large single copy region; SSC, small single copy region.

Figure 3

Comparison for border positions of LSC, SSC, and IR regions among five fine fescues and L. perenne. Genes are denoted by boxes, and the gap between the genes and the boundaries is indicated by the number of bases unless the gene coincides with the boundary. Extensions of genes are also indicated above the boxes.

Figure 4

Sequence identity plot of fine fescues chloroplast genome sequences with F. ovina (2x) as the reference using mVISTA. A cut-off of 70% identify was used for the plots, and the percent of identity varies from 50% to 100% as noted on the y-axis. Most of the sequence variations between fine fescues were in intergenic regions. Taxa in the F. ovina complex, F. brevipila, and F. ovina showed high sequence similarity. Similarly, subspecies within F. rubra complex also showed high sequence similarity.

Figure 5

(A) SSR repeat type and numbers in the five fine fescue taxa sequenced. Single nucleotide repeat type has the highest frequency. No hexanucleotide repeats were identified in the fine fescue chloroplast genomes sequenced. One penta-nucleotide repeat type (AAATT/AATTT) is unique to F. rubra ssp. rubra and F. rubra ssp. litoralis. One penta-nucleotide repeat type (ACCAT/ATGGT) is unique to F. brevipila and F. ovina. (B) Long repeats sequences in fine fescue chloroplast genomes. Complement (C) match was only identified in the F. ovina complex. Reverse (R) match has the most number variation in fine fescues.

Figure 6

Sliding window analysis of fine fescue whole chloroplast genomes. Window size: 600 bp, step size: 200 bp. X-axis: the position of the midpoint of a window (kb). Y-axis: nucleotide diversity of each window. Inverted repeat regions are highlighted in gray. rpl32-trnL region has the most nucleotide diversity followed by psbH- petB-trnL-trnF-trnS-rps4 region.

Figure 7

The alignment of rbcL-psaI intergenic sequence shows that the pseudogene accD is missing in both F. ovina (2x, 4x) and F. brevipila but present in the F. rubra complex and other species examined in this study. Species were ordered by complexes.

Figure 8

Maximum likelihood (ML) phylogram of the Festuca-Lolium complex with 1,000 bootstrap replicates. Fine fescues were grouped into previous named complexes (F. ovina and F. rubra), sister to broad leaved fescues in the Festuca-Lolium complex.