ITS sequence variation in selected taxa of Pisum

Saar, D.E. and Polans, N.O.               Dept. of Biological Sciences and Plant Molecular Biology Center

                                                                             Northern Illinois University, Dekalb, Illinois 60115

        Most investigators currently recognize only one or two legitimate species of Pisum (5). These usually include P. fulvum and a P. sativum complex comprised of two main races (humile and elatius), weedy forms and cultivated varieties (2). Two distinct isolates of humile have also been described, a “northern” form that possesses the standard sativum karyotype and a “southern” form that exhibits the same chromosomal translocation as elatius. Despite these distinctions, there is an unmistakably close genealogical affinity among all the wild and cultivated taxa of pea (4,5). One approach to characterizing the nature and degree of these genetic affinities among the various pea taxa is by comparing the nucleotide sequences of their ribosomal DNA.

       Nuclear ribosomal DNA (nrDNA) is organized as individual chromosomal units that are repeated thousands of times in most higher plant genomes. Each of these units contains the three genes that encode the 18S, 5.8S and 26S ribosomal RNA subunits, as well as several different spacer DNA regions. The nucleotide sequence variation found in both of the internal transcribed spacer regions (ITS-1 and ITS-2, Fig. 1) is used extensively for the systematic analysis of closely related taxa, at least in part due to the speedy rate of evolutionary change characterizing these DNA regions (1). In this preliminary study, ITS-1 and ITS-2 DNA sequence variation is assessed for five pairs of wild and cultivated pea taxa selected to approximate the range of Pisum. The goal of the exercise is to examine the similarity of the sequences within paired accessions, the overall level of genetic variation found across the entire genus, and the topological relationships established among the five selected groups of taxa.

 Materials and Methods

       DNA for the ITS procedure is extracted from the leaves of the individual pea plants listed in Table 1 using a CTAB protocol (7). Primers ITS2, ITS3 and ITS4 are described elsewhere (10), as are the PCR amplification cycle and the modified ITS5m primer (8). Gel purification (3) precedes DNA sequencing with an Applied Biosystems model 373 DNA sequencer. PCR is performed with Perkin Elmer (Cetus) DNA thermal cyclers. Forward and reverse DNA sequences are compared to resolve ambiguities using PC Gene software and the resulting sequences aligned with the Clustal X computer program. Sequence data are analyzed using the PAUP computer package (9).

Results and Discussion

       The pea ITS-1 and ITS-2 regions examined in this study contain 298 and 349 alignable base pairs (bp), respectively, totaling 647 bp for each of the plants analyzed. Four ambiguous pyrimidine sites are denoted by the IUPAC/IUB symbol “Y.” Of the 647 ITS bp sequenced for each individual plant, 629 (>97%) of these

Table 1. Variable ITS sites for 10 wild and cultivated taxa of pea.

*In the 5’->3’ direction (see Fig. 1), beginning with those bases nearest primer

ITS5m (for ITS-1) or primer ITS3 (for ITS-2). Complete sequences are available through GenBank for ITS-1 and ITS-2, respectively, as follows: 701(AF305582, AF305920), 702(AF305583,AF305921), 712(AF305584,AF305922), 713(AF305585,AF305923), 716(AF305586,AF305924), JI1794(AF305587,AF305925), 721(AF305588,AF305926), 722 (AF305589,AF305927), Alaska(AF305202,AF305928), JI711(AF305590,AF305929).

sites are constant among the 10 pea taxa. Only 18 of the sites are polymorphic (and only 17 are parsimony informative). Despite its smaller size, ITS-1 contains 13 of the polymorphic sites, as compared with the five found for ITS-2 (Table 1). These numbers attest persuasively to both the very close evolutionary relationships that must exist within the genus and the limited ITS information available with which to differentiate pea taxa. By contrast, when Vicia montbrettii (GenBank AF228075), a single taxon representing a sister genus to Pisum, is included in the data set for comparative purposes, more than three times as many polymorphic sites become available.

      A standard UPGMA distance analysis of the data is presented in Fig. 2. P. fulvum, phylogenetically the most diverged from the cultivars, is assigned as the outgroup. Actual nucleotide substitutions (24 in all) are placed on the phylogram branches, with approximately one-half of these base changes supporting the differentiation of fulvum from the larger sativum ingroup. Within sativum, all eight accessions pair according to their traditional taxonomic designations. The selected pairs of northern humile and elatius each displays completely identical nucleotide sequences (at 647 sites), as does the pair of fulvum lines comprising the outgroup. The cultivars differ at only one ambiguous site, and the southern humile differ at only one ambiguous and one unambiguous site. It should be noted here that accession JI1794, listed by the John Innes Institute simply as P. humile, seems to possess the morphological features of a northern humile. It is thus identified in this study in accordance with its perfect ITS sequence identity with northern humile 716.

       According to the UPGMA analysis depicted in Fig. 2, elatius is the closest taxon to the cultivated sativum, followed by northern humile. Southern humile is the taxon within the ingroup most distinct from the cultivars. The close clustering of the northern and southern forms of humile would seem intuitive, while their resolution in the phylogram supports their established distinctiveness as well. Parsimony analyses of this same small data set do not resolve these relationships as thoroughly as the distance model, although they produce many of the same branches and much of the same topology. Only the node joining the sativum ingroup with the fulvum outgroup receives strong (100%) support using parsimony methods. Neither branch-and-bound nor bootstrap searches generate high clade values among the four ingroup taxa; the single exception being a 77% bootstrap value at the node joining elatius and the cultivars.

      It has been postulated that northern humile, rather than elatius, is the closest wild progenitor of the cultivated pea, based in part on a shared chromosomal translocation (2) and detailed chloroplast studies (6). This compelling relationship, however, is inconsistent with the UPGMA findings presented in Fig. 2. Northern humile is even further removed from the cultivars in a number of the (fourteen) most parsimonious trees, in these instances reversing its position in Fig. 2 with that presently shown for southern humile. Irrespective of the relative phylogenetic positions of northern and southern humile, neither this ITS data set, nor other more extensive data sets (not shown), support northern humile as the taxon closest to the cultivars.

Conclusions

       ITS sequence variation for the selected taxa of this study suggests: 1) very close genetic affinities throughout Pisum, with P. fulvum exhibiting the greatest degree of divergence, 2) support for the established taxonomic categories of the genus based upon identical or near identical sequences within group pairs, 3) the assignment of JI1794 as a “northern” humile, 4) the validity of northern and southern humile as closely-related, but distinct, lines, 5) the apparent independent evolution of a pea chromosomal translocation and 6) a close relationship between elatius and the cultivated sativa.

Acknowledgement: We thank Scott Grayburn for his DNA sequencing skills. This work was supported by funds from the Department of Biological Sciences and the Plant Molecular Biology Center, Northern Illinois University.

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