Species Identification Confirmation of Medicago of NPGS Germplasm

Species Identification Confirmation of Medicago of NPGS Germplasm Species identification confirmation of Medicago NPGS germplasm III. Outline of specific research to be conducted Choice of taxa: Table 1 provides information on the accessions we propose to sample. Our highest priority are the crop wild relatives of alfalfa that include about 45 accessions indicated as available on the Crop Relatives in GRIN Taxonomy for alfalfa web site. Table 1 also includes other species not listed as crop wild relatives on the GRIN site. These additions are based on the taxonomic relationships described in Ernie Smalls book, Alfalfa and Relatives: Evolution and Classification of Medicago (Small, 2011). Medicago cretacea M. Bieb formerly in sect. Platycarpae, is placed in sect. Medicago subsection Medicago (with M. sativa) based on reconsideration of morphological characters; this change is supported by phylogenetic analyses of a number of molecular markers including nrDNA ITS and matK and other nuclear-encoded genes (Bena et al. 2001; Maureira-Butler et al., 2008; Steele et al. unpublished data). Earlier references also indicated a relative close relationship of M. cretacea to M. sativa and related species (e.g. Lesins and Lesins, 1979). Both Small (2011) and Lesins and Lesins (1979) placed M. suffruticosa Ramond ex DC in the same taxonomic group as M. hybrida; sect. Medicago subsect. Suffruticosae and subgenus Medicago sect. Suffruticosae respectively. Another important group of species of Medicago that we propose to sample (Table 1) are relatives of the model legume and forage species, M. truncatula in subsection Pachyspireae (Young et al. 2011). Many species in this subsection including M. italica, M. littoralis, and M. rigidula are important forage, soil improvement and companion crop species (e.g. Alfalfa Crop Germplasm Committee, 2000). Note that subsection Pachspireae is defined slightly differently here than in most references (e.g., Small, 2011) and is based on results of phylogenetic analyses of a variety of molecular markers (Steele et al, 2010) and low coverage whole genome sequence data (Yoder et al., 2013). For example, M. noeana is included in this subsection, but M. murex is not (see Figure 3, from Yoder et al., 2013). Many of the species in this subsection are difficult to identify; it will be very useful to confirm identification of selected accessions of species in this subsection (Table 1). For all accessions sampled we will coordinate our choices with the Curator for Temperate Legumes, Brian Irish, at National Temperate Forage Legume Genetic Resources Unit in Prosser, WA. When we have multiple accessions to choose among we will choose accessions to sample that have a large amount of germplasm evaluation data. Identification of accessions: We will grow approximately three to six individuals of each accession from seed in a growth chamber and isolate DNA from one or more individual plants. Voucher specimens of each accession with mature fruit will be prepared and deposited at the Arizona State University Herbarium (ASU). Sequence data for the three molecular markers described below will be obtained for each accession. Those data will be added to a data set of available sequences for each marker and a concatenated data set of the three markers. Standard methods of phylogenetic analyses, maximum parsimony, Bayesian analyses and maximum likelihood will be used to analyze those data sets (Stamatakis, 2014; Steele et al., 2010; Swofford, 2002). For most accessions in subsection Medicago (Table1), we will also estimate the ploidy level of the accession (see methods below) as part of the species identification confirmation process. We expect that most accessions will be found in a monophyletic group of accessions of the same species and will then conclude that that identification of that accession has been confirmed. For accessions suspected of being misidentified we will first re-isolate DNA from the voucher specimen and again, obtain sequence data and re-analyze those data as described above. Molecular markers: We will use the following molecular markers: the plastid-encoded ycf1 gene, matK gene and associated trnK intron sequences and the nuclear-encoded nrDNA ITS. As discussed in the introduction these markers are widely used in DNA barcoding and phylogenetic studies in angiosperms. We have considerable experience with obtaining high-quality DNA sequences of these markers and rarely have difficulty with amplification and sequencing of any of them. We have routinely obtained DNA sequence data from matK and nrDNA ITS from a wide variety of temperate legumes in addition to many species of Medicago (Steele et al., 2010; Steele et al., unpublished data). We are now sequencing ycf1 and have a number of sequences from various accessions of Medicago that show a promising level of variation. Ploidy level determination: Flow cytometry is an important and modern technique in a number of fields of biology with a number of medical applications as well as multiple uses in both molecular and plant biology (e.g. Dolezel and Bartos, 2005). The PI has used flow cytometry to obtain genome size data for 56 species and multiple accessions of a number of those species of Medicago (Zhou et al., submitted; Steele et al, in preparation). For this proposal we will use flow cytometry for ploidy level determination. Ploidy level determination using flow cytometry can be more straightforward than obtaining chromosome counts for species of Medicago which have quite small chromosomes. Figure 2 shows the results obtained for two accessions of M. cancellata with the panel on the right from PI 440491 and the panel on the left from PI 440493. Our basic protocol is to grow young plants from seed in a growth chamber to provide uniform conditions. Tissue samples will be prepared according to standard methods and will use propidium idodide (PI) as a fluorochrome (Blondon et al., 1994; Galbraith, 2009). Plant species with a known genome size will also be used as size standards, for example, the model legume, Medicago truncatula, that has a genome size of 1.15 picograms (pg) has been routinely and successfully used by us and we have also used Glycine max as a standard (Dolezel and Bartos, 2005); we have seeds available for both of those species. Although our primary goal is ploidy level determination we will also determine genome size which will be calculated as follows: [(sample G1 peak mean)/(standard G1 peak mean)] x the standard 2C DNA content (pg DNA) where G1 refers to the G1 stage of the cell cycle and standard 2C DNA content refers to the genome size of the standard (Dolezel and Bartos, 2005). The Biodesign Institute on the ASU Tempe campus has a FACSCalibur flow cytometer available for use (a core facility). This instrument has a 15-mW, 488-nm, argon-ion laser and detectors for three fluorescent parameters. Although designed for clinical use this instrument has been used successfully by plant biologists to estimate genome size and ploidy level in plants (e.g. Havanada et al., 2011).