Genetic Diversity among Forage Bermudagrass ( Cynodon spp . ) : Evidence from Chloroplast and Nuclear DNA Fingerprinting

for appropriate genetic analysis. The high value of bermudagrass as a forage crop clearly justifies new and innovaGenetic analysis of forage bermudagrasses (Cynodon spp.) lags tive approaches toward evaluating and understanding considerably behind other species, including the turf-type bermudagrasses. This research was undertaken to identify and characterize the genetic relationships among different lines of the genetic relationships within and between forage bermudagrass ecoCynodon species. types and varieties. Genetic relationships within 31 forage bermuDNA-based fingerprinting technologies have been dagrass genotypes were determined by means of 15 amplified fragment applied in genetic studies in a wide range of plant spelength polymorphism (AFLP), 10 chloroplast-specific Simple sequence cies, including turf-type bermudagrass (Cato and Richrepeat length polymorphism (CpSSRLP), 10 random amplified polyardson, 1996; Caetano-Annolles et al., 1997; Arcade et morphic DNA (RAPD), and 10 directed amplification of minisatelliteal. , 2000). An integrated study using different DNA region DNA (DAMD) primers or primer pairs. The unweighted pair fingerprinting techniques will be beneficial for the gegroup method, using arithmetic averages (UPGMA) and the bootstrap netic improvement of forage bermudagrass. Four PCRanalyses with 2000 replications, were used to calculate the relationbased DNA fingerprinting techniques including AFLP ships. Overall results indicated that forage bermudagrass genotypes have a narrow genetic base, with genetic similarity (GS) ranging from (Vos et al., 1995), CpSSRLP (Weising et al., 1998), 0.608 to 0.977. The most genetically similar forage bermudagrass lines RAPD (Costa et al., 2000), and DAMD (Bebeli et al., were ‘Tifton 78 WH’ and ‘Tifton 78’ (GS 0.977), whereas ‘McDon1997) were used to evaluate genetic relationships among ald’ and ‘Alicia’ were the most genetically diverse bermudagrass lines selected forage bermudagrass ecotypes and varieties. (GS 0.608). ‘Sumrall 007’, ‘Tanberg’, ‘Maddox’, McDonald, ‘Holly Springs’, ‘Murphy’, ‘Murphy II’, and ‘Stallings’ were distantly related MATERIALS AND METHODS to known varieties. The GS values of these genotypes were less than 0.85 compared with any other genotypes, indicating that these ecoPlant Materials types were unique in their genome structure and provided genetic Plant materials included established varieties and locally justification for their release as varieties. Close genetic relationships selected ecotypes. An ecotype is a reselection based on a between some ecotypes and varieties were detected as follows: ‘Prairie distinct phenotype from an existing variety. Forage bermuI’, ‘Prairie II’, and ‘Prairie III’ to ‘Grazer’; ‘Lott I’, ‘Lott II’, and dagrass ecotypes used in this study were first identified either ‘Lancaster’ to ‘Callie’; and Tifton 78 WH to Tifton 78. Although DNA by local farmers or extension specialists from an existing bermarkers could differentiate these ecotypes, additional molecular, cytomudagrass field (Edwards et al., 1993). A total of 31 forage logical, and phenological evidences are required to further confirm bermudagrass genotypes, including varieties, were obtained whether they could be considered as new varieties. from breeders, extension agents and farmers, and maintained in a greenhouse at Mississippi State University, Mississippi State, MS (Table 1). B varieties are planted as forage crops more than any other warm-season perennial species DNA Extraction throughout the southeastern USA (Burton and Hanna, Young and healthy plant leaf tissues (5–6 g) of each geno1995). However, current investment in genetic improvetype were collected from at least two pots (vegetative clones) ment in this crop is not commensurate with its impact on of greenhouse grown plants and mixed, washed in tap water, the U.S. economy. Bermudagrass breeders frequently dried with clean paper towels, and placed in a 80 C freezer encounter three major challenges: (i) many of the so until processed. An additional 10 plant samples (test or blind called “ecotypes” are selected from farmer fields with samples) were also collected from the greenhouse to verify limited pedigree history and source of origin; (ii) limited reproducibility and accuracy of the techniques. Genomic DNAs knowledge is known for the genome of the forage-type were extracted by a modified CTAB (hexadecyltrimethylambermudagrass, including DNA content, genome size, and monium bromide) procedure (Murray and Thompson, 1980; ploidy level; and (iii) there is a lack of suitable methods Karaca, 2001). Mehmet Karaca, Dep. of Field Crops, Akdeniz Univ., Agricultural Genomic DNA Amplification and Analysis Faculty, Antalya, 07759 Turkey; David J. Lang, Dep. of Plant and Soil Sciences, Mississippi State Univ., Mississippi State, MS 39762; Amplified Fragment Length Polymorphism (AFLP) Sukumar Saha and Johnie N. Jenkins, USDA-ARS, P.O. Box 5367, Mississippi State, MS 39762; Allan Zipf, Dep. of Plant and Soil Science, The AFLP Small Plant Mapping Protocol for Genomes, Alabama A&M Univ., Normal, AL 35762. Mention of trademark or P/N 4303051 (Perkin-Elmer, Norwalk, CT) was adapted with proprietary product does not constitute a guarantee or warranty of the modification noted. Sixty nanograms of genomic DNA, or the product by the USDA, Mississippi State Univ. and Alabama negative control without template DNA, in a 3L volume A&M Univ. and does not imply its approval to the exclusion of other were added into a 0.2-mL PCR tube containing 7 L reaction products that may also be suitable. Published as journal article no. solution consisting of 1 L MseI and EcoRI adapter pairs J9901 of the Mississippi Agric. and Forestry Exp. Stn. Received 27 (Perkin-Elmer, Norwalk, CT), 1 L 10 T4 DNA ligase buffer Aug. 2001. *Corresponding author (dlang@pss.msstate.edu). [50 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM ATP, 25 g/mL bovine serum albumin (BSA)], Published in Crop Sci. 42:2118–2127 (2002).