B.G. Abeyo 1 , D. D. Serba 1 , R.C. Shearman 1 , D. Kopec 2 , and D.J. Lee 1

1. Buffalograss False grama Blue grama ABSTRACT Buffalograss and blue grama dominate the short grass prairies of the Great Plains of North America, while false grama is found mostly in the semiarid regions of Texas and Arizona. Information about their genetic diversity or similarity is limited, and more research is needed to better understand their potential genetic diversity. This study was initiated to assess the level of genetic diversity within and among buffalograss, blue grama, and false grama genotypes, using molecular and morphological markers. Twelve genotypes of each species were evaluated; using simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) markers. In addition, morphological traits were measured. Buffalograss and false grama are stoloniferous, while blue grama is a bunch-type grass. Buffalograss is mostly dioecious, while blue grama and false grama are monoecious. The three species differ in their flower location, color and morphology. Genetic analysis based on SSR and SRAP markers showed that genotypes of each species were distinctly clustered with higher variability in buffalograss, false grama was intermediate and blue grama had the least. Principal coordinate analysis for the three species using SRAP markers also demonstrated the uniqueness of genotypes of each species. All genotypes of buffalograss, false grama and blue grama were uniquely separated and distantly related with each other, blue grama fell between buffalograss and false grama. Based on morphological and molecular markers, sufficient evidences exist among the three species to consider them under different genera. ●Amplification and polymorphism were high in buffalograss, intermediate in false gram and limited in blue grama ● UPGMA clustering identified three major clusters representing genotypes of each species ● PCoordA also demonstrated the uniqueness of genotypes of each species INTRODUCTION Native grasses are used for soil stabilization (Cooper, 1957), wild life habitat restoration (Knapp and Rice, 1994), improved forage quality (Vogel and Pedersen, 1993) and as potential source of turfgrass (Shearman, 2004). Buffalograss (Buchole dactyloides (Nutt.) Engelm), blue grama (Bouteloua gracilis [H.B.K]. ex Steud.)and false grama (Cathestecumerectum Vasey & Hack)are a few of the warm season grasses native to the US. Buffalograss is the only native grass used as a turf in North America. Its drought, heat, and cold tolerance coupled with reduced inputs requirements have made buffalograss the potential turf of the future. Information about the genetic diversity or similarity among buffalograss, blue grama and false grama is limited, and more research is needed to better understand their genetics. This study was initiated with the following objective. Objective To assess the level of genetic diversity within and among buffalograss, blue grama, and false grama genotypes, using molecular and morphological markers. Comparative Analysis of Buffalograss [Buchole dactyloides (Nutt.) Engelm], Blue grama (Bouteloua gracilis [H.B.K]. ex Steud.) and False grama (Cathestecumerectum Vasey & Hack) Fig 1. PCR amplification of genomic DNA from buffalograss, blue grama and false grama genotypes using RM 7076 SSR marker ● Cluster analysis based on 43 SSR and 48 SRAP markers showed genotypes of each species were distinctly clustered with high variability in buffalograss and least variability in blue grama Materials and Methods Plant materials and DNA Extraction Twelve genotypes of each species were used for genotyping. DNA was extracted from fresh leaf tissue samples using sap extraction method. Forty three simple sequence repeats (SSRs) and forty eight sequence related amplified polymorphism (SRAP) markers combinations were used. The Polymerase Chain Reaction (PCR) was run using 25 µl total volume and 50 ng template DNA. DNA amplifications were performed using Thermal Cycler programmed for SSR and SRAP markers at 2 minutes of initial denaturation at 940C followed by 36 cycles, each consisting of a denaturation step at 940C for one minute, an annealing step at average Tm of each primer combination for one minute, and an extension step at 720C for 1:15 minutes, with a final extension at the end of the 36 cycles at 720C for 5 minutes. SRAP markers were separated on 0.8% agarose gel while SSR markers were separated on 2.5% agarose gel. Gels were stained by adding 7.0 μL of Ethidium-bromide. Amplicons were visualized using a Gel Doc 2000 (Bio-Rad). Bands were scored as 1= present or 0= absent and used for phylogenetic analysis using NT-SYS-pc version (Rholf, 2000). Genetic similarities between genotypes were measured by the Dice similarity coefficient based on the proportion of shared alleles with SIMQUAL module (Dice, 1945; Nei and Li, 1979). PCoordA was performed. Fig 4. A three dimensional principal coordinate analysis of twelve genotypes each of buffalograss, blue grama and false grama using SRAP markers. Conclusion ● Among materials tested, buffalograss genotypes were classified into diploids, tetraploids, pentaploids and hexaploids, false grama genotypes into tetraploids and pentaploids while blue grama genotypes were all tetraploids. ● Buffalograss and false grama are stoloniferous, while blue grama is a bunch-type grass. Buffalograss is mostly dioecious while blue grama and false grama are monoecious. Buffalograss has shorter stolons than false grama. Blue grama leaf length is almost twice as long as that of buffalograss or false grama leaf lengths. ● SSR and SRAP markers showed that genotypes of each species were distinctly clustered with higher variability in buffalograss intermediate in false grama and least in blue grama. Both dendrograms revealed uniqueness of genotypes within specie with out discrepancy to taxonomic classification and the presence of distant relationship among genotypes of the three genera. Principal coordinate analysis also demonstrated the uniqueness of genotypes of each species. This complements the morphological differences observed among the species. ● Definite morphological and genetic differences exist among the three species to conclude them under three different genera. B.G. Abeyo1, D. D. Serba1, R.C. Shearman1, D. Kopec2, and D.J. Lee1 1 University of Nebraska - Lincoln, 2 University of Arizona - Tucson Fig 2. An UPGMA dendrogram of genetic relationships among buffalograss, blue grama and false grama genotypes derived from SSR markers. Results and Discussion ● Genotypes of each species were uniquely separated and distantly related blue grama fell in between ● Genetic similarities among all genotypes ranged from 0.32 - 0.98 for SRAP and from 0.25 - 0.96 for SSR markers References Columbus, J.T. 1999. An expanded circumscription of Boutelloua (Gramineae: Chloridoideae): new combinations and names. Aliso. 18:61-65. Cooper, H.W. 1957. Some plant materials and improved techniques used in soil and water conservation in the Great Plains. J. Soil water Conserv. 12:163-168. Dice L.R. 1945. Measure of the amount of ecological association between species. Ecology 26:297-302. Johnson, P.G. T.P. Riordan, k. Arumuganathan, 1998. Ploidy level determinations of buffalograss clones and populations. Crop Sci. 38:478-482. Knapp E.E., and K.J. Rice, 1994. Starting from seed: genetic issues in using native grasses for restoration. Restor. And Manage. Notes 12:40-45. Nei, M., W.H. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA. 76:5269-5273. Rohlf J.F. 2000. NTSYSpc: numerical taxonomy and multivariate analysis system. Exeter Software, Setauket, New York, USA. Shearman, R.C., T.P. Riordan, and P.G., Johnson. 2004. Buffalograss. Analysis, Monograph Series No. 43. American Society of Agronomy, Madison, WI. PP. 1003-1026. Vogel K.P., and J.F. Pedersen. 1993. Breeding systems for cross-pollinated perennial grasses. Plant Breed. Rev. 11:251-274. False grama and Blue grama Buffalograss ● False grama and blue grama are monoecious while buffalograss is dioceious ● False grama and buffalograss are stoloniferous while blue grama is bunch-type ● The three species differ in flower morphology, color, structure and location ● Buffalograss is imperfect while blue grama and false grama are perfect flowers ● Buffalograss has shorter internodes and leaf lengths than false grama Fig 3. An UPGMA dendrogram of genetic relationships among buffalograss, blue grama and false grama genotypes derived from SRAP markers.