Robert C. Shearman

Comparative analysis of seeded and vegetative biotype buffalograsses based on phylogenetic relationship using ISSRs, SSRs, RAPDs, and SRAPs

Buffalograss [Buchloe dactyloides (Nutt.) Englem.] is the only native grass that is being used extensively as a turfgrass in the Great Plains region. Its low-growth habit, drought resistance, and low-maintenance requirement make it attractive as a turfgrass species. Our objective was to obtain an overview on the genetic relatedness among and within seeded and vegetative biotype buffalograsses using inter-simple sequence repeats (ISSRs), random amplified polymorphic DNA (RAPDs), sequence-related amplified polymorphisms (SRAPs), and simple sequence repeats (SSRs) markers that were derived from related species (maize, pearl millet, sorghum, and sugarcane). Twenty individuals per cultivar were genotyped using 30 markers from each marker system. All buffalograss cultivars were uniquely fingerprinted by all four marker systems. Mean genetic similarities were estimated at 0.52, 0.51, 0.62, and 0.57 using SSRs, ISSRs, SRAPs, and RAPDs, respectively. Two main clusters separating the seeded-biotype from the vegetative-biotype cultivars were produced using UPGMA analysis. Further subgroupings were unequivocal. The Mantel test resulted in a very good fit (SRAP=0.92, ISSR=0.90) to good fit (RAPD=0.86, SSR=0.88) of cophenetic values. Comparing the four marker systems to each other, RAPD and SRAP similarity indices were highly correlated (r=0.73), while Spearman’s rank correlation coefficient between RAPDs and SSRs was r=0.24 and between ISSRs and SSRs was r=0.66. A genotype-assignment analytical approach might be useful for cultivar identification and property rights protection. Polymorphic SRAPs were abundant and demonstrated genetic diversity among closely related cultivars.

Molecular characterization of Buffalograss germplasm using sequence-related amplified polymorphism markers

Buffalograss [Buchloe dactyloides (Nutt.) Englem] germplasm has a broad resource of genetic diversity that can be used for turfgrass, forage and conservation. Buffalograss is the only native grass that is presently used as a turfgrass in the Great Plains region of North America. Its low growth habit, drought tolerance and reduced requirement for fertilizer and pesticides contribute to interest in its use. The objectives of this study were to use sequence-related amplified polymorphism (SRAP) markers in the evaluation of genetic diversity and phenetic relationships in a diverse collection of 53 buffalograss germplasms, and to identify buffalograss ploidy levels using flow cytometry. Based on their DNA contents, buffalograss genotypes were grouped into four sets, corresponding to their ploidy levels. Thirty-four SRAP primer combinations were used. This is the first report of the detection of differentiating diploid, tetraploid, pentaploid and hexaploid buffalograss genotypes, representing diverse locations of origin, using SRAP markers. Cluster analysis by the unweighted pair-group method with arithmetic averages based on genetic similarity matrices indicated that there were eight clusters. The coefficients of genetic distance among the genotypes ranged from 0.33 up to 0.99 and averaged D=0.66. The genetic diversity estimate, He, averaged 0.35. These results demonstrated that genotypes with potential traits for turfgrass improvement could readily be distinguished, based on SRAP. The use of PCR-based technologies such as SRAP is an effective tool for estimating genetic diversity, identifying unique genotypes as new sources of alleles for enhancing turf characteristics, and for analyzing the evolutionary and historical development of cultivars at the genomic level in a buffalograss breeding program.

Understanding ploidy complex and geographic origin of the Buchloe dactyloides genome using cytoplasmic and nuclear marker systems

Characterizing and inferring the buffalograss [Buchloe dactyloides (Nutt.) Engelm.] genome organization and its relationship to geographic distribution are among the purposes of the buffalograss breeding and genetics program. This buffalograss study was initiated to: (1) better understand the buffalograss ploidy complex using various marker systems representing nuclear and organelle genomes; (2) determine whether the geographic distribution was related to nuclear and organelle genome variation; and (3) compare the genetic structure of accessions with different ploidy levels. The 20 buffalograss genotypes (15 individuals from each genotype) that were studied included diploid, tetraploid, pentaploid, and hexaploid using nuclear (intersimple sequence repeat (ISSRs), simple sequence repeat (SSRs), sequence related amplified polymorphism (SRAPs), and random amplified polymorphic DNA (RAPDs)) and cytoplasmic markers (mtDNA and cpDNA). There was a significant correlation between the ploidy levels and number of alleles detected using nuclear DNA (ISSR, SSR, and SRAP, r=0.39, 0.39, and 0.41, P<0.05, respectively), but no significant correlation was detected when mitochondrial (r=0.17, P<0.05) and chloroplast (r=0.11, P<0.05) DNA data sets were used. The geographic distribution of buffalograss was not correlated with nuclear and organelle genome variation for the genotypes studied. Among the total populations sampled, regression analysis indicated that geographic distance could not explain genetic differences between accessions. However, genetic distances of those populations from the southern portion of buffalograss adaptation were significantly correlated with geographic distance (r= 0.48, P<0.05). This result supports the hypothesis that genetic relationship among buffalograss populations cannot be estimated based only on geographic proximity.

Molecular characterization of cDNA encoding resistance gene-like sequences in Buchloe dactyloides

Current knowledge of resistance (R) genes and their use for genetic improvement in buffalograss (Buchloe dactyloides [Nutt.] Engelm.) lag behind most crop plants. This study was conducted to clone and characterize cDNA encoding R gene-like (RGL) sequences in buffalograss. This report is the first to clone and characterize of buffalograss RGLs. Degenerate primers designed from the conserved motifs of known R genes were used to amplify RGLs and fragments of expected size were isolated and cloned. Sequence analysis of cDNA clones and analysis of putative translation products revealed that most encoded amino acid sequences shared the similar conserved motifs found in the cloned plant disease resistance genes PRS2, MLA6, L6, RPMI, and Xa1. These results indicated diversity of the R gene candidate sequences in buffalograss. Analysis of 5′ rapid amplification of cDNA ends (RACE), applied to investigate upstream of RGLs, indicated that regulatory sequences such as TATA box were conserved among the RGLs identified. The cloned RGL in this study will further enhance our knowledge on organization, function, and evolution of R gene family in buffalo grass. With the sequences of the primers and sizes of the markers provided, these RGL markers are readily available for use in a genomics-assisted selection in buffalograss.

Characterization of the Arthropod Community Associated with Switchgrass (Poales: Poaceae) in Nebraska

Abstract Switchgrass (Panicum virgatum L.) is a perennial warm-season grass native to the North American Great Plains. Recently, this prairie grass has received increased attention as a potential biomass energy crop. Little is known about the arthropod community affecting switchgrass grown under either managed or minimally managed conditions. This information is important for characterizing the arthropods associated with switchgrass and developing appropriate management strategies for potential pests. The objectives of this research were to identify the arthropods associated with switchgrass in Nebraska and to document the seasonal abundance of selected arthropods. In 2007 and 2008, arthropods were sampled from May through October using vacuum samples, soil cores, pitfall traps, and yellow sticky traps. Samples were collected from switchgrass stands managed for biomass production and from native switchgrass at Nine-Mile Prairie in Lancaster County, NE. Thysanoptera and Hymenoptera comprised 60% of arthropods collected. Leafhoppers, grasshoppers, grass flies, and wireworms were the most abundant potential pests, whereas beneficial arthropods included ants, rove beetles, ground beetles, parasitoid wasps, and spiders. This research provides important baseline information on the arthropods associated with switchgrass in the central Great Plains.

Cultivation Effects on Organic Matter Concentration and Infiltration Rates of Two Creeping Bentgrass ( Agrostis stolonifera L.) Putting Greens

Soil cultivation is commonly used to manage organic matter (OM) accumulation in golf course putting greens. Our objectives were to determine: (i) if hollow-tine cultivation is more effective than solidtine cultivation at managing OM and water infiltration, (ii) if venting methods are effective at managing OM and water infiltration, and (iii) if venting alters or interacts with effects of earlyor late-season cultivation. The study was a 3 ́ 5 factorial repeated on two ‘Providence’ creeping bentgrass (Agrostis stolonifera L.) research putting greens. Tine treatments were hollow-tine, solid-tine, or no-tine cultivation. Venting treatments were Hydroject, PlanetAir, quad needle tine, bayonet tine, or no venting. Soil samples were collected and analyzed for OM content using loss on ignition. Water infiltration rates were determined in situ. After 2 years, there were few consistent differences found among the tine and venting treatments, and there were no significant interactions regarding OM concentration. This response was attributed to the small amount of surface area impacted by cultivation and to the equalization of topdressing quantity across all treatment combinations. Hollowtine and solid-tine cultivation increased infiltration compared with no cultivation. In general, Hydroject treatments increased water infiltration rates more than all other venting treatments regardless of tine treatment. Organic matter accumulation in creeping bentgrass putting greens has been a concern since the innovation of sand-based root zones (Gaussoin et al., 2013). Accumulation of OM can increase thatch in a putting green, creating a soft, saturated surface that results in decreased playability (Glasgow et al., 2005). Equipment and foot traffic can also cause surface imperfection (e.g., ruts, scalping, and foot imprints) on putting greens with high OM content (Oatis, 2010). Excessive OM decreases water infiltration rates and increases surface water retention (Hurto et al., 1980). Excess surface water retention for extended periods decreases gas exchange (O2, CO2, CH4) between the soil and atmosphere, which can have a negative impact on turfgrass growth (Carrow et al., 2001; Hillel, 2004). Published in Applied Turfgrass Science DOI 10.2134/ATS-2014-0032-RS

Evaluation of Buffalograss Genotypes and Full-Sibs for Chinch Bug Resistance

ABSTRACT Fifteen buffalograss, Buchloe dactyloides (Nutt.) Engelm, genotypes and 94 diploid full-sib progeny were evaluated for western chinch bug, Blissus occiduus Barber (Hemiptera: Lygaeidae), resistance in two separate studies. The experimental design for each study was a completely randomized design. Adult chinch bugs were introduced onto caged single clone genotypes and progeny in the greenhouse. Chinch bug damage was assessed using a 1–5 visual damage rating scale with 1 = ≤ 10%; 2 = 11–30%; 3 = 31–50%; 4 = 51–70%; and 5 = ≥ 70% of the buffalograss leaf area with severe discoloration, or dead tissue. Highly significant differences were found among the genotypes and progeny for chinch bug damage. Among the genotypes, Legacy, Prestige, 184, 196, Bowie, NE 3297, NE 2769, and NE 2768 were moderately resistant with damage ratings of >1, but <3, while NE 2990, NE 2838, and 1-57-19 were moderately susceptible with damage ratings of ≥3, but <4. Among the progeny, one progeny (MP45) was highly resistant with a chinch bug damage rating of 1.0, 78 progeny (83%) had moderate resistance, with damage ratings of >1.0 and <3.0, 13 progeny (14%) were moderately susceptible with damage ratings ranging from 3.0 to 3.9, while only two were highly susceptible with damage ratings of ≥4.0. The significant variability among genotypes and progeny for chinch bug resistance indicates the ability to improve buffalograss resistance to chinch bugs through selection or hybridization of selected genotypes.