Scott E. Warnke

Association of simple sequence repeat (SSR) markers with submergence tolerance in diverse populations of perennial ryegrass.

Submergence stress can cause the death of grass plants. Identification of the association between molecular markers and submergence tolerance-related traits facilitates an efficient selection of the tolerant cultivars for commercial production. A global collection of 99 diverse perennial ryegrass (Lolium perenne L.) accessions was evaluated for submergence tolerance and analyzed with 109 simple sequence repeat (SSR) markers. Submergence significantly reduced leaf color, chlorophyll fluorescence (F(v)/F(m)), maximum plant height (HT), and relative growth rate (RGR). Significant variations in these trait values were observed among the accessions under submerged conditions. Rapid linkage-disequilibrium (LD) decay was identified within 4cM. The analysis of population structure (Q) identified four subpopulations in the collection, but no obvious relative kinship (K) was found. The Q model was the best to describe associations between SSR and traits, compared to the simple linear, K, and Q + K models. Fifteen SSR markers were associated with a reduction in leaf color, F(v)/F(m), HT, and RGR under submergence stress using the Q model. These markers can be used for genetic improvement of submergence tolerance of perennial ryegrass after further validation. The diverse populations of perennial ryegrass is a valuable resource for association mapping of stress tolerance-related physiological traits.

Inheritance of superoxide dismutase (Sod-1) in a perennial × annual ryegrass cross and its allelic distribution among cultivars

Abstract.Identifying annual ryegrass contamination in perennial ryegrass seed lots has been of major interest in seed-testing laboratories and for seed regulatory agencies in the USA for many years. This study was conducted to characterize a superoxide dismutase locus (Sod-1) and determine its potential to distinguish cultivated ryegrass species. The inheritance of Sod-1 was evaluated in a three-generation annual × perennial ryegrass mapping population and segregation fitted an expected 1:2:1 ratio for a single locus with two alleles. The molecular form of the Sod-1 locus was determined by H2O2 and KCN inhibitor assays which indicated that the Sod-1, and a second independently segregating Sod-2, locus were both Cu/ZnSod enzymes. The common alleles at the Sod-1 locus were scored in 13 annual and 24 perennial ryegrass cultivars to determine the potential of using this locus for species separation. The Sod-1b allele was homozygous in 98% of perennial ryegrass individuals from 24 cultivars, but those not 100% homozygous for Sod-1b were seed lots with unknown contamination from annual ryegrass. These results indicate that the Sod-1b allele in the homozygous condition is a good indicator of perenniality. All eight annual ryegrass cultivars originating in Europe or Asia had a low frequency of Sod-1b homozygous individuals or none at all. The five cultivars originating in the Western Hemisphere, however, had genotype frequencies for homozygous Sod-1b of up to 56%. The potential of the Sod-1 locus to serve as a test to separate the two growth forms depends on the source of the annual-type contamination.

DNA fingerprinting and anastomosis grouping reveal similar genetic diversity in Rhizoctonia species infecting turfgrasses in the transition zone of USA.

Rhizoctonia blight is a common and serious disease of many turfgrass species. The most widespread causal agent, Thanatephorus cucumeris (anamorph: R. solani), consists of several genetically different subpopulations. In addition, Waitea circinata varieties zeae, oryzae and circinata (anamorph: Rhizoctonia spp.) also can cause the disease. Accurate identification of the causal pathogen is important for effective management of the disease. It is challenging to distinguish the specific causal pathogen based on disease symptoms or macroscopic and microscopic morphology. Traditional methods such as anastomosis reactions with tester isolates are time consuming and sometimes difficult to interpret. In the present study universally primed PCR (UP-PCR) fingerprinting was used to assess genetic diversity of Rhizoctonia spp. infecting turfgrasses. Eighty-four Rhizoctonia isolates were sampled from diseased turfgrass leaves from seven distinct geographic areas in Virginia and Maryland. Rhizoctonia isolates were characterized by ribosomal DNA internal transcribed spacer (rDNA-ITS) region and UP-PCR. The isolates formed seven clusters based on ITS sequences analysis and unweighted pair group method with arithmetic mean (UPGMA) clustering of UP-PCR markers, which corresponded well with anastomosis groups (AGs) of the isolates. Isolates of R. solani AG 1-IB (n = 18), AG 2-2IIIB (n = 30) and AG 5 (n = 1) clustered separately. Waitea circinata var. zeae (n = 9) and var. circinata (n = 4) grouped separately. A cluster of six isolates of Waitea (UWC) did not fall into any known Waitea variety. The binucleate Rhizoctonia-like fungi (BNR) (n = 16) clustered into two groups. Rhizoctonia solani AG 2-2IIIB was the most dominant pathogen in this study, followed by AG 1-IB. There was no relationship between the geographic origin of the isolates and clustering of isolates based on the genetic associations. To our knowledge this is the first time UP-PCR was used to characterize Rhizoctonia, Waitea and Ceratobasidium isolates to their infra-species level.

Dideoxy polymorphism scanning, a gene-based method for marker development for genetic linkage mapping

One of the fastest growing areas of biotechnology research today is marker-assisted breeding of crops. As a prerequisite to marker assisted breeding, genetic linkage maps are currently being developed for many species. For many purposes gene-based markers are the marker type of choice. The biggest problem in genetic linkage mapping using gene-based markers is the identification of polymorphisms between the parents of the population. To improve the efficiency of marker generation, we have developed a simple, and reasonable-cost method of polymorphism detection termed dideoxy polymorphism scanning. Since most of the time required to develop a gene-based linkage map is spent in identification of useful polymorphisms, this method will significantly shorten the time required for map generation and therefore reduce the overall cost.

Colonial Bentgrass Genetic Linkage Mapping

Colonial bentgrass (Agrostis capillaris) is an important turfgrass species used on golf courses in temperate regions, although the related species, creeping bentgrass (A. stolonifera), is often preferred. One of the major management problems for creeping bentgrass is the fungal disease called dollar spot. Colonial bentgrass as a species has good resistance to dollar spot and may be a source of novel genes or alleles that could be used in the improvement of creeping bentgrass. As one approach to ultimately identifying the genes in colonial bentgrass that confer dollar spot resistance, we are developing a genetic linkage map of colonial bentgrass. To provide tools for mapping genes, we have generated expressed sequenced tag (EST) resources for both colonial and creeping bentgrass, and have developed a new approach to gene-based marker development.

Comparative genome analysis between Agrostis stolonifera and members of the Pooideae subfamily, including Brachypodium distachyon.

Creeping bentgrass (Agrostis stolonifera, allotetraploid 2n = 4x = 28) is one of the major cool-season turfgrasses. It is widely used on golf courses due to its tolerance to low mowing and aggressive growth habit. In this study, we investigated genome relationships of creeping bentgrass relative to the Triticeae (a consensus map of Triticum aestivum, T. tauschii, Hordeum vulgare, and H. spontaneum), oat, rice, and ryegrass maps using a common set of 229 EST-RFLP markers. The genome comparisons based on the RFLP markers revealed large-scale chromosomal rearrangements on different numbers of linkage groups (LGs) of creeping bentgrass relative to the Triticeae (3 LGs), oat (4 LGs), and rice (8 LGs). However, we detected no chromosomal rearrangement between creeping bentgrass and ryegrass, suggesting that these recently domesticated species might be closely related, despite their memberships to different Pooideae tribes. In addition, the genome of creeping bentgrass was compared with the complete genome sequence of Brachypodium distachyon in Pooideae subfamily using both sequences of the above-mentioned mapped EST-RFLP markers and sequences of 8,470 publicly available A. stolonifera ESTs (AgEST). We discovered large-scale chromosomal rearrangements on six LGs of creeping bentgrass relative to B. distachyon. Also, a total of 24 syntenic blocks based on 678 orthologus loci were identified between these two grass species. The EST orthologs can be utilized in further comparative mapping of Pooideae species. These results will be useful for genetic improvement of Agrostis species and will provide a better understanding of evolution within Pooideae species.

Comparative Analysis of Disease Resistance Between Ryegrass and Cereal Crops

Perennial ryegrass (Lolium perenne L.) is one of the important forage and turf grasses in temperate zones in the world. Gray leaf spot caused by the fungus Pyricularia oryzae has recently become a serious problem on perennial ryegrass for golf course fairways. The causal agent also causes rice blast disease on rice, as well as foliar diseases on wheat and barley. Crown and stem rust caused by Puccinia spp. are also important for forage- and turf-type perennial ryegrass and seed production. In addition, foliar diseases caused by Bipolaris species, are common and widespread on graminaceous plants. Despite a recent advancement of molecular markers for forage and turf grasses, effective utilization of genetic information available in cereal crops will significantly lead to better understanding of the genetic architecture of disease resistance in ryegrass. Quantitative trait loci (QTL) analysis based on a three-generation interspecific ryegrass population detected a total of 16 QTLs for resistance to the four pathogens. Those QTL were compared with 45 resistance loci for the same or related pathogens previously identified in cereal crops, based on comparative genome analysis using a ryegrass genetic map and a rice physical map. Some pathogen-specific QTLs identified in ryegrass were conserved at corresponding genome regions in cereals but coincidence of QTLs for disease resistance in ryegrass and cereals was not statistically significant at the genome-wide comparison. In conclusion, the conserved synteny of disease resistance loci will facilitate transferring genetic resources for disease resistance between ryegrass and cereals to accommodate breeding needs for developing multiple disease resistance cultivars in ryegrass.