Distinguishing Between Four Perennial Ryegrass Cultivars in Monoculture and in Mixture Using SSR

1. Distinguishing Between Four Perennial Ryegrass Cultivars in Monoculture and in Mixture Using SSR By: Dr. Liu J., Dr. D. Barker

2. Species diversity Darwin (1859) first proposed a connection between biodiversity and ecosystem functioning. This topic resurfaced recently (since 1982, over 100 studies on it). Existing experiments indicate that biodiversity enhances production, though exceptions exist. (Tilman, et al, 1997) But biodiversity should also include within-species diversity Cultivar diversity (within species) Greater and more stable yield of blends than the components pure line have been found in many other crop species (Smithson, 1996). Mixtures of perennial ryegrass cultivars were found to give higher yield than monoculture at some harvests (Thomson, 1969) Introduction Plant biodiversity and production

3. Introduction cont’ More complete utilization of resources “Niche complementality” (Tillman, 2001) Stability in face of environmental fluctuations “Buffering effect” (Helland, 2001 ) Species diversity mechanisms: Cultivar diversity mechanisms : ?

4. Perennial ryegrass (Lolium perenne  L.)- Family: Poaceae; genus: Lolium- Forage & turf - Bunch type, easy to clone - Self-incompatible, usually synthetic variety - BG34 is a blend of 4 ryegrass cultivars

5. Why SSR? Widely dispersed and genetically well defined, ideal for diversity measurement Highly polymorphic and this polymorphism is easily assayed by PCR, useful for distinguishing among closely related varieties SSR locus is co-dominant, and very genetically informative

6. Simple Sequence Repeats (SSR) • Short stretches of tandomly repeated nucleotide (2, 3 or 4) motifs • SSR polymorphism is the variation in number of repeats • The length polymorphism can be monitored with PCR primers that flank the SSR

7. ISSR: Inter-simple Sequence Repeats, a modification of SSR • Single primer targets the repeat per se. • No flanking genomic sequence info required • Highly reproducible Reproducibility evaluation Two leaf blades of one plant were sampled for DNA extraction and following PCR amplification to evaluate the reproducibility and accuracy of panel assay 1000 500

8. Sample gel bands results ---Primer (AG)10C Barnhem 1 -5 Barmaco 1 - 5 Barlet 1 - 5 Mara1 - 5 Polymorphic bands were scored for presence(1) or absence (0) Discriminant analysis (of SAS) was used to calculate similarity distance 1000 500 B1 - B5 Mark C1 - C5 D1 - D5 Mark E1 - E5

9. SSR primer proven to be useful (out of many published data for ryegrass): • Reference population: • Unknown populations are from three dairy farms (Holmes/Wayne Co, OH) sampled Sep 2004:

10. Analysis of panel population Accuracy of assignment improved as number of loci increased. For Mara and Barlet, in-lines were more similar For Barnhem and Barmaco, in-lines were different

11. Analysis of field samples • By estimation, farms have different cultivar structure • % of confidence is based on the average of largest discriminant scores All these farms were planted with BG34, composed of %:

12. Acknowledgement We thank: • Dr J.C. Jang for providing his lab and all facilities needed for SSR work. • Dr Mark Sulc and Dr Guo-liang Wang for technical advice and support • Barenbrug seed Co. for providing seed of BG34 cultivars and lines