By: Dr. Liu J., Dr. D. Barker

1
Distinguishing Between Four Perennial Ryegrass
Cultivars in Monoculture and in Mixture Using SSR

• By Dr. Liu J., Dr. D. Barker

2
Introduction Plant biodiversity and production

• 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)

3
Introduction cont

• Species diversity mechanisms

More complete utilization of resources Niche
complementality (Tillman, 2001) Stability in
face of environmental fluctuations Buffering
effect (Helland, 2001 )
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
Cultivar Description in BG34 Country of origin
Barlet Diploid. Bred from very diverse genetic base 40 Netherlands
Barmaco Diploid. Late heading variety. Very winter-hardy and persistent 25 Netherlands
Barnhem Diploid. Forms an extremely dense sward 25 Netherlands
Mara Diploid. Most winter hardy 10 Romania
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)
Primer of alleles  Sequence Ref.
M2 7 GGTCTGGTAGACATGCCTC TACCAGCACAGGCAGCAGC Kubic (1999)
10C 9 (AG)10C Ghariani (2003)
10T 10 (AG)10T Ghariani (2003) 

• Reference population

Cult. Barlet Barlet Barmaco Barmaco Barnhem Barnhem Mara Mara
Line 1 2 1 2 1 2 1 2
screened 50 50 50 50 50 50 50 50

• Unknown populations are from three dairy farms
  (Holmes/Wayne Co, OH) sampled Sep 2004

Farm Noyes Gessell Kozak
sampled 100 100 100
10

Analysis of panel population
of each cult has been correctly classified of each cult has been correctly classified of each cult has been correctly classified of each cult has been correctly classified of each cult has been correctly classified
 Loci used Barnhem Barmaco Barlet Mara
10C 66.7 40.0 61.5 56.4
10T 49.4 63.2 60.6 78.0
M2 50.5 46.4 67.0 28.9
10C10T 72.0 71.4 75.6 78.3
10C10TM2 84.0 81.1 86.7 80.9
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

1. By estimation, farms have different cultivar
   structure
2. of confidence is based on the average of
   largest discriminant scores

Estimated number of plants of each cultivar Estimated number of plants of each cultivar Estimated number of plants of each cultivar Estimated number of plants of each cultivar Estimated number of plants of each cultivar Estimated number of plants of each cultivar
Barnhem Barmaco Barlet Mara of confidence
Noyes 37 39 10 14 75.2
Gessell 32 22 39 7 70.3
Kozak 13 37 31 19 78.7
All these farms were planted with BG34, composed
of
Barnhem Barmaco Barlet Mara
25 25 40 10
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