Post on 30-Dec-2015
description
transcript
By: Dr. Liu J., Dr. D. Barker
Distinguishing Between Four Distinguishing Between Four Perennial Ryegrass Cultivars in Perennial Ryegrass Cultivars in
Monoculture and in Mixture Using Monoculture and in Mixture Using SSRSSR
Introduction 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)
Introduction cont’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 :
?
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
Why SSR?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
Amplify repeat region by polymerase chain reaction (PCR)
Analyze PCR products by agrose gel electrophoresis
Number of repeats is highly variable among individuals
= Repeat (e.g., ag)Unique flanking regions Structure
Design primers ( ) complementary to flanking regions
Simple Sequence Repeats Simple Sequence Repeats (SSR)(SSR)
Short stretches of tandomly repeated nucleotide (2, 3 or 4) motifsSSR polymorphism is the variation in number of repeatsThe length polymorphism can be monitored with PCR primers that
flank the SSR
ISSR: Inter-simple Sequence Repeats, a ISSR: Inter-simple Sequence Repeats, a modification of SSRmodification 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
Sample gel bands results ---Primer (AG)10C
Barnhem 1 -5 Barmaco 1 - 5
B1 - B5 Mark C1 - C5 D1 - D5 Mark E1 - E5
Polymorphic bands were scored for presence(1) or absence (0)
Discriminant analysis (of SAS) was used to calculate similarity distance
A 1 A 2 …Barnhem 1 1 1 …Barnhem 2 0 0 …… … … …Barmaco 1 1 0 …Barmaco 2 0 1 …… … … …Barlet 1 1 0 …Barlet 2 1 1 …… … … …Mara 1 1 1 …Mara 2 1 1 …… … … …
1000
500
Barlet 1 - 5 Mara1 - 5
Cult. Barlet Barmaco Barnhem Mara
Line 1 2 1 2 1 2 1 2
# screened 50 50 50 50 50 50 50 50
Farm Noyes Gessell Kozak
# sampled 100 100 100
Primer # of alleles Sequence Ref.
M2 7 GGTCTGGTAGACATGCCTCTACCAGCACAGGCAGCAGC
Kubic (1999)
10C 9 (AG)10C Ghariani (2003)
10T 10 (AG)10T Ghariani (2003)
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:
% 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
10C+10T 72.0 71.4 75.6 78.3
10C+10T+M2 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
Analysis of panel population
Analysis of field samples
Barnhem Barmaco Barlet Mara
25 25 40 10
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%
1) By estimation, farms have different cultivar structure
2) % of confidence is based on the average of largest discriminant scores
All these farms were planted with BG34, composed of %:
AcknowledgementAcknowledgementWe 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