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Microsatellite markers in Bermudagrass: linkage and QTL analysis
Sameer Khanal
PGML
docstoc.com
Introduction
• Warm-season perennial grasses
• Resilient nature suitable for turf purposes
Google images
Biology
• Family: Poaceae
• Subfamily: Chloridoideae (PACC clade)
Biology
• Family: Poaceae
• Subfamily: Chloridoideae (PACC clade)
• Genus: Cynodon
• Eight extant species and 10 varieties
(Harlan 1970)
• Major cultivars are triploid hybrids
Germplasm
• Common bermudagrass (Cynodon dactylon)
2n=4x=36
• African bermudagrass (Cynodon transvaalensis)
2n=2x=18
• Hybrids: triploids 2n=3x=27
Framework linkage map
113 F1 mapping population
Marker system: RFLP
Mapping strategy: Two-way pseudo-testcross
African
Bermudagrass (T574)
Common
Bermudagrass (T89)
RFLP markers
• Bermudagrass derived or heterologous RFLP probes
• Restriction digestion gels were blotted with labeled probes
RFLP markers
• Polymorphic fragments were scored
• Parental origin of polymorphic fragments were ascertained
Two-way pseudo-testcross
• Open pollinated/heterozygous parents were crossed
African
Bermudagrass (T574)
Common
Bermudagrass (T89)
Two-way pseudo-testcross
• Fragments originating from one parent and absent in the other parent, but still segregating
T574
Two-way pseudo-testcross
• Fragments originating from one parent and absent in the other parent, but still segregating
T89
Two-way pseudo-testcross mapping
Marker segregation patterns in autotetraploids
Single dose marker: showing 1:1 segregationFor example: Parent 1: oo x Aooo :Parent 2
F1: Aoo:ooo (1:1)
Marker segregation patterns in autotetraploids
Double dose marker: showing 5:1 segregationFor example: Parent 1: oo x AAoo :Parent 2
F1: AAo /Aoo :ooo (5:1)
Framework linkage map
T574 (Diploid): 77 single dose restriction fragments on 18 linkage groups
Seven homologous groups
T89 (Tetraploid): 155 single dose + 17 double dose markers on 35 linkage groups
Four homologous and 4 partial homologous groups identified
Further development
Twenty microsatellite loci + 4 resistant gene analogs were added to this framework
Yet,
Marker coverage is less than 70%
Homologous groups not completely linked
My research
Addition of simple sequence repeat markers to the framework linkage map of bermudagrass
QTL analysis of morphological and turf quality traits
SSR resource
Primers from sugarcane EST templates available (total no. of EST-SSRs: 1,240)
Sugarcane markers have been mapped to several grass species
Further, EST-SSRs are shown to be highly transferable among different grass species
Phase I: parental screening
• Two genotypes: T574 and T89
Phase II: screening a subset
• Parents and 10 F1 progenies were genotyped
T89
T574 F1 progenies
Phase III: population screening
• A total of 96 available primer pairs were screened against two parents and 92 F1s
T89
T574F1 progenies
Marker scoring
• Fragments originating from one parent and absent in the other parent, but segregating in the progeny were scored
T89
Marker scoring
• Fragments originating from one parent and absent in the other parent, but segregating in the progeny were scored
T574
Marker scoring
• Fragments originating from both the parents and segregating (biparental markers) were also scored
Biparental
Marker scoring
• A total of 120 SSR marker loci were scored
Markers # SSR loci # RFLP loci
T89 73 270 SDRF + 64 DDRF
T574 36 103
Biparental 11 0
Mapping
• Software tools used:
• TetraploidMap
• OneMap (R environment)
TetraploidMap
• Handles two-way pseudo testcross data
• Dominant and codominant markers
• Single/double dose, and biparental markers
• Four steps: 1. Segregation analysis
2. Cluster analysis
3. Linkage analysis
4. Linkage phase and maps
Segregation analysis
Cluster analysis
Linkage analysis
Phase determination
Drawing maps
TetraploidMap: T574
• A total of 116 (30 SSRs) markers were mapped
- Framework linkage map had 77 markers
• Map is composed of 20 linkage groups, 17 of them organized into 8 homologous groups
- Framework linkage map had 18 linkage groups, 15 of them organized into 7 homologous group
TetraploidMap: T574
Lack of repulsion phase linkages in one of the groups
TetraploidMap: T574• Map covers a total of 932 cM with 116 markers
separated by an average of 9.6 cM
- Framework map: 973.4 cM with 77 markers separated by an average of 16.5 cM
• Linkage groups are defined by about 6 markers and cover ~50 cM
- Framework map: 4 markers and cover ~54 cM
TetraploidMap: T89
• A total of 282 (62 SSRs, 24 DDRFs) markers were mapped
- Framework linkage map had 155 SDRFs and 17 DDRFs
TetraploidMap: T89
• Map is composed of 39 linkage groups, 34 of them organized into 15 partial homologous groups (of 2-3 linkage groups each)
- Framework map: 35 linkage groups, 16 organized into four complete homologous groups and eight into partial homologous groups
Inferring complete homologous groups Multilocus probe linked in repulsion
Repulsion phase linkage
Multilocus probe
TetraploidMap: T89
• Map covers a total of 1,165 cM with 282 markers separated by an average of 9 cM
- Framework map: 1837.3 cM with 155 markers separated by an average of 15.3 cM
• Linkage groups are defined by about 9 markers and cover ~70 cM
- Framework map: 4 markers and cover ~52.5 cM
Inferring ancestral chromosomes
T574Consensus 1
T89Consensus 4
Inferring ancestral chromosomes
T574Consensus 7
T89Consensus 8
Inferring ancestral chromosomes
T574LG 9
T89Consensus 4
OneMap
• R language based program
• Suitable for diploid outcrossing species
• Does not handle double dose markers
• Routinely used in sugarcane mapping studies
OneMap: T574
# mapped markers
# linkagegroups Total cM
OneMap 115 13 1,364
TetraploidMap 116 20 932
Framework Map
77 18 973
OneMap: T89
# mapped markers
# linkage groups Total cM
OneMap 260 32 3,355
TetraploidMap 282 39 1,165
Framework Map
155 35 1,837
Future Works
• Updating the maps (rescored SSRs, changing input formats)
• Defining homologous groups
• QTL studies of morphological and turf quality traits
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