Used TetraploidMap to build linkage groups, results were not complete. Used other approaches instead.

1. Microsatellite markers in
Bermudagrass: linkage and QTL
analysis
Sameer Khanal
PGML
docstoc.com

2. Introduction
• Warm-season perennial grasses
• Resilient nature suitable for turf purposes
Google images

3. Biology
• Family: Poaceae
• Subfamily: Chloridoideae (PACC clade)

4. Biology
• Family: Poaceae
• Subfamily: Chloridoideae (PACC clade)
• Genus: Cynodon
• Eight extant species and 10 varieties
(Harlan 1970)
• Major cultivars are triploid hybrids

5. Germplasm
• Common bermudagrass (Cynodon dactylon)
2n=4x=36
• African bermudagrass (Cynodon
transvaalensis)
2n=2x=18
• Hybrids: triploids 2n=3x=27

6. Framework linkage map
113 F1 mapping population
Marker system: RFLP
Mapping strategy: Two-way pseudo-testcross
African
Bermudagrass (T574)
Common
Bermudagrass (T89)

7. RFLP markers
• Bermudagrass derived or heterologous RFLP
probes
• Restriction digestion gels were blotted with
labeled probes

8. RFLP markers
• Polymorphic fragments were scored
• Parental origin of polymorphic fragments were
ascertained

9. Two-way pseudo-testcross
• Open pollinated/heterozygous parents were
crossed
African
Bermudagrass (T574)
Common
Bermudagrass (T89)

10. Two-way pseudo-testcross
• Fragments originating from one parent and
absent in the other parent, but still
segregating
T574

11. Two-way pseudo-testcross
• Fragments originating from one parent and
absent in the other parent, but still
segregating
T89

12. Two-way pseudo-testcross mapping

13. Marker segregation patterns in
autotetraploids
Single dose marker: showing 1:1 segregation
For example:
Parent 1: oo x Aooo :Parent 2
F1: Aoo:ooo (1:1)

14. Marker segregation patterns in
autotetraploids
Double dose marker: showing 5:1 segregation
For example:
Parent 1: oo x AAoo :Parent 2
F1: AAo /Aoo :ooo (5:1)

15. 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

16. 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

17. My research
Addition of simple sequence repeat markers to
the framework linkage map of bermudagrass
QTL analysis of morphological and turf quality
traits

18. 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

19. Phase I: parental screening
• Two genotypes: T574 and T89

20. Phase II: screening a subset
• Parents and 10 F1 progenies were genotyped
T89
T574
F1 progenies

21. Phase III: population screening
• A total of 96 available primer pairs were
screened against two parents and 92 F1s
T89
T574
F1 progenies

22. Marker scoring
• Fragments originating from one parent and
absent in the other parent, but segregating in
the progeny were scored
T89

23. Marker scoring
• Fragments originating from one parent and
absent in the other parent, but segregating in
the progeny were scored
T574

24. Marker scoring
• Fragments originating from both the parents
and segregating (biparental markers) were
also scored
Biparental

25. 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

26. Mapping
• Software tools used:
• TetraploidMap
• OneMap (R environment)

27. 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

28. Segregation analysis

29. Cluster analysis

30. Linkage analysis

31. Phase determination

32. Drawing maps

33. 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

34. TetraploidMap: T574
Lack of repulsion phase linkages in one of the groups

35. 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

36. TetraploidMap: T89
• A total of 282 (62 SSRs, 24 DDRFs) markers
were mapped
- Framework linkage map had 155 SDRFs and
17 DDRFs

37. 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

38. Inferring complete homologous groups
Multilocus probe linked in repulsion
Repulsion phase linkage
Multilocus probe

39. 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

40. Inferring ancestral chromosomes
T574
Consensus 1
T89
Consensus 4

41. Inferring ancestral chromosomes
T574
Consensus 7
T89
Consensus 8

42. Inferring ancestral chromosomes
T574
LG 9
T89
Consensus 4

43. OneMap
• R language based program
• Suitable for diploid outcrossing species
• Does not handle double dose markers
• Routinely used in sugarcane mapping studies

44. OneMap: T574
# mapped
markers
# linkage
groups Total cM
OneMap 115 13 1,364
TetraploidMap 116 20 932
Framework
Map
77 18 973

45. OneMap: T89
# mapped
markers
# linkage
groups Total cM
OneMap 260 32 3,355
TetraploidMap 282 39 1,165
Framework
Map
155 35 1,837

46. Future Works
• Updating the maps (rescored SSRs, changing
input formats)
• Defining homologous groups
• QTL studies of morphological and turf quality
traits