2. SEMINAR –II
DOUBLE HAPLOIDS (DH)
Presenter: Shilpa v malaghan.
Class : Sr. MSc(Agri).
Agriculture collage Raichur.
Date: 7/12/2012.

3. Contents

4. Introduction
Doubled Haploid : An individual with the doubled
chromosome number of the haploid
(Gupta., 2006)

5. What is a doubled haploid plant?
Each cell contains 2 sets of genetic
information which are
(but not exactly) identical most.
For example, one gene set may carry a
gene for disease resistance when the
other set does not.
Doubled haploid plant has
cells containing 2 gene sets
which are exactly identical.
If one gene set has the disease
resistance gene the other gene
also having resistance.

6. History
• Blakeslee et al. (1922) - Datura stramonium
• Guha and Maheswari (1964) - Anther culture technique
for the production of haploids in the laboratory
• Niizeki and Oono (1968) - Production of rice haplpoids
• wide crossing
Kasha and Kao, (1970) - Barley
Burk et al., (1979) - Tobacco
Doubled haploid methodologies have now been applied to
over 250 species - Forster and Thomas, 2007

7. 1) Haploid production
- In vitro versus in vivo
- Maternal versus Paternal
2) Haploid identification
- Markers: Morphological or molecular
- Cytological / flowcytometry
3) Genome doubling
- Colchicine
- Other
Technologies of DH production

9. Identification of haploid kernels using an R1--‐nj marker system

10. Haploid identification
Figure . Flow cytometric analysis of the ploidy level. The x-axis of the
histogram represents the intensity of DNA fluorescence in relative units; the y-
axis represents the number of nuclei counted per histogram channel. (A) A
representative peak set for diploid or doubled haploid material. (B) Peaks
corresponding to a typical haploid individual.
Payam et al., 2007

11. Mechanisms of genome doubling
Genome doubling

12. (Seguí-Simarro et al., 2008)

13. Genome doubling methods.
Spontaneous Chromosome doubling
Artificial Chromosome doubling

14. Spontaneous Chromosome doubling
Lianquan et al., 2011

15. Anti-microtubule drugs
Colchicine
Oryzalin
Amiprophosmethyl(APM)
Trifluralin
Pronamide

16. Stages of application of anti mitotic in DH production
Anther treatment
Microspore treatment
Haploid embryo treatment
Young haploid seedling
treatment
Young haploid Root tip
treatment

17. Fig. The interaction between the colchicine concentration in the pre-
treatment medium and the duration of colchicine pre-treatment on the
ELSs production in the ETH-M82 genotype.
Payam et al., 2007

18. Fig; Haploid embryos in cotyledonary stage (a and b)
normal regenerated plant (c) normal doubled
haploid plant (d)
Haploid embryos treated with colchicine and inoculated in
colchicine free NLN-13 medium for regeneration
Payam et al., 2011

19. Table : Effects of colchicine concentration and duration
treatment on regeneration and recovery of doubled
haploid plants of oilseed rape.
Colchicine
treatment
concentration
(mg/L)
Colchicine
treatment
duration (h)
Number of
regenerated
plants
Number
of doubled
haploid plants
0 (control) 0 53 0
125 12 49 10
24 37 9
36 18 8
250 12 41 13
24 42 27
36 15 9
500 12 17 7
24 14 8
36 9 6
1000 12 0 0
24 0 0
36 0 0

20. Table :percentage rates of doubled haploid (DH) plants
derived from individual treatments
Genotype Total no.
of plants
tested
mean Colchicine
in vivo
Colchicine
In vitro
Oryzalin
In vitro
Trifluralin
In vitro
SL-3/04 560 55.53 71.09 76.62 86.02 80.24
OP-41/1 615 39.34 55.65 77.29 69.80 88.07
SL-2/04 534 31.88 39.94 68.38 43.78 88.67
Mean 1709 42.25 55.56 74.10 66.53 85.66
Miroslav et al., 2008

21. Diploidization frequency of individual treatments
Miroslav et al., 2008

22. Genetics of doubled haploid populations
• Only two types of genotypes – pair of alleles – A ,a
- Frequency – ½ AA and ½ aa
- Diploid – ¼ AA,1/2Aa,1/4aa
• Probability of getting desired genotype is (1/2)n
- Diploid – (¼)n
Kunzel et al., 2000

23. • Selfing in autogymous spp.
• Vegetative propagation
• Bud pollination
• Late or early pollination methods

24. Breeding Using Doubled Haploid System

25. Conventional Breeding System

26. Difference between DH method and conventional method.
Particulars DH method Conventional method
Time required for
developing pure line
1 year or 1 crop
season
3-5 year
Time required for
cultivar development
2-3 year 7-8 year
Fixation of heterosis possible Not possible
Expenditures More than
Conventional method
Less than DH method
Identification of
recessive mutation
Very easy Difficult
Singh., 2007

27. Applications of DHs in Plant Breeding
Mapping Quantitative Trait Loci (QTL)
Backcross breeding
 Bulked segregant analysis (BSA)
Hybrid sorting
 Genetic maps
 Genetic studies
 Elite crossing
 Cultivar development
Fixation of heterosis

28. QTL MAPPING
Type of
Population
Strength Weakness
F2:3
- Speed of production
- d and a estimates
- Heterogeneous families
RIL - Homogeneous families
- Power of QTL detection
- Slow production
DH - Speed of producing
homogeneous families
- Power of QTL detection
- Laborious production process
- Lower recombination (>RIL)
BC - Speed of production - Heterogeneous families

29. QTLs controlling six traits determining root morphology and
distribution in a IR6429 X Azucena doubled-haploid rice population
Yadav et al., 1997

30. Conti….

31. Comparison between QTLs identified in IR64 x
Azucena and Co39 x Moroberekan

32. RFLP linkage map showing chromosomal locations
of QTLs for the three traits
Liu et al., 2006

33. Cont….
Liu et al., 2006

34. Phenotypic performance of parents and the DH population
for biomass yield (BY), straw yield (SY) and grain yield(GY) in
two growth seasons
Liu et al., 2006

35. Backcrossing or Gene Pyramiding
Thomas et al., 2003

36. Trait / Gene Stacking
X
Line 1 Line 2
or
Selfing (F2) DH Induction
Goal: Fixation of target alleles
No. of genes F2 DH
1 0.25 0.5
2 0.0625 0.25
4 0.004 0.0625
8 0.00002 0.004
16 0.00000000002 0.00002
Probability for Fixation of Target Genes

37. Resistant genotype their pedigree, resistance genes and
linked molecular markers Kay et al., 2005

38. strategy I: Scheme of pyramiding BaYMD resistance genes rym4, rym9
and rym11 by two haploidy steps. Kay et al., 2005

39. Strategy II: pyramiding of in one haploid step Kay et al., 2005

40. Total DH lines
lines
Target gene No DH lines
107 DH
(Strategy 1)
rym4 rym9 rym11 20 DH
187 DH
(Strategy 2)
rym4 rym9 rym11 27 DH
DH- plants carrying all possible two gene combinations
Kay et al., 2005

41. Pyramiding the stem rust resistance genes Sr24, Sr26, and
SrR in Westonia background
Mago, et al., 2011

42. pyramiding the stem rust resistance genes Sr24, Sr26, Sr31
and SrR in Pavon background
Mago, et al., 2011

43. Doubled haploid lines with multiple stem rust resistant
genes
Gene combinations Background
cultivar
No. of putative
haploids received
No. of haploids
with genes
No. of DHs
with genes
Sr24–Sr26–SrR Westonia 11 3 0
Sr24–Sr26 Westonia 34 2 2
Sr24–SrR Westonia 30 2 1
Sr26–SrR Westonia 31 4 3
Sr24–Sr31–SrR Pavon 37 10 3
Sr24-Sr26-Sr31 Pavon 20 4 2
Sr24–Sr31 Pavon 40 4 3
Sr24–SrR Pavon 40 2 2
Sr26–Sr31 Pavon 25 2 2

44. Bulked segregant analysis (BSA)
• BSA is dependent on accurate phenotyping and the DH
population has particular advantage in that they are true
breeding and can be tested repeatedly.
• DH populations are commonly used in bulked segregant
analysis, which is a popular method in marker assisted
breeding. This method has been applied to rapeseed and
barley.

45. A linkage map of RAPD markers in a DH population drived
from 'Quantum‘ X 'China A' cross.
Samizadeh et al., 2007

46. PCR profiles produced by RAPD analysis in B. napus with PL
18 (A) and PL 2 (B) primers.
Samizadeh et al., 2007

47. Analysis of variance for pod length marker
Samizadeh et al., 2007
Marker MS R2 Mean ± SE
Long Short
PL2 2202.4 14.60 105.5 ± 22.01 92.10 ±10.03
PL9 712.42 4.70 101.6 ± 20.33 94.10 ±13.27
PL 14 863.62 6.00 102.2 ± 20.22 93.90 ±13.56
PL 17 522.39 3.40 101.7 ± 18.98 95.14 ± 16.12
PL 18 1468.3 9.70 105.2 ± 19.46 93.94 ±12.24
PL 26 1027.4 7.80 100.6 ± 18.98 89.94 ± 8.19
PL 2 × PL 18 1129.6 22.40 112.2 ± 21.86 89.23 ± 5.23

48. Hybrid sorting
• Hybrid sorting - Selection of superior plants among hapliods
derived from F1 through anther culture
• Selection of recombinant superior gametse
• Superior over pedigree & bulk method
- frequency of superior gametes – higher than the
corresponding F2 generations
- Reduces the time required to release a variety
• Successful in china & Japan

49. Varieties bred through hybrid sorting
crop Varieties developed Attributes
Rice Tanfong 1, xin xion,
late keng 76,
shanyou 63
Good quality, high
yield
Wheat Yunhua 1, yunhua 2 Rust resistance,cold
resistance,lodging
resistant
Tobacco Tanyu 1, Tanyu 2,
Tanyu 3
Disease resistant,
Mild smoking

50. SELECTION OF SALT TOLERANCE GENOTYPES FROM
DOUBLED HAPLOIDS IN RICE
Dang et al., 2004

51. Table : Relative response of anther culture lines and
the tolerance checks at salinity of 6dS/m
and 15dS/m.

52. Rice anther culture to obtain DHs with multiple resistance
Bambang et al., 2010
• Released varieties: Way Rarem, Jatiluhar.
• Accessions tolerant to aluminum toxicity( Al): Dupa, Krowal
• Accessions tolerant to shade: Dodakan, ITA-247
Results
• 5 lines – tolerant to Al toxicity, and shade and tolerant to
4 races of blast.
• 11 lines – tolerant to Al toxicity, and tolerant to 4 races
of blast.
• 1 lines – tolerant to shade and tolerant to 4 races of
blast.
• 2 lines – sensitive to Al toxicity, and shade but tolerant
to 4 races of blast.

53. Linkage maps of F2 populations derived from the
cross between two temperate japonica cultivars
‘Koshihikari’ and ‘Akihikari’
Masumi Yamagishi et al.,2010

54. Linkage maps of DH populations derived from the cross between
two temperate japonica cultivars ‘Koshihikari’ and ‘Akihikari’
Masumi Yamagishi, et al

55. Genetic studies - mutation genetics
• Genetic ratios and mutation rates can be read directly
from haploid populations.
• A small doubled haploid (DH) population was used to
demonstrate that a dwarfing gene in barley is located
chromosome 5.
• In another study the segregation of a range of markers
has been analyzed in barley.

56. Means ± standard errors and ranges for erucic acid (% of total fatty
acids) in seeds of the M2 and M3 generations of six doubled haploid
mutant lines of Brassica carinata Barro et al.,2002
Line Plants analysed Generation
M1 M2
Control 10 42.8 ± 0.7 43.5 ± 0.5
40.2–44.4 41.4–44.7
BC2.6.1 8 48.5 ± 0.3 50.6 ± 0.4
47.8–48.7 49.7–51.5
BC2.8.1 6 48.3 ± 0.4 49.6 ± 0.2
48.1–48.9 49.4–49.9
BC3.3.2 8 48.1–48.9 50.3 ± 0.6
47.8–49.8 49.2–51.1
BC5.2.2 10 48.7 ± 0.4 49.7 ± 0.3
48.3–50.1 49.1–50.7
BC6.19 8 49.5 ± 0.6 48.5 ± 0.2
48.8–50.2 48.0–48.8
BC1.5 10 48.7 ± 0.6 48.7 ± 0.1
48.4–49.6 48.5–48.9

57. Dendrogram showing the relationship among 102 DH
wheat based on gliadins bands.
Ojaghi and Akhundova., 2010

58. Based on morphology.

59. Figure . Dendrogram showing the relationship among 102 doubled
haploid wheat based on RAPD
markers

60. Doubled haploid varieties
Crop Varieties Country
Rice Xin-Xin, Hua-Hau-Zao china
Wheat Jing Hua 1,3,5 china
Barley Mingo Canada
Chickpea Quantum, Q2, Duplo, Mingo
Cabbage Orange queen Japan
Brocolli Three man Japan
Tobacco Dan-yu1,2,3 China
Hot pepper Haihua 19 China
Sweet pepper Haihua 29 China
Raina ,1997

61. Elite crossing:
• Traditional breeding methods are slow and take
10–15 years for cultivar development.
• Another disadvantage is inefficiency of selection in
early generations because of heterozygosity.
• These two disadvantages can be over come by DHs,
and more elite crosses can be evaluated and
selected within less time.
• Improved genetic gain can be achieved with DHs .

62. Obtained DH lines from spring and winter
wheat hybrids
Grauda et al.,2010

63. Application of Doubled haploid in Cultivar
development
Singh., 2007
Name of crop No of cultivar released Developed by
Barley 115 Anther culture
Rape seed 47 Anther culture
Wheat 21 Anther culture
Melon 9 irradiation
Capsicum annum 8 Anther culture
Rice 8 Anther culture
Asparagus 7 irradiation
Tobacco 6 Anther culture
Egg plant 5 Anther culture

64. Rao, 2006

65. Double haploid rice varieties in India.
AICRIP and PH-43
has performed well (2000- 05)
CRAC 2224 -1041 (early duration )
PHB71, PA6201, DRRH1, KRH2, Pusa RH 10,
CRHR 4 and Rajlaxmi
2000 DH lines developed

67. G.J.N.Rao 2006

68. Rao, 2006

69. Speed in Line Development
Founder line 1 x Founder line 2
F1
Selfing
Inbred line
DH
Inbred line

70. Advantages
of Doubled Haploid Techniques
• No risk of herterozygosity - Based on gamete selection
• Develop immediate homozygosity, shorten the time to cultivar release -
additive & additive x additive variances
• Provide greater efficiency of selection in plant breeding
cytogenetics
• Production of aneuploids & determine the basic chromosome number
• Improve the precision of genetic and mapping studies
• Accelerate gene pyramiding
• Improve efficacy and efficiency in screening for resistance

71. Some Drawbacks
with Doubled Haploid Production
GENERAL:
– More expensive: expertise, facilities
– Restriction on number of crosses
• SPECIFIC:
– Mutagenic treatment
– Genotype dependent haploid induction
– Low haploid regeneration frequency

73. Video clip