Post on 15-Jul-2015
SEMINAR –IIDOUBLE HAPLOIDS (DH)
Presenter: Shilpa v malaghan.
Class : Sr. MSc(Agri).
Agriculture collage Raichur.
Date: 7/12/2012.
Contents
Introduction
Doubled Haploid : An individual with the doubled
chromosome number of the haploid
(Gupta., 2006)
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.
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
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
Identification of haploid kernels using an R1--‐nj marker system
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
Mechanisms of genome doubling
Genome doubling
(Seguí-Simarro et al., 2008)
Genome doubling methods.
Spontaneous Chromosome doubling
Artificial Chromosome doubling
Spontaneous Chromosome doublingLianquan et al., 2011
Anti-microtubule drugs
Colchicine
Oryzalin
Amiprophosmethyl(APM)
Trifluralin
Pronamide
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
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
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
Table : Effects of colchicine concentration and duration treatment on regeneration and recovery of doubled
haploid plants of oilseed rape.
Colchicinetreatment
concentration(mg/L)
Colchicinetreatment
duration (h)
Number ofregenerated
plants
Numberof 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
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
OryzalinIn vitro
TrifluralinIn 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
Diploidization frequency of individual treatments
Miroslav et al., 2008
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
• Selfing in autogymous spp.
• Vegetative propagation
• Bud pollination
• Late or early pollination methods
Breeding Using Doubled Haploid System
Conventional Breeding System
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
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 developmentFixation of heterosis
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
QTLs controlling six traits determining root morphology and distribution in a IR6429 X Azucena doubled-haploid rice population
Yadav et al., 1997
Conti….
Comparison between QTLs identified in IR64 x
Azucena and Co39 x Moroberekan
RFLP linkage map showing chromosomal locations of QTLs for the three traits
Liu et al., 2006
Cont….
Liu et al., 2006
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
Backcrossing or Gene Pyramiding
Thomas et al., 2003
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
Resistant genotype their pedigree, resistance genes and linked molecular markers Kay et al., 2005
strategy I: Scheme of pyramiding BaYMD resistance genes rym4, rym9 and rym11 by two haploidy steps. Kay et al., 2005
Strategy II: pyramiding of in one haploid step Kay et al., 2005
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
Pyramiding the stem rust resistance genes Sr24, Sr26, and SrR in Westonia background
Mago, et al., 2011
pyramiding the stem rust resistance genes Sr24, Sr26, Sr31 and SrR in Pavon background
Mago, et al., 2011
Doubled haploid lines with multiple stem rust resistant genes
Gene combinations Backgroundcultivar
No. of putativehaploids received
No. of haploidswith genes
No. of DHswith 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
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.
A linkage map of RAPD markers in a DH population drivedfrom 'Quantum‘ X 'China A' cross.
Samizadeh et al., 2007
PCR profiles produced by RAPD analysis in B. napus with PL 18 (A) and PL 2 (B) primers.
Samizadeh et al., 2007
Analysis of variance for pod length markerSamizadeh 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
Hybrid sorting
• Hybrid sorting - Selection of superior plants among hapliodsderived 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
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
SELECTION OF SALT TOLERANCE GENOTYPES FROM DOUBLED HAPLOIDS IN RICE
Dang et al., 2004
Table : Relative response of anther culture lines and the tolerance checks at salinity of 6dS/m
and 15dS/m.
Rice anther culture to obtain DHs with multiple resistanceBambang 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.
Linkage maps of F2 populations derived from the cross between two temperate japonica cultivars
‘Koshihikari’ and ‘Akihikari’
Masumi Yamagishi et al.,2010
Linkage maps of DH populations derived from the cross between two temperate japonica cultivars ‘Koshihikari’ and ‘Akihikari’
Masumi Yamagishi, et al
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.
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
Dendrogram showing the relationship among 102 DH wheat based on gliadins bands.
Ojaghi and Akhundova., 2010
Based on morphology.
Figure . Dendrogram showing the relationship among 102 doubled haploid wheat based on RAPD
markers
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
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 .
Obtained DH lines from spring and winter wheat hybrids
Grauda et al.,2010
Application of Doubled haploid in Cultivar development
Singh., 2007Name 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
Rao, 2006
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
G.J.N.Rao 2006
Rao, 2006
Speed in Line Development
Founder line 1 x Founder line 2
F1Selfing
Inbred line
DH
Inbred line
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
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
Video clip