1. Single Seed Descent and the Doubled Haploid Method

2. SSD
Single seed descent can be used in self or cross pollinated crops. It is a method of inbreeding a segregating population that is quite conducive to environments that are not typical : good news for off- season nurseries!!

3. • Goulden (1941) proposed a similar system (without calling it SSD) : rapidly inbreed populations before evaluating individual lines. • He noted that a wheat breeding program could be divided into the development of pure lines from a segregating populations and selection among the best of those lines.

4. He emphasized that with the pedigree method, plants had to be grown in an environment in which genetic differences would be expressed for the characters under selection; and thus probably limited to one generation per year.

5. The pedigree method is based on the premise that selection progress can be made at the same time as the lines are being inbred to homozygosity. Goulden’s alternative was to separate the inbreeding and selecting generations in order to speed the process along.

6. The number of progeny grown from a plant in each generation should be one or two only, and two generations can be grown in the greenhouse and one in the field. He proposed the model with spring-sown cereals. In this manner, he could attain the F6 generation in 2 years, as opposed to 5 years as with the pedigree method.

7. Example: Spring Barley
June: Make Initial Cross
Sept: Grow F1 in GH
Dec: Grow F2 in GH
March: Grow F3 in GH
June: Grow F4 in GH
Sept: Grow F5 in GH
April: Grow F6 in field

8. After the desired level of homozygosity was achieved, the lines could then be tested for desired characteristics.

9. Single Seed Procedure This is the classic procedure of having a single seed from each plant, bulking the individual seeds, and planting out the next generation.

10. Season 1: F2 plants grown. One F3 seed per plant is harvested and all seeds are bulked. Collect a reserve sample of 1 seed/plant. Brim suggested harvesting the 2-3 seeded soybean pod and using 1 seed for planting and 1-2 for reserve.

11. Season 2: Bulk of F3 seed is planted
Season 2: Bulk of F3 seed is planted. One F4 seed per plant is harvested and all seeds are bulked. Collect a reserve sample of 1 seed/plant. Season 3: Repeat.

12. Season 4: Grow bulk of F5 seed and harvest individual plants separately. Season 5: Grow F5:6 lines in rows; select among rows and harvest selected rows in bulk. Season 6: Begin extensive testing of F5 derived lines.

13. Population Development in a Winter Wheat Cross
Pioneer 25R26 (red) / Pioneer 25W60 (white) Random F3 F3:4 Planted in GH 2004 F3:4 Heads Harvested
R=Red
W=White
Talk about more later—looking at both R and W--
Of 786 lines characterized at Wooster-2 extremes=ideal pop’n for investigation of gluten strength
SSD was used to rapidly advance population while maintaining genetic variation
SSD not common in soft winter wheat-but in order to accommodate PhD project was used to rapidly produce homozygous lines—therefore
have progeny that can be replicated
1 seed randomly selected from each head and planted

14. Population Development
2004 (cont) F4:5 Planted in GH
F4:5 Plants Harvested
F 4:6 Seed Planted
Lexington, KY
F4:6 lines planted in single rep as head hills at 1 location

15. Population Development
2005 F 4:6 Head Hills Harvested F4:7 Seed Planted Lexington & Princeton, KY F4:7 Inbred Lines Evaluated
F4:7 seed planted in single head rows as RCB at 2 locs w/ 2 reps

16. Population size will decrease with each generation You want 200 F4 plants; assume 70% of the seeds in each generation will produce plants with at least one seed. By working back to the F2 generation, you need to plant 584 F2 plants.

17. Each single seed traces back to a single F2 plant
Each single seed traces back to a single F2 plant. If you start with a large enough F2 sample, then by the F5 generation you will still have an adequate sample of variability from the cross.

18. As stated previously, the breeder must expect the genotypic frequencies in a bulk population to change during the propagation period. To eliminate, or at least reduce, shifts in genotypic frequencies in bulk populations, Brim (1966) proposed using the Modified Pedigree Method – a modification of the SSD.

19. The true single seed descent method maintains the total genotypic array. The modified pedigree is similar but allows some selection during inbreeding.

20. SSD’s Bonus Points: Rapid generation advance, maintenance of an unbiased broad germplasm base, labor and time efficient, able to handle large number of samples, and easily modified!

21. Single Hill Procedure It can be used to ensure that each F2 plant will have progeny in the next generation of inbreeding. Progeny from individual plants are maintained as separate lines during each generations by using a few seeds per hill and harvesting those to plant back the following year.

22. Multiple Seed Procedure To avoid starting with a large F2 population that compensates for loss of seed over generations, bulk 2-3 seeds per plant at harvest.

23. Genetic Considerations 1) Additive genetic variation among individuals increased at a rate of (1+F)2A where F=0 in F2. There is little natural selection, except for seed germination potential or where the environment prevents some genotypes from setting seed.

24. In multiple seed procedure, there may be a variation associated with sampling of seed from a bulk samples to plant the next generation. This sampling results in exclusion of progeny from some plants, and multiple representation of progeny from others.

25. There may be a reduction in 2G but is slight
There may be a reduction in 2G but is slight. Multiple seed descent indicated about 18% of the lines were due to repetitive sampling and did not represent independent lineages. (See Keim et al., 1994, Crop Sci. 34:55-61).

26. Pros Easy way to maintain pops during inbreeding
Natural selection does not influence pops
Well suited to GH and off season nurseries

27. Cons
Selection based on individual phenotype rather than progeny performance
Natural selection cannot influence pop in a positive manner

28. Doubled Haploids What are they?
Homozygous diploid lines that come from doubling the chromosome number of haploid individuals.
Heterozygous haploid individuals are produced, the chromosome number doubled, and an array of inbred homozygotes results.

29. Doubled Haploids Where do the haploids come from? Naturally occurring
Maternally derived
Paternally derived

30. Interspecific Crosses
Concept: Make a very wide cross
Use the species of interest as female
Fertilization Occurs
Chromosomes of wild species are eliminated
Use embryo rescue to recover haploid embryo

31. Interspecific Crosses
Hordeum bulbosum method:
Emasculate H. vulgare (2n=2x=14)
Pollinate with H. bulbosum (2n=2x=14)
Treat with hormones
Culture embryo
Treat seedlings with colchicine
Place in pots, harvest selfed seed

32. Interspecific Crosses
Wheat x maize method
Emasculate wheat plant
Pollinate with fresh maize pollen
After several days maize chromosomes eliminated
Rescue embryo and place in culture
Treat seedling with colchicine, harvest selfed seed

33. Anther culture
Anthers, or in some cases, microspores (pollen cells) can generate haploids
Haploids are grown in tissue culture
Callus is induced to differentiate through hormone treatments
Plantlets are obtained and treated with colchicine
Selfed seed harvested

34. Anther culture
In tobacco, found that anther derived di-haploids (doubled haploids) were more variable and less fit than SSD lines
Why?
Residual heterozygosity?
Alterations brought about through tissue culture?

35. Pros Homozygosity achieved rapidly
Selection among homozygotes more efficient than selection among heterozygotes
Homozygous, homogeneous seed source available for release
Dominance not a problem when selecting among haploids

36. Cons Requires a “well-oiled machine” method of producing haploids
Evaluation of inbred lines will require at least as much time as usual
May be problems among the DH ( tobacco example)
Not feasible to use with all of your populations
Frequency of haploid production impossible to predict

37. From the web:
Great Lakes Hybrids, a division of AgReliant Genetics LLC,
has accelerated hybrid development with innovative doubled-haploid
breeding technology. This approach produces pure parent lines
in one year, compared to three to five years using conventional
methods.
The doubled-haploid method is much faster and can produce a new,
genetically stable inbred line in one year. The plants in the genetic
population (germplasm pool) are pollinated with a haploid inducer.
When the harvested kernels are planted, they produce haploid plants.

38. Haploid direct planting

39. Use of DH in Recurrent Selection
Griffing (TAG 46:367 -) shows that if an efficient DH extraction method can be devised DH based selection will be much more efficient than diploid selection
In the case of individual selection, given certain parameter values, theory says that individual DH selection can be ~ 6 times as efficient as individual diploid selection