Single Seed Descent and the Doubled Haploid Method

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 offseason nurseries!!

• 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.

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.

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.

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 F 6 generation in 2 years, as opposed to 5 years as with the pedigree method.

Example: Spring Barley • June: Make Initial Cross • Sept: Grow F 1 in GH • Dec: Grow F 2 in GH • March: Grow F 3 in GH • June: Grow F 4 in GH • Sept: Grow F 5 in GH • April: Grow F 6 in field

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

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.

Season 1: F 2 plants grown. One F 3 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.

Season 2: Bulk of F 3 seed is planted. One F 4 seed per plant is harvested and all seeds are bulked. Collect a reserve sample of 1 seed/plant. Season 3: Repeat.

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

Population Development in a Winter Wheat Cross Pioneer 25 R 26 (red) / Pioneer 25 W 60 (white) 2003 300 Random F 3: 4 Planted in GH 2004 F 3: 4 Heads Harvested

Population Development 2004 (cont) F 4: 5 Planted in GH F 4: 5 Plants Harvested F 4: 6 Seed Planted Lexington, KY

Population Development 2005 F 4: 6 Head Hills Harvested F 4: 7 Seed Planted Lexington & Princeton, KY 2006 116 F 4: 7 Inbred Lines Evaluated

Population size will decrease with each generation You want 200 F 4 plants; assume 70% of the seeds in each generation will produce plants with at least one seed. By working back to the F 2 generation, you need to plant 584 F 2 plants.

Each single seed traces back to a single F 2 plant. If you start with a large enough F 2 sample, then by the F 5 generation you will still have an adequate sample of variability from the cross.

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.

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

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!

Single Hill Procedure It can be used to ensure that each F 2 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.

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

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

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.

There may be a reduction in 2 G 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).

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

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

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.

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

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

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

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

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

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?

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

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

From the web: Great Lakes Hybrids, a division of Ag. Reliant 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.

Haploid direct planting

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