Single Seed Descent

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!!

Goulden (1941) proposed a similar system (without calling it SSD) and it resulted from the interest of plant breeders to 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.

Also, the pedigree method is based on the premise that progress in obtaining the lines with the required characteristics can be made at the same time as the lines are being selected for homozygosity. His alternative was to separate the inbreeding and selecting generations in order to speed the process along.

By doing this, 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. 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: 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.

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.

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.

In reality, the population size will decrease with each generation (due to lack of germination, lack of seed set, etc.). So if you want 200 F4 plants and 70% of the seeds in each generation will produce plants with at least one seed. Then, by working back to the F2 generation, you need to plant 584 F2 plants. Be sure to take this into account when selecting the number of F2 seeds.

Each single seed traces back to a single F2 plant Each single seed traces back to a single F2 plant. Theoretically, if you start with a large enough F2 sample, then by the F5 generation you will still have a broad representation 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 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.

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.

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. 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 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).

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 Haploid Breeding

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

Maternally Derived Haploids Maize - cross normal color (recessive) female parent x purple color male parent Germinate the F1 seeds Purple seedlings are F1’s Green seedlings are haploids (or selfs)

Paternally Derived Haploids Occur at a very low frequency Not practical for use in a breeding progam

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 (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

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

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