Kermit

1. Single Seed DescentDoubled HaploidsRapid Generation Advancement Stephen Harrison AGRO 4064 Oct 2012 “It’s like taking two finely tuned automobiles– a Ferrari and a Porsche - disassembling them, mixing the parts together, reassembling the mix into one complete vehicle, and expecting it to run better than either original.” (Unknown) Kermit Slide show borrows heavily from: • Dave Van Sanford – University of Kentucky • Wayne Smith – Texas A&M

2. “When one starts tugging at a single thing in nature he finds it attachedto the rest of the world” John Muir

3. 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!! VanSanford - UKY

4. 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. VanSanford - UKY

5. 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.

6. 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.

7. Single Seed Descent Inbreed with one seed from each plant in each generation Select superior line after F6 Crosses with no high heritability traits segregating

8. In our discussions of Pedigree Selection, Mass Selection, and Backcross breeding it is apparent that the primary focus of the first few generations is to inbreed segregating populations and obtain homozygous and homogeneous lines for more focused selection in later generations. • Plant breeding can be divided into two phases: • Generation of a segregating population followed by inbreeding to homozygosity • Selection of elite homozygous lines and seed increase • It takes about 10 generations to develop, test, seed increase, and release a new variety. This often means 10 years. • Plant breeders have always grappled with methods to decrease the time required for variety development. ISSUE:

9. Todays discussion is about methods to obtain pure lines for testing in the shortest amount of time. • The breeder has to decide when a line is pure enough. • Remember that with selfing, heterozygosity within a single plant decreases by 1/2 each generation. • Likewise, with single plant selection each generation, heterogeneity decreases by 1/2, trailing heterozygosity by a generation.

10. 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.

11. 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 • 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.

12. SSD • Charlie Brim (soybean breeder at NC State) popularized the method (modified pedigree system, 1966) with soybean breeders in the 1970s. • SSD as described by Brim is widely used by soybean breeders in conjunction with off-season nurseries for Rapid Generation Advancement. • Frank Kaufman concluded SSD is superior to PS in oats when yield is the objective because: • Larger numbers of selections can be evaluated more thoroughly. • Discuss the LSU oat breeding program of RGA

13. SSD

14. Single Seed Descent • SSD is a system to rapidly develop pure lines followed by selection among those pure lines. • SSD normally involves at least two generations per year with one of those generations in an off-season nursery or greenhouse. • The inbreeding phase of SSD does not have to occur in an environment typical of the one where the final product will be grown. • 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! • There are 3 basic types of SSD • 1. single seed 2. single hill 3. multiple seed

15. Single Seed Descent Variations • Single seed (SSD) • from each plant in a segregating population, e.g. F2, one selfed seed is obtained and bulked within parental population • Single hill (SHD) • several segregating plants, e.g. F2, are grown in a “hill.” Selfed seed are harvested from each plant in the hill and used to establish a hill the following generation; THUS hill identity must be maintained • Multiple seed (MSD) • simply means that the breeder collects > 1 seed per plant, e.g.: • soybean breeders may collect a single pod rather than a single seed • cotton breeders may collect a single boll rather than a single seed.

16. Brim suggested harvesting the 2-3 seeded soybean pod and using 1 seed for planting and 1-2 for reserve. - discuss maturity groups and SSD segregation SSD: 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. • 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.

17. SSD: • 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.

18. Single Seed Descent

19. Single Seed Descent • Genetic Considerations: • expected genotypic frequencies are those of an idealized diploid population without selection • i.e., for a given locus, heterozygosity decreases at (1/2)n • additive genetic variability among plants, i.e. plant to plant variability, increases at the rate of (1+F)(additive genetic variance), where F is the inbreeding coefficient and is equal to 0 in the F2, 0.5 in the F3, 0.75 in the F4, etc. (Recall that the inbreeding coefficient (F) is the probability that the two alleles at a given locus are identical by descent, i.e both inherited from the same parent • no natural selection (unless environment affects germination) • yield potential doesn’t affect SSD since 1 seed (or a few) represents a plant regardless of it’s yield potential

20. Single Seed Descent • Advantages of SSD, SHD, MSD • easy to maintain populations • no natural selection pressure • well suited to greenhouse or winter nursery advancement of gen. • Disadvantages • artificial selection is based on “pure line” or individual plants and not on the progeny performance. THUS there is no accumulation of desirable progeny. • natural selection CAN NOT influence the population in a positive way

21. Winter Rice Nursery in Puerto Rico

22. Winter Soybean Nursery in Puerto Rico

23. Summer Oat Nursery in Puerto Rico

24. Summer Oat Nursery in Puerto Rico

25. Summer Oat Nursery in Puerto Rico

26. Summer Oat Nursery in Puerto Rico

27. Advantages of SSD per se: • requires less time and land than SHD or MSD • max. genetic variability within population since every plant traces to a different F2 plant • Rapid generation advance, maintenance of an unbiased broad germplasm base, labor and time efficient, able to handle large number of samples, and easily modified! • Useful to develop RILs for mapping • Disadvantages of SSD per se: • every F2 plant may not be advanced due to germination failure • must adjust size of populations for germination percent • requires more time at harvest than MSD because you must obtain one sample to plant the following generation and one reserve • Must be sure and harvest seed to plant the following generation AND seed to to hold in reserve in case of crop failure. This is an issue in SSD and SHD but usually less so in MSD.

28. SSD • 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

29. Advantages of Single Seed Descent Method • Requires little space • Saves time. Extensive field trials are not required. • Little record keeping • Fast especially with multiple generations / year • Easy method to handle populations, handles large number of crosses • Good for traits with low heritability • Natural selection has little influence • Disadvantages of Single Seed Descent Method • Little scope for natural selection • Maximum productivity is established in F2 generations, superior plants may be lost • No recombination occurrence among selected lines • Must evaluate large numbers of inbred lines to find out superior ones

30. Multiple Seed Procedure • 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 2Gbut 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).

31. Single Hill Descent (modified pedigree)

32. Single Hill Descent (modified pedigree) • 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.

33. Advantages of SHD per se: • every plant traces to a different F2 and thus variability is maximized • Lots of seed as remnant • Disadvantages of SHD per se: • requires more time at planting and harvest • requires more land than SSD or MSD • requires more record keeping (e.g. identification of individual hills rather than simply a bulk within each parental combination)

34. http://www.kursus.kvl.dk/shares/plantbreed/previous%20years%20(Danish)/200_aktiviteter2004/_50_Background/SelfBreed/Dias.ppt#256,1,Breeding self-pollinated crops Sven Bode Andersen Double Haploid Inbreed via Doubled haploids Select superior lines directly from DH offspring Preselection if DHs expensive

35. 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.

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

37. 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 • H Bulbosum • Corn pollen with wheat

38. Interspecific Crosses Hordeumbulbosummethed: • 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

39. 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

40. 42 21 Colchicine

42. Heartland Plant Innovations http://www.youtube.com/watch?v=zwPrj05eEk8&feature=player_embedded#!

43. 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 Commonly used with rice

44. Anther Culture

45. 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? Somaclonal Variation

46. 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

47. 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

48. 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 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.

49. Haploid direct planting

50. Points to Consider • The advantages of techniques such as anther culture are minimized when several generations per year can be grown in a RGA system. • DH techniques require a lab staffed with experienced technicians and need to run full-time to be efficient. • Some species are much easier than others. • It cost about $3 per DH plant at Heartland Plant Innovations. That is about $5,000 per populations. The average breeding program has 250 – 500 new populations every year. • The LSUAC rice breeding program essentially abandoned antler culture after 10 years. It worked fairly well but was expensive to maintain and was no more effective than a South American winter nursery.