1. Marker Assisted Selection in Soybean Brian Diers University of Illinois

2. Mike Lee states in Advances in Agronomy (1995): “The challenges loom large for marker assisted selection (MAS); in many crops, conventional selection has had several decades to evolve into a very effective technology.”

3. Outline Overview of the soybean industry. Marker assisted selection (MAS) in the private sector. Experiences mining for new genes using MAS in my program. –Soybean cyst nematode (SCN) –Aphid resistance –Yield Intellectual property concerns. Conclusions.

4. Take Home Messages MAS is being done in the private sector on a large scale. Public breeders are using it successfully in discovery, prebreeding, and limited cultivar development. Pick your trait well, MAS is being used successfully for simply inherited traits (defensive traits).

5. US Soybean Germplasm has a Narrow Base Breeders in North America are selecting within an narrow germplasm base. –80% can be traced to 13 ancestral lines. –50% of the northern US germplasm can be traced to 3 ancestral lines. There are likely genes in plant introductions (PIs) that could be used to improve soybean yield and other traits. –The germplasm collection in Urbana contains over 18,000 PIs.

6. From Specht et al. Crop Sci. 39:1560-1570

7. Soybean Cultivars Purchased by Farmers are Mostly From Private Industry In the Midwest, less than 5% of the acreage is planted to public cultivars. Over 90% of the acreage is planted to Roundup Ready cultivars. Reduced emphasis of cultivar development in the public sector. Acres of Certified Seed Produced of Public Cultivars in Illinois

8. MAS in soybean is being done on an industrial scale in private industry

9. MAS in Private Sector Monsanto and Pioneer making 100,000’s of selections / year. Pioneer has used an allele specific hybridization system. Largely being done for disease resistance genes.

10. Marker Assisted Selection In Public Sector MAS on the order of thousands of genotypes a year can be done with relatively inexpensive technology. We use simple sequence repeat (SSR or microsatellite) markers to do several thousand selections / year. –There are a 1,000 mapped SSR markers publicly available.

11. Collect Leaf Tissue into 96 Well Plate

12. Add Extraction Solution

13. Grind Tissue with Paint Shaker

14. Do PCR and Load, Run and Score Gels

15. MAS for Resistance to Soybean Cyst Nematodes (SCN) Most important disease of soybean. Estimate yield loss is 174 million/bu annually. Resistant cultivars are available to growers, perception that they yield less. Phenotype can be highly heritable but is tedious and expensive to test.

16. MAS Selection for SCN Resistance can be Effective PI 88788 source selected with Satt309

17. SCN Resistance from G. soja Can we identify new sources of SCN resistance genes? G. soja is a logical species to look for new resistance genes. In collaboration with Prakash Arelli (USDA-ARS) we mapped to major SCN resistance QTL from PI 468916, a G. soja introduction.

18. LOD Plots of SCN QTL

19. SCN Resistance from G. soja The G. soja resistance QTL on LG E and G were backcrossed into soybean through MAS. BC4 population was tested for SCN resistance and agronomic performance. Selecting for SCN resistance from G. soja in breeding populations.

20. Greenhouse Resistance of Lines in a BC4 Population Segregating for G. soja Resistance

21. Yield Test of a BC4 Population Segregating for G. soja Resistance 5 Environments Across 2 Years Yield Linkage Group P>FG. sojaG. max Other significance E0.00646.645.4 Later mat. (0.75 days) G0.00446.845.4 Greater lod. (0.15 units)

22. SCN Germplasm Release Release of LDX01-1-65 germplasm line. –The line has two new SCN resistance genes from wild soybean crossed into it. –Line was released and requested by 16 private breeders from 12 companies. SCN Female Index YieldMat.Lod.Ht.HG bu/adatescoreIn.Type 0Type 2.5.7 A81-35602238.89-151.64195- LDX01-1-6542.69-182.641143 Dwight49.19-111.039255

23. Soybean Aphid Resistance Soybean aphids became a new soybean pest in North America in 2000. Losses in Illinois and Minnesota were estimated to be in excess of $170 million in 2003. Resistance to soybean aphid was identified in Dowling, a maturity group VIII cultivars released in Texas in 1978. Dowling is not adapted to the Midwest.

24. Aphid Resistance Crossed Dowling with Loda (susceptible adapted cultivar) and tested a F2 population and F2:3 lines for resistance. Made bulks of resistant and susceptible plants and did a bulk segregant analysis to identify linked markers. Found resistance to be single gene and mapped the gene. Using marker assisted selection to aggressively develop cultivars with this resistance.

25. Backcrossing Aphid Resistance x Loda (Susc) Dowling (Res) Test F2 for resistance, map resistance Grow F1plant March 2004

26. Select phenotypically resistant F2 plants that also are homozygous resistant for linked markers. Backcrossing Aphid Resistance RS SS RS RS RR Loda Produce BC1F1 (March 2004) X X Select heterozygous BC1F1 plants based on markers. Loda Produce BC2F1 (July 2004)

27. Backcrossing Aphid Resistance X Select heterozygous BC3F1 plants based on markers. Loda Produce BC4F1 (January 2005) X Select heterozygous BC2F1 plants based on markers. Loda Produce BC3F1 (October 2004) Grow BC2F2 plants, select resistant ones

28. Backcrossing Aphid Resistance Select heterozygous BC5F1 plants based on markers. (June 2005) X Select heterozygous BC4F1 plants based on markers. Loda Produce BC5F1 (March 2004) Grow BC4F2 plants, select resistant ones

29. Backcrossing Aphid Resistance With modest resources, we have been able to produce BC5F1 plants. –March 2004 to June 2005 In the field in 2005 we have: –BC1F2-derived and F2-derived lines in yield tests and resistance evaluations in 4 states. –BC3F2-derived to F2-derived lines in plant rows. –BC5F1, BC4F1s (four backgrounds) and F1s from several other backgrounds.

30. Backcrossing Aphid Resistance Did not use markers to more quickly recover the recurrent parent. –According to theory with a selection of 5% of the BCF1s each generation, we could have selected BC2F1 with a similar amount of genome recovered as a BC3F1. –Theory meets practicality. Difficult to product large number of F1s in soybean.

31. Mapping of Yield QTL Narrow genetic base of soybean makes it likely that there are new genes in soybean germplasm that could increase yield. How to mine these genes? In collaboration with Randy Nelson, my program is mapping yield increasing QTL from exotic germplasm.

32. Mapping of Yield QTL IA 2008 x PI 468916 (Glycine soja) BC2 populations. –Set of 5 backcross 2 populations. Each is predicted to segregate for 25% of the G. soja genome. –Did not use advances backcross methods because of the difficulty in producing F1s. Tested populations for yield across 4 environments in 2 years.

33. Positive Yield QTL from the Adapted Parent a Estimated effect of substituting one allele of one allele of PI 468916 with one allele of IA2008. Analysis based on field evaluations across four environments.

34. Positive Yield QTL from G. soja a Estimated effect of substituting one allele of one allele of PI 468916 with one allele of IA2008.

35. Confirmation Testing Confirmation populations were developed prior the the completion of the study. Random BC2 F 2 plants from the population were crossed with another cultivar. Populations of F 4 -derived lines were developed from these F 1 s. Four populations had regions carrying three ‘moderately significant’ G. soja regions with a positive effect. The G. soja regions were not significant in three populations but was significant in the fourth.

36. Confirmation Testing Linkage Group L QTL The confirmation population was small (50 F4-derived lines)

37. NIL Development Determine which of the F 4 -derived lines from are heterogeneous for the yield QTL region based on markers. F 4 plant would be fixed for 7/8 of the genome

38. Linkage Group L QTL in NIL Two populations of near isogenic lines segregating for the LG L QTL were developed. These were tested in 2003 and no significant marker-yield associations were found.

39. Linkage Group E QTL LG E QTL was backcrossed into A81- 356022 because this region is associated with SCN resistance. The additive effect of the G. soja QTL allele was 1.2 bu/acre yield increase across two environments in 2004. Increase because of SCN resistance? Need to quantify the SCN pressure in the fields.

40. Mining New Genes from Germplasm Collections Difficulty Less Complex Inheritance More Complex Inheritance Difficulty Trait at a low level in elite Trait at a high level in elite

41. Yield QTL vs. Resistance QTL Selection in Elite Germplasm SCN Resistance Yield Know how the QTL entered the gene pool and understand its movement into elite germplasm? YesProbably not Know the allele carried by parents based on phenotype? YesNo

42. Intellectual Property Non-science issues can cloud the use of MAS in breeding programs. Major company has a patent on the major SCN resistance gene. –The royalty rate for using this technology makes it not economical. Public sector moving in the same direction. –Pressure from central administration for Ag Colleges to increase there revenue.

43. Intellectual Property Is the public sector moving to the dark side with patenting? Young plant breeder Intellectual Property Manager I must be true to my profession and work for the public good. You will have riches if you join me and patent your gene.

44. Conclusions Private sector is doing MAS on an industrial level. Public sector does MAS in research and breeding programs. Discovery of useful alleles and MAS for these alleles has been more successful for simply inherited traits (resistance) than complex traits (yield). Intellectual property issues may both hinder and help science.

45. Acknowledgements Funding from: United Soybean Board Contributors: Shawn Carlson Illinois Soybean Checkoff Board Randall Nelson Glen Hartman North Central Soybean Research Program Eileen Kabelka Dechun Wang USDA-NRI

46. Sources of Resistance in Northern Varieties (MG I-IV) Data from Marion Shier “Soybean varieties with soybean cyst nematode resistance, January 2002.”