1. What are gametophytic factors? And why is there an interest in them? Gametophytic factors limit the types of pollen that can effect fertilization. They can be used to screen out unwanted pollen sources.

2. Examples include: Eliminating pollen of yellow corn from white corn production fields. Preventing field corn from pollinating popcorn. Preventing undesirable sweet corn combinations (su/sh2, etc.). Preventing transgenics from crossing with organics.

3. The best known and most widely used such factor is Ga1-s. It has been used to protect popcorn quality and is often referred to as “Dent Sterile”. Ga1-s effectively blocks normal pollen when homozygous.

4. NC State has released 24 Ga1-s inbreds since 1990. NC296 has been described as the line with the highest yield potential of all lines that allow “freedom to operate”. NC296 is Ga1-s.

5. NC296 x 5 Pioneer sister-line testers (2 yr., 8 env.)YLD H 2 O EP HYBRIDS t/ha % % 3 PHI SS x NC2969.40 20.0 98 2 PHI NS x NC2969.06 19.9 97 DeKalb 697 8.76 19.6 93 Pioneer 31D588.81 19.2 99 Pioneer 31G669.10 19.1 99 Pioneer 31P418.92 19.0 100 Pairwise.05 LSD0.69 0.7 5 C.V.% 8 4 5.

6. NC Line Source NC Line Source. NC296 105.5 NC444 258.296*4 NC296A 105.5 NC448 105.5x155 NC302 105.5x101 NC460 298.296 NC336 105.5 NC462 304.296 NC338 105.5x304.504 NC464 300.348 NC346 105.5 NC466 298.7905 NC348 105.5x155 NC470 B73p.296)2 NC352 105.5 NC490 258.296 NC354 105.5x304.101 NC494 258.'352' NC396 105.5x101 NC514 258.296*3 NC400 300.348 NC520 258.296 NC440 B73.296*4 NC522 258.296.

7. To test for cross contamination of Ga1-s hybrids, we grew enough various homozygous Ga1-s hybrids in an iso to produce a minimum of 220,000 kernels if pollinated well. The male ‘parent’ was P33M54. All Ga1-s plants were detasselled. Only 110 kernels were harvested for a contamination rate of 0.05%.

8. But can Ga1-s combinations be found that yield (and stand up)? We had never knowingly crossed Ga1-s lines until our organic folks decided that gametophytic hybrids might be an alternative to the 108 day organic hybrids being used for isolation from 115 to118 day transgenics.

9. Data were available for the 24 NC- Ga1-s lines. We took that data and tested various Ga1-s hybrids of all- NC origin. The results were not surprising.

10. (2yr, 10 env) YLD % % PEDIGREE B/A H2O EP NC522 x NC338 100 21.0 71 DeKalb 697 131 18.9 81 Garst 8288 118 19.8 86 LH132 x LH51 109 17.9 81 NC320 x NC368 133 18.8 77 Pioneer 31D58 130 19.1 86 Pioneer 31G66 136 18.2 83 Pioneer 31P41 136 19.2 86 Pioneer 33M54 125 18.9 86 LSD.05 ENTRY x LOC) 11 0.8 8 C.V.% (ENTRY x LOC) 12 5 12

11. What are we trying now: Crossing our better Ga1-s lines with other materials to lower the average coefficient of parentage. OTHER MATERIALS INCLUDE: “Better” ex-PVP lines Ga1-s synthetics from Frank Kutka Ga1-s lines from Walter Goldstein

12. (2013, 5 locations) YLD % % PEDIGREE B/A H 2 O EP 296xG35.296 S3 x T11 153 17.6 95 P3737.NC354BC3S2 x P33M54159 19.5 94 NC302.NC354 x BR71PM50 146 19.3 99 P3737.NC492*2 x NC464 130 19.8 90 Blue River 71PM50 119 17.2 98 DeKalb 697 161 18.5 98 DKHBA1.NC476xNC368.320 146 17.5 96 Pioneer 31G66 162 18.4 99 Pioneer 33M54 154 17.9 100 LSD.05 (ENTRY x LOC) 13 0.5 7 C. V. % (ENTRY x LOC) 7 2 6 T11 = FR1064.LH132

13. At that point, Jesus Sanchez from the University of Guadalajara reported a very high rate of Ga1-M in Mexican land races and Mexican hybrids. Ga1-M is a ‘promiscuous’ allele. Its carriers pollinate all and are receptive to pollen from all other Ga1 sources.

14. Ga1-M in a hybrid would easily overcome Ga1-s isolation. Our initial estimates were that this might happen in 20 to 40 years. BUT we have released 10+ Ga1-M inbreds, there is one ex-PVP inbred with Ga1-M, and Tom Hoegemeyer (who holds a Ga1 patent himself) reports that there is a 114 day Pioneer hybrid in Nebraska with Ga1-M.

15. NC Line Ga1-M Source 300 306.5x105 340 306.5x105 356 TROPHY 390 TROPHY 396 105.5x101 416 '300'.262A 442 258.296*4 458 KU2301.PM703 468 TROPHY 512 QK37x105.155 358 & 394, both from TROPHY, vary.

16. As a possible alternative to Ga1-s, Jerry Kermicle has developed (and patented) Tcb1, a dominant crossing barrier from teosinte. We are still backcrossing Tcb1 into numerous lines, but the initial yield trials suggest serious yield drag.

17. 2 yr, 10 env YLD % % PEDIGREE B/A MOIS EP P4639.Tcb1*2 x P33M54 116 17.9 95 NC522.Tcb1*2 x P33M54 106 18.1 95 NC320 x NC368 157 18.5 98 Pioneer 33M54 144 18.1 100 LSD.05(ENTRY x LOC) 10 0.3 4 C.V.% (ENTRY x LOC) 10 2 5

18. Our situation is roughly as follows: Source % Yield Best Commercial Hybrids 100 Best NCSU Single Cross 95+ Best NC State Double-crosses 85+ Best NC State Ga1-s SC 75 Typical NC State Ga1-s SC 60 Best NC State Tcb1 Cross 60 Best NC State Ga1-s x +/+ SC 100

19. The problem is NOT yield potential of Ga1-s materials; it is high coefficients of parentage among our Ga1-s lines. What have we tried (& so far basically failed): 1. Outside materials carrying Ga1-s 2. Sources of Tcb1 (from teosinte in a maize background)

20. What are we trying now: Crossing (and backcrossing) dominant gametophytic factors from Mexican maize accessions. Jesus Sanchez of the University of Guadalajara hypothesized that specialty corns (popcorns and sweetcorns, for example) might have unstudied isolating mechanisms.

21. Some of accessions we used were suggested by Sanchez: Jal78, Jal300, Jal304, Zac182. Others have been discovered by Matt Krakowsky as he developed BC lines in the Allelic Diversity Project of GEM.

22. We shared some of our earliest- maturing materials with Jerry Kermicle. His conclusions, based on crosses with his testers, were that no new loci were involved (other than Ga1-s and Tcb1), but one accession, Jal78, had both Ga1-s and Tcb1.

23. The material sent to Jerry included derivatives of Jal78 and Jal304, two of Jesus Sanchez’s suggestions. The initial crosses with these two accessions (plus those of Jal300) were made possible by seed donated by Jim Holland and winter nursery F 1 s arranged by Randy Holley of Pioneer.

24. The accessions from Matt’s Allelic Diversity work include: Gto141, Gto181, Gto100, Zac40, Bnz875, Z07011, Bnz878, and Mich412 in order of frequency of apparent discovery.

25. We are in our 2 nd season with the GEM Allelic Diversity materials (the 3 rd if you include Matt’s efforts), and the fourth with the Sanchez materials (5 th if you include Pioneer’s crosses). There are many false negatives (dead pollen, aged silk, heat, wet bags, etc.). But there’s little doubt that Dominant Gametophyte Factors exist in populations of Maíz Dulce.

26. The fishing expedition we follow varies somewhat, but here is one cast: 1. Cross Accession x PHB47 2. Use individual F 1 plants as males in backcrosses to B47 3. Pollinate those F 1 s with normal pollen (all such seed are eventually junked) 4. Keep only those crosses with no seed set on the corresponding F 1 s 5. Repeat process with BC 1 plants 6. Try to obtain selfed seed on 2 nd ear 7. Topcross and yield test 2 nd ears

27. We are crossing and backcrossing these to DKHBA1, NC296, NC320, NC368, NC476, PHP38, PHN46, and 1274-1 (an NC320 relative with fusarium resistance) and several other semi-random lines (mostly ex-PVPs) which happened to be flowering when not much else was.

28. Whether dominant factors are single genes or gene complexes (and all essentially identical to Ga1-s and/or Tcb1) is unknown. Will the dominance be lost during backcrossing? Are any resistant to Ga1-M? Several (1222-2 and 1228-2) seem to be. Will know more in January.

29. We have released 1222-2 from Jal78 as a genetic stock. It appears to be completely dominant, and is classified by Jerry Kermicle as Tcb1-s. Several attempts to cross it as female by Ga1-M males failed. Further tests have been made in our winter nursery and we should have definitive results by mid-January.

30. And why didn’t Ga1-M reach the US with other Mexican corn “exports”? Tcb1 may limit interchange between maize and teosinte, and hence have a limited distribution. But Ga1-M is all over Mexico at high frequencies. Why didn’t it get here?