1. Use of the Roundup Ready TM Trait to Estimate Selfing in Hand Crosses of Alfalfa Mark McCaslin and Stephen Temple Forage Genetics International

2. Background Alfalfa is predominately cross pollinated % selfing is difficult to determine without simple and effective genetic markers Transgenic traits offer unique advantages in studying alfalfa genetics and reproductive biology. –Cross vs self pollination –Pollen flow studies

3. Advantages of the Roundup Ready Trait CP4 gene inherited as a single gene dominant trait Easily identified phenotype –Efficient high-throughput screening Multiple transgenic events available with event- specific PCR primers. PromoterCP4 GeneTPTerminator Event specific PCR

4. Experimental Design Two populations of plants (~50 plants each) –Pop B – simplex plants containing transgenic event B (Bxxx). –Pop D – simplex plants containing transgenic event D (Dxxx). Non-emasculated hand crosses were made in greenhouse Pop D (female parent) x Pop B

5. Experimental Design Seed harvested from Pop D plants Progeny were sprayed with Roundup RR progeny were tested with event- specific PCR to identify the RR genotype –BD dihomogenic –D event only –B event only

6. Results 74.4% of the progeny were tolerant to Roundup (75% expected). Genotypes of the RR progeny were established with event-specific PCR and frequency of each genotype was compared to theoretical value. Genotype# plants% actual% expected BD dihomo206830.533.3 B only192328.433.3 D only277640.933.3

7. Discussion Increased frequency of D event only progeny and decreased frequency of B event only and BD dihomogenic is likely due to unintentional selfing during hand crossing. Data suggests ~10% selfing in this cross Similar results from other crosses over multiple years, locations and genetic backgrounds.

8. Discussion Other pollination related experiments planned or in process: –Cross vs self-fertility with leafcutter bees in commercial seed production setting. –Pollen flow studies with honeybees