Use of the Roundup ReadyTM Trait to Estimate Selfing in Hand Crosses

of Alfalfa

Mark McCaslin and Stephen Temple

Forage Genetics International

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

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.

Promoter CP4 Gene TP Terminator

Event specific PCR

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

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

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 dihomo 2068 30.5 33.3

B only 1923 28.4 33.3

D only 2776 40.9 33.3

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.

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