K. Uchiyama, S. I. Ivashuta, M. Gau and S. Isobe

Hokkaido Natl. Agric. Exp. Sta., Hitsujigaoka, Toyohira-ku, Sapporo 062-8555, Japan

We are trying to find DNA markers associated with freezing tolerance and/or fall dormancy of alfalfa using F₂ and BC₁F₁ populations between cv. Rambler (freezing tolerant) and cv. Moapa 69 (freezing sensitive). To reduce labor and cost in analyzing the segregating populations, in which we have to deal with a lot of plants, we preliminarily screened the RAPD markers (Operon) using parental clones and cultivars, and other cultivars of contrasting freezing tolerance. We are trying to find the DNA markers of greater or lower frequency in freezing tolerant cultivars or sensitive ones or specific to them. We have done several approaches for that purpose.

1) We used 2 genotypes each from Moapa 69 and Rambler including parental clones for producing F₂ and BC₁F₁ populations. We screened RAPD markers common in one cultivar and absent in the other one. 251 out of 316 primers showed polymorphism possible to analyze and 804 polymorphic bands were observed. There were 131 bands (16.3%) specific to Moapa 69 and 117 bands (14.6%) specific to Rambler.

2) Using 36 cultivars (10 plants bulk) of contrasting freezing tolerance, we screened 20 primers for the bands of greater frequency in cold tolerant cultivars or sensitive ones. We observed 220 total bands and 169 polymorphic ones (76.8%). We found 2 markers each of greater frequency in cold tolerant cultivars or in sensitive ones.

3) Using 10 plants each from 9 cultivars (each 3 cultivars from cold sensitive, moderately tolerant and tolerant ones), we screened 5 primers for the bands of higher frequency or lower frequency in cold sensitive or tolerant cultivars. We observed 77 total bands and 5 bands were adapted to our purpose.

Using F₂ progeny between Rambler and Moapa 69 and BC₁F₁ progeny backcrossed to Rambler, now we are analyzing the segregation of freezing tolerance and DNA markers. We will check whether our pre-screening strategies are effective or not in the segregating populations.