Potential of trispecies bridge crosses and random amplified polymorphic DNA markers for introgression of Medicago daghestanica and M. pironae germplasm into alfalfa (M. sativa).

This report describes the production and cytology of the first interspecific hybrids between cultivated alfalfa (Medicago sativa L.) at the diploid level (2n = x = 16) and the diploid (2n = 2x = 16) perennial species M. daghestanica and M. pironae. An ovule-embryo culture technique was required to rescue hybrid embryos and all hybrids were diploid. Predominately bivalent chromosome pairing was observed at meiotic metaphase. All F1 hybrids were male and female sterile and no species backcross progeny could be produced. We discovered that trispecies hybrids could be efficiently recovered via crossing diploid F1 interspecific hybrids of M. sativa x M. rupestris with either M. daghestanica or M. pironae. Ovule-embryo culture was also required to recover these trispecies hybrids with recovery efficiency of trispecies hybrids about 10 times greater than for bispecies hybrids. Most chromosomes paired as bivalents in the trispecies hybrids. Importantly, progeny can be recovered from crossing the trispecies hybrids with M. sativa. Therefore, the M. sativa x M. rupestris hybrids provide a bridge cross to potential introgression of M. daghestanica or M. pironae germplasm. Analysis of randomly amplified polymorphic DNA (RAPD) markers in the trispecies hybrids indicates that RAPD markers offer considerable potential for assaying germplasm introgression following complex hybridization of the type reported here.