The Hybridization Barrier between Herbaceous Medicago sativa and Woody M. arborea Is Weakened by Reproductive Abnormalities in M. sativa Seed Parents.

Historically, crosses between Medicago sativa (alfalfa) and M. arborea with alfalfa as the seed parent failed, as did crosses using M. arborea as the seed parent. Thus, a reproductive barrier kept the two species isolated until early in this century. The breakthrough came when alfalfa seed parents were identified in Wisconsin USA and Queensland AU that produced partial hybrids (hereafter hybrids). The hybrids were obtained by making large numbers of crosses on selected alfalfa parents. This was the first level of weakening the crossing barrier as reported in Plants in 2013. Further weakening of the barrier is reported herein whereby more hybrids were obtained with fewer crosses. This was accomplished by pedigree selection for new alfalfa seed parents and by using a product of the first hybrids called Alborea. New alfalfa seed parents were crossed with M. arborea, and Alborea parents were backcrossed to M. arborea. Hybrid plants were produced with fewer crosses in both cases. These hybrids, like the first hybrids, have mostly alfalfa traits but also have traits from M. arborea. It was theorized early on that the alfalfa component could be explained by 2n eggs in the alfalfa parents that were fertilized by normal n gametes from M. arborea. Evidence that the Wisconsin alfalfa and Alborea seed parents did in fact produce 2n eggs was reported in Plants in 2022. Moreover, they produced 2n eggs at approximately the same frequency that they produced hybrids. As reported herein, Alborea parents produced the highest frequency of hybrids and thus had the weakest barrier. Importantly, they also have the highest frequency of 2n eggs. It was determined that alfalfa and Alborea parents that produce 2n eggs and hybrids, also produce 2n pollen. In effect, an experiment was undertaken in reverse showing that 2n pollen could be used to screen for plants that produce hybrids. In the thousands of crosses made over the years, fertilization of normal n eggs in alfalfa parents always failed. Normal meiosis appears to be the main barrier to producing interspecific hybrids in our case. Fertilization of abnormal 2n eggs ensures sufficient alfalfa genetic material to continue embryogenesis. Evidently, the meiotic abnormality of 2n eggs is the major factor that weakens the crossing barrier.

Evidence That 2n Eggs Explain Partial Hybrids between Medicago sativa and Medicago arborea.

Selected genotypes of alfalfa (Medicago sativa) produce partial hybrids in sexual crosses with Medicago arborea, as reported in Plants (2013). The hybrids contain mostly alfalfa DNA and traits, but also contain DNA and traits from M. arborea. It was proposed in 2008 that the partial hybrids could be explained by fertilization of 2n eggs in alfalfa by normal pollen from M. arborea, followed by partial loss of M. arborea chromosomes during embryogenesis. In this paper, we confirm the presence of 2n eggs in the first alfalfa parents that produced hybrids. The test for 2n eggs involved pollinating 4x alfalfa with pollen from 8x alfalfa. The production of 8x progeny in the cross proved that selected alfalfa parents produced 2n eggs. Thus, 2n eggs appear to explain how the partial hybrids (hereafter hybrids) contain mostly alfalfa DNA and traits. However, two of the six alfalfa plants that did not hybridize with M. arborea also had 2n eggs. Thus, although 2n eggs explain the alfalfa content of hybrids, 2n eggs are not the only factor involved in weakening the hybridization barrier, and in transferring genes to alfalfa from M. arborea.

The Hybridization Barrier between Herbaceous Medicago sativa and Woody M. arborea Is Weakened by Selection of Seed Parents.

Medicago sativa, alfalfa or lucerne, and M. arborea were considered reproductively isolated until recently. Then, in 2003, an alfalfa genotype was identified that produced a few seeds and progeny with hybrid traits after a large number of pollinations by M. arborea. A derivative of this alfalfa genotype also produced a low frequency of progeny with hybrid traits. Thus, the hybridization barrier was weakened by selection of seed parents. Hybrids from both events expressed traits from M. arborea and M. arborea-specific DNA bands, although more of the M. sativa genome was retained, based on the DNA results. Thus, there was chromatin elimination during embryogenesis, resulting in partial hybrids (hereafter hybrids). However, more than 30 hybrids with an array of M. arborea traits have been obtained thus far, and research continues on the nature of the hybrids. Traits have been genetically transmitted in crosses, and selected traits are in use for alfalfa breeding. This paper reviews the first hybrids and then focuses on further weakening of the hybridization barrier with the discovery of a more efficient hybridizer derived from crossing Medicago sativa subspecies, sativa, coerulea and falcata. This genotype was found to have reproductive abnormalities associated with its complex subspecies origin that are best described as hybrid breakdown. In effect, this subspecies derivative is a bridge-cross parent that consistently produces hybrids. Reproductive abnormalities in the bridge-cross parent are reported and discussed.