AGRONOMIC AND MOLECULAR ANALYSIS OF HETEROSIS IN ALFALFA

Double 'free-hybrids' (DH) in alfalfa, a polyploid, allogamous and perennial forage crop, were obtained by crossing in a diallelic scheme six simple hybrids (SH), multiplied for two generations (2S2Syn3), derived from four partly inbred (S2) constituents (Rotili et al., 1999). Specific Combining Ability (SCA) source of variation for dry matter yield (DMY) resulted highly significant and larger than General Combining Ability (GCA) component and supported heterosis values of DHs vs the best parent of +45% on average, ranging from +76 to +5%. The investigation at the molecular level was carried out by means of SSR marker analysis on the 6 parental SHs and the 15 DH progenies and by comparison of gene expression profiles of a single DH and the respective parental plants. The parental genetic diversity, estimated by SSR markers, showed a significant relationship with heterosis and SCA effects (r= 0.70 and 0.76, respectively). The variation of heterozygosity estimates of the DHs explained a little part (about 20%) of their variation in DMY (r = 0.45 n.s.) while the number of alleles was significantly related to DM performance (r = 0.61 P < 0.05). A microarray analysis of the transcriptome was conducted in a highly performing hybrid (DH) compared to its parents (2S2Syn3). T-test enabled to identify genes with additive/non additive (according to the nomenclature as reviewed in Hochholdinger and Hoecker, 2007) value of expression in the hybrid compared to the parents (P < 0.05, Benjamini and Hochberg False discovery rate). Q-PCR validated the level of expression from microarray analysis for a few selected genes. In our experiment, most of the variation in gene expression was additive (87%). Interestingly among the genes with non-additive pattern of expression the greater proportion of probe sets (86%) fell outside the parental range, namely above/below the level of expression of the high/low parent; among such genes ontology classes highly represented are metabolism and genetic information processing. Prevalent additive mode of gene expression has been observed in a few expression analysis of heterotic hybrid vs. parents in different organisms (maize, Arabidopsis). Rotili P, 1999, www.naaic.org/TAG/TAGpapers/rotili/rotili.html.