Inheritance and mapping of 2n-egg production in diploid alfalfa

The production of eggs with the sporophytic chromosome number (2n eggs) in diploid alfalfa (Medicago spp.) is mainly associated with the absence of cy tokinesis after restitutional meiosis. The formation of 2n eggs through dip losporic apomeiosis has also been documented in a diploid mutant of M. sati va subsp. falcata (L.) Arcang. (2n = 2x = 16), named PG-F9. Molecular taggi ng of 2n-egg formation appears to be an essential step towards marker-assis ted breeding and map-based cloning strategies aimed at investigating and ma nipulating reproductive mutants of the M. sativa complex. We made controlle d crosses between PG-F9 and three wild type plants of M. sativa subsp. coer ulea (Less.) Schm. (2n = 2x = 16) and then hand-pollinated the F-1 progenie s with tetraploid plants of M. sativa subsp. sativa L. (2n = 4x = 32). As a triploid embryo block prevents the formation of 3x progenies in alfalfa be cause of endosperm imbalance, and owing to the negligible selfing rate, see d set in 2x-4x crosses was used to discriminate the genetic capacity for 2n -egg production. F-1 plants that exhibited null or very low seed sets were classified as normal egg producers and plants with high seed sets as 2n-egg producers. A bulked segregant analysis (BSA) with RAPD (random amplified p olymorphic DNA), ISSR (inter-simple sequence repeat), and AFLP (amplified f ragment length polymorphism) markers was employed to identify a genetic lin kage group related to the 2n-egg trait using one of the three F-1 progenies . This approach enabled us to detect a paternal ISSR marker of 610 bp, gene rated by primer (CA)(8)-GC, located 9.8 cM from a putative gene (termed Tne (1), two-n-eggs) that in its recessive form determines 2n eggs and a 30% re combination genomic window surrounding the target locus. Eight additional R APD and AFLP markers, seven of maternal, and one of paternal origin, signif icantly co-segregated with the trait under investigation. The minimum numbe r of quantitative trait loci (QTLs) controlling seed set in 2x-4x crosses w as estimated by ANOVA and regression analysis. Four maternal and three pate rnal independent molecular markers significantly affected the trait. A pate rnal RAPD marker allele, mapped in the same linkage group of Tne(1), explai ned 43% of the variation for seed set in 2x-4x crosses indicating the prese nce of a major QTL. A map of the PG-F9 chromosome regions carrying the mino r genes that determine the expression level of 2n eggs was constructed usin g selected RAPD and AFLP markers. Two of these genes were linked to previou sly mapped RFLP loci belonging to groups 1 and 8. Molecular and genetic evi dence support the involvement of at least five genes.