High-resolution physical mapping in Pennisetum squamulatum reveals extensive chromosomal heteromorphism of the genomic region associated with apomixis

Abstract

Gametophytic apomixis is asexual reproduction as a consequence of parthenogenetic development of a chromosomally unreduced egg. The trait leads to the production of embryos with a maternal genotype, i.e. progeny are clones of the maternal plant. The application of the trait in agriculture could be a tremendous tool for crop improvement through conventional and nonconventional breeding methods. Unfortunately, there are no major crops that reproduce by apomixis, and interspecific hybridization with wild relatives has not yet resulted in commercially viable germplasm. Pennisetum squamulatum is an aposporous apomict from which the gene(s) for apomixis has been transferred to sexual pearl millet by backcrossing. Twelve molecular markers that are linked with apomixis coexist in a tight linkage block called the apospory-specific genomic region (ASGR), and several of these markers have been shown to be hemizygous in the polyploid genome of P. squamulatum. High resolution genetic mapping of these markers has not been possible because of low recombination in this region of the genome. We now show the physical arrangement of bacterial artificial chromosomes containing apomixis-linked molecular markers by high resolution fluorescence in situ hybridization on pachytene chromosomes. The size of the ASGR, currently defined as the entire hemizygous region that hybridizes with apomixis-linked bacterial artificial chromosomes, was estimated on pachytene and mitotic chromosomes to be approximately 50 Mbp (a quarter of the chromosome). The ASGR includes highly repetitive sequences from an Opie-2-like retrotransposon family that are particularly abundant in this region of the genome.

Figure 1.

High resolution FISH mapping of BACs linked with apomixis on pachytene chromosomes of apomictic backcross (BC3) and P. squamulatum accessions (PS24, PS26). a, Dual-color FISH of pachytene chromosomes in BC3 using low-copy BACs containing apomixis-linked markers. b, 4× magnification of image in a. c, The ASGR-carrier chromosome at pachytene in PS24 hybridized with low- and high-copy BACs containing apomixis-linked markers. Red arrow indicates centromere. d, 4× magnification of image c. e, Dual-color FISH of pachytene chromosome spread from PS26 using labeled BACs P208 and P800 and blocked with DNA from BAC P602. f, Schematic of the BAC order as inferred from multiple FISH experiments. The bars in a, c, e and b, d = 10 μm and 2.5 μm, respectively.

Figure 2.

A, FISH analysis of subclone AA5 from P602. B, Comparison of an open reading frame from subclone AA5 (AY375366) to AAN60494 (rice) and Opie-2 of maize (AAC49501).

Figure 3.

a, c, and e, The ASGR-carrier chromosome in PS24 hybridized with BAC P602 (green), which contains the highest frequency of Opie-2-like sequences, and a centromeric probe (red, arrowheads). b and d, The DNA distribution pattern based on DAPI fluorescence intensity. The green area indicates the hemizygous region. f, Inverted DAPI image of e. Green arrowhead indicates the hemizygous region. a and b, Mitotic chromosome. c and d, Pachytene chromosome. e and f, chromosomes at diplotene. Bars = 10 μm.