Deletion mapping of genetic regions associated with apomixis in Hieracium

Abstract

Although apomixis has been quoted as a technology with the potential to deliver benefits similar in scale to those achieved with the Green Revolution, very little is currently known of the genetic mechanisms that control this trait in plants. To address this issue, we developed Hieracium, a genus of daisies native to Eurasia and North America, as a genetic model to study apomixis. In a molecular mapping study, we defined the number of genetic loci involved in apomixis, and we explored dominance and linkage relationships between these loci. To avoid difficulties often encountered with inheritance studies of apomicts, we based our mapping effort on the use of deletion mutagenesis, coupled with amplified fragment length polymorphism (AFLP) as a genomic fingerprinting tool. The results indicate that apomixis in Hieracium caespitosum is controlled at two principal loci, one of which regulates events associated with the avoidance of meiosis (apomeiosis) and the other, an unlinked locus that controls events associated with the avoidance of fertilization (parthenogenesis). AFLP bands identified as central to both loci were isolated, sequenced, and used to develop sequence-characterized amplified region (SCAR) markers. The validity of the AFLP markers was verified by using a segregating population generated by hybridization. The validity of the SCAR markers was verified by their pattern of presence/absence in specific mutants. The mutants, markers, and genetic data derived from this work are now being used to isolate genes controlling apomixis in this system.

Fig. 1.

Apomixis in Hieracium follows the developmental mechanism of apospory. Three critical deviations from sexual reproduction are apparent: an avoidance of meiosis (apomeiosis), an avoidance of fertilization before embryo formation (parthenogenesis), and an avoidance of fertilization before endosperm formation (autonomous endospermy).

Fig. 2.

AFLP markers in a selection of 81 mutants in which apomeiosis and/or parthenogenesis was dysfunctional. Vertical columns represent mutants, horizontal rows represent markers, a light square represents a marker that is present, and a dark square represents a marker that is absent. Phenotypic data are shown in the bar at the top of each group. The controls were subjected to irradiation and regeneration, but they did not express a mutant phenotype with respect to apomixis.

Fig. 3.

AFLP markers in an advanced selection of mutants displaying small deletions in either the LOA or LOP loci. Vertical columns represent mutants, horizontal rows represent markers, a light square represents a marker that is present, and a dark square represents a marker that is absent. Phenotypic data are shown in the bar at the top of each group. The control was subjected to irradiation and regeneration, but it did not express a mutant phenotype with respect to apomixis. Dotted lines indicate apparent midpoints for the consensus deletions, and therefore they are the most probable sites of critical loci. An asterisk represents a data point that was inferred from a previous experiment but not directly tested. A group of 17 lop markers was mapped to a site between markers lop 279 and lop 302. No further ordering is possible for this group because discriminating breakpoints were not available.

Fig. 4.

Demonstration of SCAR marker use against the panel of mutants. In each gel lane 1 is a wild-type plant, lanes 2–8 are seven mutants in LOA (134, 125, 165, 152, 146, 135, and 132), lanes 9–15 are seven mutants in LOP (179, 138, 143, 116, 171, 144, and 156), and lane 16 is the dual mutant 168. The markers used are listed to the left of each gel.