Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress
Salsomaggiore Terme, Italy - 26/29 September, 2001
ANALYSIS OF APOMIXIS IN HYPERICUM PERFORATUM L. COMBINING MOLECULAR MARKERS AND FLOW CITOMETRY
ARZENTON F.*, BRAKE M.*, SGORBATI. S.**, VAROTTO S.*
* Dipartimento di Agronomia Ambientale e Produzioni Vegetali Università degli Studi di Padova,
Via Romea 16, 35020 Legnaro (PD)
** Dipartimento Scienze dell’Ambiente Territorio Università di Milano Piazza della Scienza 1, 20126 Milano
Hypericum perforatum, apomixis, molecular markers, DNA fingerprinting
St. John’s Worth (Hypericum perforatum L.) is a medicinal plant which provides a natural antidepressant in phytotherapy and represents a valuable source of pharmaceuticals. In particular, the anthraquinone hypericin and its derivatives are widely studied, especially due to their antiviral and anticancer activities. Traditionally, plant materials of H. perforatum have been collected from wild growing populations. Nowadays this species is object of breeding programs to develop cultivars which respond to the distinctiveness, uniformity and stability requisites.
H. perforatum is allotetraploid (2n=4x=32) and reproduces by facultative apomixis involving the parthenogenetic development of unreduced eggs from aposporic embryo sacs that arise from a somatic cell of the nucellus. Apomictic reproduction in this species is pseudogamous and leads to progenies of maternal origin in the vast majority of cases. Sexual reproduction through either selfing or outcrossing is also possible and gives rise to segregating, offtype progenies.
Little information is available on the reproductive biology of this species and a deeper knowledge of the genetic aspects of the apomictic system is needed for its exploitation in breeding programs.
DNA fingerprinting of H. perforatum ecotypes was performed using 2 Eco/Mse and 2 Pst/Mse primer combinations, 14 10-mer primers and 7 Inter-SSR primers. Ordination analysis based on 89 RAPD, 30 Inter-SSR and 152 AFLP polymorphisms allowed to cluster 12 ecotypes and the cultivar Topas into three groups showing a mean Dice’s similarity estimate of 0.62 and to distinct three off-type (1, 6 and 13) from the rest of the ecotypes. Five ecotypes (2, 5, 7, 9 and 15) and the cultivar Topas were positioned into the first subgroup that had a mean similarity estimate of 0.680. In the second, that is ecotypes 8, 11, 12 and 14, the mean similarity estimate was 0.698. The ecotypes 3, 4 and 10, included in the third subgroup, revealed the highest mean similarity estimate (GS = 0.852).
One of the ecotypes, 4 Cellarda (BL), displayed highly variable molecular fingerprints suggesting the presence of individuals characterized by sexual reproduction. The dendrogram related to the ecotype 4 separated the 10 plants into two main subgroups. Five plants (2, 5, 6, 8 and 10) of the first subgroup did not show any significant difference in the fingerprints produced. These data suggest an apomictic origin of these four plants. Instead, genotypes 3 and 7, even though positioned in the first group, could be distinguished from the other plants of this group. These plants were probably originated from selfing or haploid parthenogenesis. Finally, the second subgroup included genotypes 1, 4 and 9. These plants did not share with the remaining plants of the Cellarda ecotype any marker alleles and therefore they were considered to have a probable hybrid origin.
Principal coordinates analysis (PCOORDA) allowed to define the centroids of the 15 ecotypes, the cultivar Topas and the 10 plants of the Cellarda ecotype. Centroids analysis clustered the 26 genotypes into three distinct groups.
The first coordinate was able to distinguish the plants of the Cellarda ecotype (except for the off-types 1, 4 and 9) and the ecotypes 3 and 10 from the rest of samples. The second coordinate discriminated the ecotypes 11 and 14 and the off-types of the Cellarda ecotype from the rest of the ecotypes.
Preliminary analyses confirm that the predominant mode of reproduction in this species is apomixis but, also that cross fertilization or selfing may occur in H. perforatum as a minor event.
Occurrence of haploid parthenogenesis and fertilization of unreduced (aposporic) eggs giving rise to (poly)aploids and (poly)triploids (Biii) was confirmed by preliminary investigations of nuclear DNA contents of seed pools from all H. perforatum ecotypes by using flow cytometry.