Discussion
The combined approach proposed here has proved its usefulness for allowing the rapid cloning and characterization of specific conserved genes, as well for providing plant genomic fingerprinting information. An orthologous element for the Arabidopsis More Axillary Branching gene was successfully cloned and sequenced, for the first time, from several H. rosa-sinensis cultivars and from Hibiscus wild species. This general approach is particularly useful when dealing with plant species for which no or poor information is available at the genomic level. Minor changes could be made to the suggested protocol, i.e. the choice of the restriction enzyme could likely increase the efficiency of the method.
The high conservation sequence degree observed among commercial varieties and the Hibiscus species sexually compatible with H. rosa-sinensis are in agreement with the cytogenetic evidence produced by Singh and Khoshoo (1989), which showed that these inter-fertile species have contributed to the extensive genetic variability of H. rosa-sinensis. The revealed HibMAX2-like sequence analysis is consistent with secondary ranks of taxa (Sections) proposed by Pfeil and Crisp (2005) through chloroplast DNA analysis. In fact, the highest similarity values for the target sequence were achieved among the analyzed H. rosa-sinensis cultivars and the sexually compatible species, all belonging to the Lilibiscus Section, while the lower values were observed for species of different taxonomic Sections such as H. syriacus, H. panduriformis, H. moscheutos, H. tiliaceus and H. calyphyllus. Concerning the H. cannabinus, it could likely either do not possess the HibMAX2 gene, or possess a highly differentiated element, therefore not amplified. Kenaf is one of the fast-growing plants classified in the Furcaria Section of Hibiscus; it has both annual and biennial genotypes, often not branched. This differentiates from the other examined species (shrubs or small trees) characterized by perennial life cycle, with complex vegetative morphologies (Craven et al 2003). Moreover, a previous study (Braglia et al 2010) had revealed the lowest genetic similarity value between H. rosa- sinensis cultivars and kenaf defining this latter as the most distantly related species within the Hibiscus genus.
The occurrence of LRRs in the Hibiscus isolated fragment assigns this sequence among the F-box genes, one of the largest multigene superfamilies involved in shoot lateral branching growth. Members of this protein family function as subunits of the multiprotein Skp-Cullin-Fbox for polyubiquitination and degradation by the 26S proteasome (Xu et al 2009). In particular, the F-box LRR proteins confer substrate specificity to the SCF complex via their two distinct functional domains: the first domain (F-box) binds to another subunit of the SCF complex, the second domain (LRR repeats) interacts with specific proteins to be polyubitquinated (Stirnberg et al 2007).
Although the present results are the first step in the isolation of the whole Hibiscus specific element for MAX2 gene, the cloned fragment can be already investigated for association to the branched trait, to evaluate its utility in marker-assisted breeding schemes. Further studies including the isolation of a cDNA fragment (working back to the full length through RACE-PCR technique), the mRNA expression analysis and the functional variant identification are in turn necessary to better characterize the HibMAX2-like sequence, as well as to clarify its involvement in the axillary branch proliferation mechanisms.
Acknowledgements
We wish to thank Mr. Cesare Bianchini and Dr. Marco Ballardini for their support in managing the germplasm collection. Research founded by the Italian Ministry of Agriculture in the framework of the project “Risorse tecniche e genetiche per il florovivaismo (FLORIS)”.
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