Evaluation of reproductive barriers contributes to the development of novel interspecific hybrids in the Kalanchoe genus

Background: Interspecific hybridization is a useful tool in ornamental breeding to increase genetic variability and introduce new valuable traits into existing cultivars. The successful formation of interspecific hybrids is frequently limited by the presence of pre- and post-fertilization barriers. In the present study, we investigated the nature of hybridization barriers occurring in crosses between Kalanchoe species and evaluated possibilities of obtaining interspecific hybrids. Results: The qualitative and quantitative analyses of pollen tube growth in situ were performed following intra-and interspecific pollinations. They revealed occurrence of pre-fertilization barriers associated with inhibition of pollen germination on the stigma and abnormal growth of pollen tubes. Unilateral incongruity related to differences in pistil length was also observed. The pollen quality was identified as a strong factor influencing the number of pollen tubes germinating in the stigma. In relation to post-fertilization barriers, endosperm degeneration was a probable barrier hampering production of interspecific hybrids. Moreover, our results demonstrate the relation of genetic distance estimated by AFLP marker analysis of hybridization partners with cross-compatibility of Kalanchoe species. At the same time, differences in ploidy did not influence the success of interspecific crosses. Conclusions: Our study presents the first comprehensive analysis of hybridization barriers occurring within Kalanchoe genus. Reproductive barriers were detected on both, pre- and post-fertilization levels. This new knowledge will contribute to further understanding of reproductive isolation of Kalanchoe species and facilitate breeding of new cultivars. For the first time, interspecific hybrids between K. nyikae as maternal plant and K. blossfeldiana as well as K. blossfeldiana and K. marnieriana were generated.

Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida)

Background: Gene expression studies are a prerequisite for understanding the biological function of genes. Because of its high sensitivity and easy use, quantitative PCR (qPCR) has become the gold standard for gene expression quantification. To normalise qPCR measurements between samples, the most prominent technique is the use of stably expressed endogenous control genes, the so called reference genes. However, recent studies show there is no universal reference gene for all biological questions. Roses are important ornamental plants for which there has been no evaluation of useful reference genes for gene expression studies. Results: We used three different algorithms (BestKeeper, geNorm and NormFinder) to validate the expression stability of nine candidate reference genes in different rose tissues from three different genotypes of Rosa hybrida and in leaves treated with various stress factors. The candidate genes comprised the classical "housekeeping genes" (Actin, EF-1α, GAPDH, Tubulin and Ubiquitin), and genes showing stable expression in studies in Arabidopsis (PP2A, SAND, TIP and UBC). The programs identified no single gene that showed stable expression under all of the conditions tested, and the individual rankings of the genes differed between the algorithms. Nevertheless the new candidate genes, specifically, PP2A and UBC, were ranked higher as compared to the other traditional reference genes. In general, Tubulin showed the most variable expression and should be avoided as a reference gene. Conclusions: Reference genes evaluated as suitable in experiments with Arabidopsis thaliana were stably expressed in roses under various experimental conditions. In most cases, these genes outperformed conventional reference genes, such as EF1-α and Tubulin. We identified PP2A, SAND and UBC as suitable reference genes, which in different combinations may be used for normalisation in expression analyses via qPCR for different rose tissues and stress treatments. However, the vast genetic variation found within the genus Rosa, including differences in ploidy levels, might also influence expression stability of reference genes, so that future research should also consider different genotypes and ploidy levels.