Genetic analysis of the genus Clivia

Abstract

The genus Clivia Lindl. currently consists of four species (C. miniata, C. gardenii, C. caulescens and C. nobilis) Observations on karyotypes with orcein staining showed that all species are diploid with the same chromosome number (2n=2x=22) and similar basic karyotypes make up of four pairs of metacentric chromosomes and seven pairs of acrocentric chromosomes including three pairs of large, five pairs of medium size and three pairs of small chromosomes. Karyotype analysis showed that all four species can be distinguished by their chromosome banding patterns using Giemsa C-banding, fluorochrome banding (DAPI and CMA), Ag-NOR staining and the locations of 45S rRNA gene sites by in situ hybridization. The 45S rRNA gene sites were located on one pair of chromosomes in C. gardenii, two pairs in C. caulescens and C. miniata and three pairs in C. nobilis. One pair of 5S rRNA gene sites was found on chromosome 8 in all Clivia species. Five pairs of chromosomes in C. miniata and C. gardenii and seven pairs in C. nobilis and C. caulescens can be identified using these chromosome landmarks. Genomic in situ hybridization and C-banding were used successfully to identify the parental genomes of putative cultivars and artificial interspecific hybrids. Parental genome analysis showed that C. x cyrtanthiflora introduced to New Zealand from different sources was F1 interspecific hybrid between C. miniata and C. nobilis, confirming the origin of this cultivar. The "German hybrid" and the "Belgian hybrid" were identified as selections or intraspecific hybrids from C. miniata rather than interspecific hybrids. Five artificial interspecific hybrids with different combinations were confirmed as true hybrids. The investigation of parental genome disposition in five interspecific hybrids using Giemsa C-banding and genomic in situ hybridization showed that parental genomes were spatially separted in a concentric fashion in all five hybrids. This is the first report of genome separation in the Amaryllidaceae. This suggested that parental genome separation might be a common phenomenon in plants. Sequences of the internal transcribed spacer (ITS) of 45S rDNA and the non-transcribed spacer of 5S rDNA were used to resolve phylogenetic relationships among the species of the genus. Similar phylogenetic trees were constructed from the two gene regions. The phylogeny showed that Clivia has a monophyletic origin and that all the extant species are closely related. Among them C. miniata is more closely related to C. gardenii than to the other species, C. nobilis is relatively distant from these two species and C. caulescens occupies an intermediate position. In 46 accessions of the species and cultivars the genetic relationships between species and within species were investigated using RAPD. The result has provided additional evidence to support the species phylogeny inferred from sequence data. Within C. miniata five genetically distinct groups were found among 32 accessions. The variations between cultivars and wild populations are larger than those of within cultivars and within wild populations. The result also suggested that some popular cultivars such as the "German hybrid" and Daruma may have been developed from the same genetic background, but they are distinct from "Belgian hybrid". This suggests there is a potential to develop new varieties if different genetic resources of the same species can be used. "Robust gardenii", also known as "Swamp gardenii", despite some morphological differences, has been thought to be a form of C. gardenii. The genetic analysis in this study provided strong evidence that "Robust gardenii" is a new species distinct from the present four species. It differs from the other species in its karyotype banding patterns, and 45S rDNA sites. RAPD analysis indicated that "Robust gardenii" was closely related to C. gardenii and C. miniata, but differed from them. Sequence data analysis confirmed this, placing it in a separate clade in the phylogenetic trees.