Investigation of interspecific genome-plastome incompatibility in Oenothera and Passiflora

Interspecific genome-plastome incompatibility is a widely observed
phenomenon but its primary causes are still unknown. It reflects
genome-plastome interactions that play a direct role in speciation
processes, such interspecific combinations of nuclear genomes and
plastomes that fail to develop fully autotrophic plants which then
are usually eliminated by natural selection. We have investigated
two plant models displaying genome-plastome incompatibility,
Oenothera and Passiflora, using strategies of molecular biology in
order to contribute to an analysis of primary causes of
interspecific genome-plastome incompatibility. 1. Expressed
sequence tags in Oenothera: In this study we present the first
analyzed EST data set for Oenothera. 3,532 cDNA sequences derived
from 9-week-old Oenothera plantlets were the analysed and assembled
into 1,621 nonredundant clusters, including 1,133 singletons and
488 multi-member unigenes which contain a total of 875,940
nonredundand nucleotides. EST sequences were analysed by Sputnik
algorithm. They were also used in the development of gene-specific
PCR-based codominant markers (SNPs, CAPS, micro-satellites). The
cDNA library could be directly used for macroarray applications
including gene expression studies and for physical mapping. 2.
Genotyping analyses in Oenothera using AFLP technology: The
comparison of AFLPs from Oenothera with AFLPs from Arabidopsis was
used to obtain an approximation of the genome size. The genotyping
data provide evidence that genome of Oenothera is only six times
larger than that of Arabidopsis corresponding to a size of about
750 Mb. The AFLP markers were also successfully applied to
construct first genetic maps using F2 mapping population of
interspecific hybrids between Oenothera elata ssp. hookeri, line
johansen, AA-III, x Oenothera grandiflora ssp. tuscaloosa, BB-III.
The linkage maps contain 88 AFLP markers covering a total map
length of 154.4 cM for dominant markers in johansen, AA-III and 104
AFLP markers and a total size of 155.3 cM for dominant markers in
grandiflora, BB-III. In addition, it was possible to assign
genome-plastome incompatibility locus to the margin of coupling
group 2B with 13 cM distance to the next AFLP marker. SUMMARY 91
The EST project followed by genotyping analysis increases knowledge
and requirements in discovering primary causes of genome-plastome
incompatibility. Oenothera with genome-plastome incompatibility,
chromosomal translocations and many chromosomal arrangements
provides an elegant tool in the study of genomeplastome
interactions, speciation processes and species evolution. 3.
Investigation of genome-plastome incompatibility in Passiflora: We
present the first evidence of hybrid bleaching in this genus. The
hybrid between Passiflora menispermifolia x Passiflora oerstedii
showed bleaching regions during plant development. Reciprocal
crosses have also shown hybrid bleaching but as well significant
differences in leaf shape. Molecular analyses of cpDNA showed that
Passiflora plastids are inherited bi-parentally and that the P.
menispermifolia plastome is incompatible in F1 hybrids with P.
oerstedii. This is the first evidence of genome-plastome
incompatibility in Passiflora, which differ from Oenothera
incompatibilities. The analysis of plastid ultrastructure showed
that green tissues in the F1 generation have fully developed
chloroplasts with thylakoids and grana; the incompatible material
in F1 hybrids lacks differentiated plastids and contains plastids
with only rudimentary membranes. An unexpected plastid
ultrastructure was found in P. menispermifolia. The leaf from plant
growing at greenhouse conditions contains plastids in different
development stages including etioplasts, which normally develop
from proplastids in darkness. Electron micrographs also indicated
retardation of grana formation in P. menispermifolia which shows
that vesicles could deliver parts of thylakoid components and that
they may directly participate in the formation grana stacks.
Northern and Western analyses demonstrated that genome-plastome
incompatibility affects both transcription and translation, but
with differences for nuclear and plastome encoded genes.