The use of low-copy nuclear genes in the radiation of the Macaronesian Crassulaceae Sempervivoideae - Phylogeny and evolutionary processes

Speciation and evolution of species are two of the most exciting topics in biology. Radiations, with their wide morphological and physiological variety, provide a promising tool to understand speciation and diversity of species. Numerous studies have revealed that the high morphological diversity of radiated species is not represented at the molecular level. Neutral markers like rDNA nrITS and chloroplast (cp) DNA evolve slowly compared to speciation in radiations and thus, may not provide enough information to resolve phylogenetic relationships. In contrast, low-copy nuclear genes evolve faster and may help to resolve relationships. This is supported by the hypothesis that accelerated changes in regulatory genes, as opposed to structural genes, can explain the evolution of species. To contribute to this ongoing discussion, the radiation of the Macaronesian Crassulaceae Sempervivoideae (MCS) was studied. The polyploid species of the MCS are mainly distributed on the Canary Islands and comprise more than 70 species in three genera (Aeonium, Aichryson, and Monanthes) that display a huge morphological (e.g., flower color, number of floral organs, growth-form) and physiological (e.g., CAM activity) variety. Two regulatory genes, homologs of the floral homoeotic genes APETALA1 and APETALA3, and the structural gene encoding for phosphoenolpyruvate carboxylase (PEPC) were analyzed with respect to the following aims: 1) to evaluate the use of the low-copy nuclear genes to reconstruct phylogenies and to compare genealogies with the species phylogeny; 2) to estimate the impact of the studied genes in the speciation process and elucidate differences between the roles of regulatory and structural genes; 3) to determine if gene duplications occurred and to distinguish duplicates into orthologs and paralogs, and 4) to calculate the selection pressure (Ka/Ks-values) acting on the respective gene copies. The three analyzed low-copy nuclear genes both support and contradict the phylogenetic relationships inferred by other markers. The selection acting on the studied low-copy genes is in contrast with the neutral evolution of nrITS and cpDNA markers and may explain observed differences. In particular APETALA3 seems to be a promising marker for resolving species relationships. In addition, the studied genes may have had an influence in speciation since individually they exhibit accelerated Ka/Ks-values compared to mean Ka/Ks-values estimated for regulatory and structural genes. Their Ka/Ks-values are also much higher than those obtained for other genes in studies with comparable experimental designs. Accelerated evolutionary rates were estimated for the regulatory genes as opposed to the structural gene PEPC. However, summarizing all observations, the impact of these genes may be limited. Further study is recommended to evaluate their true impact. For all studied genes duplications were observed and emphasize the greatest challenge of working with low-copy nuclear genes – the differentiation of orthologs and paralogs. The observed duplication pattern suggests that the gene duplications are the result of polyploidization, a phenomenon to which the island colonization of the MCS species was connected previously. In addition, all gene copies were under purifying selection pressure, even if the estimated Ka/Ks-values for the respective copies varied. Rate differences were estimated for PEPC and APETALA3; the latter also showed significant differences in the Ka/Ks-values comparing copy A and copy B. For APETALA1 similar evolutionary rates and highest Ka/Ks-values were found. Altogether, this thesis offers a promising approach to study speciation and evolution in the radiation of the MCS and is a valuable basis for further studies

Genetic architecture of a pollinator shift and its fate in secondary hybrid zones of two Petunia species.

BACKGROUND Theory suggests that the genetic architecture of traits under divergent natural selection influences how easily reproductive barriers evolve and are maintained between species. Divergently selected traits with a simple genetic architecture (few loci with major phenotypic effects) should facilitate the establishment and maintenance of reproductive isolation between species that are still connected by some gene flow. While empirical support for this idea appears to be mixed, most studies test the influence of trait architectures on reproductive isolation only indirectly. Petunia plant species are, in part, reproductively isolated by their different pollinators. To investigate the genetic causes and consequences of this ecological isolation, we deciphered the genetic architecture of three floral pollination syndrome traits in naturally occurring hybrids between the widespread Petunia axillaris and the highly endemic and endangered P. exserta. RESULTS Using population genetics, Bayesian linear mixed modelling and genome-wide association studies, we found that the three pollination syndrome traits vary in genetic architecture. Few genome regions explain a majority of the variation in flavonol content (defining UV floral colour) and strongly predict the trait value in hybrids irrespective of interspecific admixture in the rest of their genomes. In contrast, variation in pistil exsertion and anthocyanin content (defining visible floral colour) is controlled by many genome-wide loci. Opposite to flavonol content, the genome-wide proportion of admixture between the two species predicts trait values in their hybrids. Finally, the genome regions strongly associated with the traits do not show extreme divergence between individuals representing the two species, suggesting that divergent selection on these genome regions is relatively weak within their contact zones. CONCLUSIONS Among the traits analysed, those with a more complex genetic architecture are best maintained in association with the species upon their secondary contact. We propose that this maintained genotype-phenotype association is a coincidental consequence of the complex genetic architectures of these traits: some of their many underlying small-effect loci are likely to be coincidentally linked with the actual barrier loci keeping these species partially isolated upon secondary contact. Hence, the genetic architecture of a trait seems to matter for the outcome of hybridization not only then when the trait itself is under selection

Genetic architecture of a pollinator shift and its fate in secondary hybrid zones of two Petunia species

Background: Theory suggests that the genetic architecture of traits under divergent natural selection influences how easily reproductive barriers evolve and are maintained between species. Divergently selected traits with a simple genetic architecture (few loci with major phenotypic effects) should facilitate the establishment and maintenance of reproductive isolation between species that are still connected by some gene flow. While empirical support for this idea appears to be mixed, most studies test the influence of trait architectures on reproductive isolation only indirectly. Petunia plant species are, in part, reproductively isolated by their different pollinators. To investigate the genetic causes and consequences of this ecological isolation, we deciphered the genetic architecture of three floral pollination syndrome traits in naturally occurring hybrids between the widespread Petunia axillaris and the highly endemic and endangered P. exserta. Results Using population genetics, Bayesian linear mixed modelling and genome-wide association studies, we found that the three pollination syndrome traits vary in genetic architecture. Few genome regions explain a majority of the variation in flavonol content (defining UV floral colour) and strongly predict the trait value in hybrids irrespective of interspecific admixture in the rest of their genomes. In contrast, variation in pistil exsertion and anthocyanin content (defining visible floral colour) is controlled by many genome-wide loci. Opposite to flavonol content, the genome-wide proportion of admixture between the two species predicts trait values in their hybrids. Finally, the genome regions strongly associated with the traits do not show extreme divergence between individuals representing the two species, suggesting that divergent selection on these genome regions is relatively weak within their contact zones. Conclusions: Among the traits analysed, those with a more complex genetic architecture are best maintained in association with the species upon their secondary contact. We propose that this maintained genotype–phenotype association is a coincidental consequence of the complex genetic architectures of these traits: some of their many underlying small-effect loci are likely to be coincidentally linked with the actual barrier loci keeping these species partially isolated upon secondary contact. Hence, the genetic architecture of a trait seems to matter for the outcome of hybridization not only then when the trait itself is under selection

Semi-dwarfism and lodging tolerance in tef (Eragrostis tef) is linked to a mutation in the α-Tubulin 1 gene

The semi-dwarf and lodging-tolerant kegne mutant linked to defects in microtubule orientation has the potential to enhance the productivity of an African orphan crop tef (Eragrostis tef

Technology generation to dissemination:lessons learned from the tef improvement project

Indigenous crops also known as orphan crops are key contributors to food security, which is becoming increasingly vulnerable with the current trend of population growth and climate change. They have the major advantage that they fit well into the general socio-economic and ecological context of developing world agriculture. However, most indigenous crops did not benefit from the Green Revolution, which dramatically increased the yield of major crops such as wheat and rice. Here, we describe the Tef Improvement Project, which employs both conventional- and molecular-breeding techniques to improve tef\u2014an orphan crop important to the food security in the Horn of Africa, a region of the world with recurring devastating famines. We have established an efficient pipeline to bring improved tef lines from the laboratory to the farmers of Ethiopia. Of critical importance to the long-term success of this project is the cooperation among participants in Ethiopia and Switzerland, including donors, policy makers, research institutions, and farmers. Together, European and African scientists have developed a pipeline using breeding and genomic tools to improve the orphan crop tef and bring new cultivars to the farmers in Ethiopia. We highlight a new variety, Tesfa, developed in this pipeline and possessing a novel and desirable combination of traits. Tesfa\u2019s recent approval for release illustrates the success of the project and marks a milestone as it is the first variety (of many in the pipeline) to be released

Bringing high-throughput techniques to orphan crop of Africa: Highlights from the Tef TILLING Project

Orphan- or understudied-crops are mostly staple food crops in developing world. They are broadly classified under cereals, legumes, root crops, fruits and vegetables. These under-researched crops contribute to the diet of a large portion of resource-poor consumers and at the same time generate income for small-holder farmers in developing countries, particularly in Africa. In addition, they perform better than major crops of the world under extreme soil and climatic conditions. However, orphan crops are not without problems. Due to lack of scientific investigation, most of them produce low yields while others have a variety of toxins that affect the health of consumers. Here, we present some highlights on the status and future perspectives of the Tef Biotechnology Project that employs modern improvement technique in order to genetically improve tef (Eragrostis tef), one of the most important orphan crop in Africa. A reverse genetics approach known as TILLING (Targeting Induced Local Lesions IN Genome) is implemented in order to tackle lodging, the major yield limiting factor in tef.Key words: Orphan crops, underresearched crops, Eragrostis tef, TILLING, semi-dwarf

Application of TILLING for Orphan Crop Improvement

People in developing countries mostly depend for their diet on special staple crops, so called orphan crops. These crops play a key role in food security since they are grown by many resource-poor farmers and consumed locally. Despite their huge importance in the economy and livelihood of the developing world, orphan crops have received little attention in terms of scientific improvement. Although conventional breeding is widely implemented to improve crop plants, alternative methods such as marker-assisted breeding and reverse genetics approaches have proved to be efficient in developing crop cultivars. In this review, we present detailed description of a non-transgenic and reverse genetics technique called TILLING (Targeting Induced Local Lesion IN Genomes). The method was originally optimized in the model plant Arabidposis thaliana and subsequently applied to crops such as maize, wheat, and rice. We also present detailed procedures for several TILLING strategies and discuss their benefits and drawbacks. The application of the technique for orphan crop improvement is also discussed based on several TILLING platforms currently carried-out on these understudied crops of the world

Complex evolution of novel red floral color in Petunia

Red flower color has arisen multiple times and is generally associated with hummingbird pollination. The majority of evolutionary transitions to red color proceeded from purple lineages and tend to be genetically simple, almost always involving a few loss-of-function mutations of major phenotypic effect. Here we report on the complex evolution of a novel red floral color in the hummingbird-pollinated Petunia exserta (Solanaceae) from a colorless ancestor. The presence of a red color is remarkable because the genus cannot synthesize red anthocyanins and P. exserta retains a nonfunctional copy of the key MYB transcription factor AN2. We show that moderate up-regulation and a shift in tissue specificity of an AN2 paralog, DEEP PURPLE (DPL), restores anthocyanin biosynthesis in P. exserta. An essential shift in anthocyanin hydroxylation occurred through re-balancing the expression of three hydroxylating genes. Furthermore, the down-regulation of an acyltransferase promotes reddish hues in typically purple pigments by preventing acyl group decoration of anthocyanins. This study presents a rare case of a genetically complex evolutionary transition towards the gain of a novel red color