Cultivated lettuce (Lactuca sativa L.) is an important leafy vegetable worldwide. However, the phylogenetic relationships between domesticated lettuce and its wild relatives are still not clear. In this thesis, I focus on the phylogenetic relationships within Lactuca L., including an analysis of the wild Lactuca species that are endemic to Africa for the first time. The genetic variation of responses to salinity in a recombinant inbred line population, derived from a cross between the lettuce crop (L. sativa Salinas) and wild species (L. serriola), was investigated and the candidate gene in the identified QTL regions was further studied.
In Chapter 1, I introduce and discuss topics related to genetic diversity and evolution in Lactuca, including an overview of lettuce cultivars and uses, its hypothesized domestication history, the taxonomic position of Lactuca, current status of molecular breeding in lettuce and mechanisms of salinity tolerance in plants, especially the High-affinity K+ Transporter (HKT) gene family.
In Chapter 2, the most extensive molecular phylogenetic analysis of Lactuca was constructed based on two chloroplast genes (ndhF and trnL-F), including endemic African species for the first time. This taxon sampling covers nearly 40% of the total Lactuca species endemic to Africa and 34% of all Lactuca species. DNA sequences from all the subfamilies of Asteraceae in Genbank and those generated from Lactuca herbarium samples were used to elucidate the monophyly of Lactuca and the affiliation of Lactuca within Asteracaeae. Based on the subfamily tree, 33 ndhF sequences from 30 species and 79 trnL-F sequences from 48 species were selected to infer phylogenetic relationships within Lactuca using Randomized Axelerated Maximum Likelihood (RAxML) and Bayesian Inference (BI) analyses. In addition, biogeographical, chromosomal and morphological character states were analysed based on the Bayesian tree topology. The results showed that Lactuca contains two distinct phylogenetic clades - the crop clade and the Pterocypsela clade. Other North American, Asian and widespread species either form smaller clades or mix with the Melanoseris species in an unresolved polytomy. The newly sampled African endemic species probably should be excluded from Lactuca and treated as a new genus.
In Chapter 3, twenty-seven wild Lactuca species and four outgroup species were sequenced using next generation sequencing (NGS) technology. The sampling covers 36% of total Lactuca species and all the important geographical groups in the genus. Thirty chloroplast genomes, including one complete (partial) large single copy region (LSC), one small single copy region (SSC), one inverted repeat (IR) region, and twenty-nine nuclear ribosomal DNA sequences (containing the internal transcribed spacer region ) were successfully assembled and analysed. A methodology paper for which I am co-author, but is not included in this thesis, of the sequencing pipeline was published: Herbarium genomics: plastome sequence assembly from a range of herbarium specimens using an Iterative Organelle Genome Assembly (IOGA) pipeline. These NGS data helped resolve deeper nodes in the phylogeny within Lactuca and resolved the polytomy from Chapter 2. The results showed that there are at least four main groups within Lactuca: the crop group, the Pterocypsela group, the North American group and the group containing widely-distributed species. I also confirmed that the endemic African species should be removed and treated as a new genus.
In Chapter 4, quantitative trait loci (QTLs) related to salt-induced changes in Root System Architecture (RSA) and ion accumulation were determined using a recombinant inbred line population derived from a cross between cultivated lettuce and wild lettuce. I measured the components of RSA by replicated lettuce seedlings grown on vertical agar plates with different NaCl concentrations in a controlled growth chamber environment. I also quantified the concentration of sodium and potassium in replicates of greenhouse-grown plants watered with 100 mM NaCl. The results identified a total of fourteen QTLs using multi-trait linkage analysis, including three major QTLs associated with general root development (qRC9.1), root growth in salt stress condition (qRS2.1), and ion accumulation (qLS7.2).
In Chapter 5, one of the identified QTL regions (qLS7.2) reported in Chapter 4 was found to contain a homolog of the HKT1 from Arabidopsis thaliana. I did a phylogenetic analysis of Lactuca HKT1-like protein sequences with other published HKT protein sequences and determined transmembrane and pore segments of lettuce HKT1;1 alleles, according to the model proposed for AtHKT1;1. Gene expression pattern and level of LsaHKT1;1 (L. sativa Salinas) and LseHKT1;1 (L. serriola) in root and shoot were investigated in plants growing hydroponically over a time-course. The measurements of Na+ and K+ contents were sampled at the same time as the samples used for gene expression test. In addition, I examined the 5 promoter regions of the two genotypes. The results showed low expression levels of both HKT1;1 alleles in Lactuca root and relatively higher expression in shoot, probably due to the negative cis-regulatory elements of HKT1 alleles found in Lactuca promoter regions. Significant allelic differences were found in HKT1;1 expression in early stage (0-24 hours) shoots in and in late stage (2-6 days) roots. shoot HKT1;1 expression/root HKT1;1 expression was generally consistent with the ratios of Na+/K+ balance in the relevant tissues (shoot Na+/K+ divided by root Na+/K+).
In Chapter 6, I summarize and discuss the results from previous chapters briefly. The implications of Chapter 2 and 3 for Lactuca phylogenetics are discussed, including some key characters for the diagnosis of species within Lactuca, the use of herbarium DNA for NGS technology, and perspectives into Lactuca phylogeny. Future perspectives of genome-wide association mapping for lettuce breeding were also discussed. Lastly, I propose to integrate phylogenetic approaches into investigations of allelic differences in lettuce, not just associated with salinity stress but also with other stressed and beneficial characters, both within and between species.