An important goal of backyard orchard culture is to maintain relatively small trees to facilitate pruning, thinning, pest management, and harvesting. By heading the newly planted tree at knee height, about 18 to 24 inches , you force the tree to develop low branches. However, if access under the tree is important, head the tree higher, up to 36 inches . Small trees, those with a trunk diameter of 3 ⁄8 inch or less, usually have no lateral branches on their trunks worth saving, so remove all side branches . Larger trees, 1 ⁄2-inch diameter or larger, often have large lateral branches along their trunks. Some of these branches can be removed completely, but a few that are well spaced vertically and radially around the trunk can be headed back, leaving 3-inch outward-growing stubs with two or three lateral buds . Fleshy fruits are agriculturally and economically important plant organs that have evolved from dry fruits many times during angiosperm evolution. However, the genetic changes that are required for this shift to occur are as yet unknown . In the agriculturally, pharmacologically, and horticulturally important plant family Solanaceae , hydroponic nft gully there was a shift to fleshy fruit in the subfamily Solanoideae from plesiomorphic dry fruit . In the family two independent transitions to fleshy fruits have also occurred in the genera Duboisia and Cestrum , as well as a reversal to dry fruit in the genus Datura .

Evidence from tomato indicates that FRUITFULL transcription factors have novel functions in fleshy fruit development compared to Arabidopsis and Nicotiana . FUL is a MADS-box TF that plays pleiotropic roles in both reproductive and vegetative development in the model plant Arabidopsis thaliana . FUL controls cell proliferation in the fruit valves and spatially limits the formation of the dehiscence zone in the dry silique of A. thaliana, enabling the mature fruits to dehisce . Overexpression of a Nicotiana tabacum FUL ortholog in woodland tobacco resulted in indehiscent fruits with reduced lignification at the dehiscence zones, suggesting a role similar to that observed in silique development in A. thaliana . Several groups have examined the function of euFUL genes, the core-eudicot clade to which FUL belongs, in tomato . All studies showed defects in fruit pigmentation during ripening when FUL ortholog expression was down regulated, and some studies also suggested roles in ethylene production and pericarp and cuticle thickness . These data indicate that euFUL genes are controlling different processes in dry and fleshy fruits in the Solanaceae. Early in the diversification of core-eudicots, there was a duplication in the euFUL gene clade, which resulted in the euFULI and euFULII clades . The A. thaliana FUL gene belongs to the euFULI clade while its paralog, AGL79 which plays a role in lateral root development, branching, leaf morphology, and transition to flowering, belongs to the euFULII clade . The euFULI clade has duplicated in Solanaceae resulting in two subclades, designated here as FUL1 and FUL2; likewise the euFULII clade has two Solanaceae-specific subclades, here designated MBP10 and MBP20 .

We studied the evolution of euFUL genes in Solanaceae to characterize patterns of selection, duplication, and sequence evolution to identify changes that might be correlated with the shift to fleshy fruit. We tested the following hypotheses: following the duplication of euFUL genes, there was a relaxation of selection in some or all of the resulting clades that resulted in sequence diversification; changes in amino acid sequences are correlated with the origin of fleshy fruit. Although we found several sites showing changes in amino acid residues that might have resulted in changes in protein function, none of these were associated with the evolution of fleshy fruit. Consistent with our hypothesis, we found that the FUL1 and MBP10 genes are evolving at significantly faster rates in comparison to FUL2 and MBP20. In combination with the relatively weak expression of MBP10 and loss of potential regulatory elements, our data suggest that the MBP10 lineage may be undergoing pseudogenization.RNA was extracted from fruit, floral/inflorescence or leaf tissue using RNeasy Plant Mini Kits according to the manufacturer’s protocol. For Grabowskia glauca, Dunalia spinosa, Fabiana viscosa, and Salpiglossis sinuata RNA extractions, lysis buffer RLC was used instead of RLT and 2.5% polyvinylpyrrolidone was added. The RLT buffer was used for extracting RNA from all other species. RNA quality was checked using a BioSpectrometer Basic and stored at −80◦C. cDNA was synthesized using SuperScript III Reverse Transcriptase according to the manufacturer’s protocol and the product was checked by amplifying ACTIN. Clade-specific degenerate primers were designed to target specific euFUL gene homologs based on conserved regions in Solanaceae euFUL gene alignments .

PCR was run for two initial cycles with an annealing temperature between 40 and 45◦C followed by 30 cycles at 55◦C annealing temperature. The PCR products were visualized on a 1% agarose gel. If multiple amplicon band sizes were present, the annealing temperature of the first two cycles was increased until only one product size was achieved.PCR products were purified using QIAquick PCR Purification Kit according to the manufacturer’s protocol. The purified product was then cloned using TOPO TA Cloning Kit according to the manufacturer’s protocol, and the ligated plasmids were transformed into chemically competent TOP10 strain of Escherichia coli. Transformants were plated on LB plates with kanamycin selection coated with 40 µL of 25 mg/mL X-Gal and IPTG, and incubated at 37◦C overnight. Individual positive colonies were used as templates in amplification with M13F and M13R primers to identify those colonies with inserts of the expected size between 500 bp and 1 kb. These were grown overnight in 5 mL liquid LB medium supplemented with kanamycin in an incubator-shaker at 250 RPM and 37◦C. Plasmids were extracted from the liquid cultures using Plasmid Miniprep Kit according to the manufacturer’s protocol, and sequenced using M13 reverse primer at the Institute for Integrative Genome Biology at UCR or Eton Bioscience, Inc. . For library preparation, RNA quality was checked using a Bioanalyzer . Each assembled transcriptome was then used to create a custom Basic Local Alignment Tool  database. The BLAST database for each species was queried on the HPCC with both blastn and tblastx using all available sequences in our euFUL sequence file using a UNIX command line that sequentially matched each sequence in our query file against the database . BLAST analyses were also conducted on the NCBI2 and oneKP3 databases using A. thaliana FUL and various Solanaceae FUL homologs as query. Matching output sequences from both transcriptomes assemblies and database mining were further confirmed by compiling a gene tree as described below. We confirmed the accuracy of our sequences using gene specific primers and Sanger sequencing. Unless specified otherwise, all sequences referred to in this manuscript are the full or partial mRNA sequences.The Multiple Sequence Comparison by Log-Expectation  tool was used to align euFUL sequences . The appropriate model for tree building, GTR+G, was determined with jModelTest 2.0 . Ten independent maximum likelihood analyses starting with random trees were performed using GARLI v2.1 . euFUL genes from Convolvulaceae , which were retrieved from the oneKP database4 , dutch buckets for sale were designated as the out group in each analysis, which meant these sequences were automatically excluded from the in group clades. Each ML run was set to terminate when there was no significantly better scoring topology for 20,000 consecutive generations. The ten resulting trees were checked for agreement by calculating the pairwise Robinson–Foulds distance using ‘ape’ and ‘phangorn’ packages on R . The tree with the largest ML value was chosen as the starting tree in a bootstrap analysis involving 1,000 replicates. The results of the replicates were summarized and bootstrap values were calculated using SumTrees tool of DendroPy package on Python ver. 2.7 or Geneious 10.2 . Any sequences that did not group with any of the subclades were aligned with the paralogs to investigate whether these may have been splice isoforms. Any such isoform was expected to have large insertions/deletions at splice junctions.The CODEML program within the Phylogenetic Analysis by Maximum Likelihood  v 1.35 software package was run on the HPCC at UCR to analyze the selection pressure acting on euFUL genes. These analyses were performed to test if different gene lineages as well as sub-groups within those lineages were evolving at significantly different rates. Further scenarios were considered in which each gene, the transition branches from dry to fleshy fruit trait, or specific sites in the sequences were tested for significantly different rates of evolution. Model 0 was used to estimate a single evolutionary rate for all genes when the clades being analyzed encompassed the entire dataset. Model 2 was used when two groups encompassing the entire data set have different rates or when two groups that are being compared do not encompass the entire data set.

In the latter case, the two clades being compared were grouped together to obtain a single evolutionary rate in comparison to the rate for the remaining data . This single rate for the two clades grouped together was then compared to the rates for each clade separately to determine if the separate rates were significantly different from the combined rate. The test statistic, 21 L , and the degrees of freedom , were then used in chi-squared tests to check for statistical significance. In any comparison where the P-value was less than 0.05, the second hypothesis was considered to have the better fit than the first, implying there is statistical power to support that the gene clades are evolving at different rates. Since Solanaceae has a well-supported phylogeny , for PAML analyses, the branches of the gene-tree described above were adjusted to match the phylogenetic relationships of the species included in the analysis. In the euFUL gene groups that are evolving faster,sites undergoing positive selection were analyzed using mixed effects model of evolution 6 . The gene alignments for the euFUL subclades that are evolving at statistically significantly faster than the other subclades were translated using AliView . In these protein alignments, the sites that changed from hydrophilic to hydrophobic or vice versa were identified manually. Those changes that might have been functionally deleterious versus those that might have been neutral were identified using the PROVEAN Protein tool7 . MADS , intervening/interacting and keratin-like domains of the proteins were identified using a published MADSbox protein model . The structure of M, I, and K domains of tomato FUL1 and MBP10 were predicted using PHYRE2 server8 .Our analysis consisted of 106 sequences from 45 species in 26 genera obtained from direct amplification, transcriptomes, and online genomic databases . Of these, 64 sequences belonged to species from the Solanoideae, characterized by the derived fleshy fruit, whereas the other 42 sequences were from species with the ancestral dryfruit trait. We designated euFUL genes from Convolvulaceae, the sister-group of Solanaceae, as the out group . For many species in the analysis, we have an incomplete set of paralogs; however, we had substantial and diverse representation from across the phylogeny, which allows us to test hypotheses regarding the evolution of this gene lineage in Solanaceae. We used maximum likelihood methods  to reconstruct the relationships of Solanaceae euFUL genes . The resulting tree shows two major lineages of euFUL genes, with 80% and 100% bootstrap support, respectively, that correspond to the previously identified core eudicot euFULI and euFULII lineages . A Solanaceae whole-genome triplication has been proposed , which would suggest that all Solanaceae should have three euFULI and three euFULII genes. However, others have suggested a duplication . Our data and other studies, as well as searches of the tomato genome have shown that tomato has four euFUL genes: two euFULI and two euFULII instead of the six predicted by a triplication. Additional genome sequencing , transcriptome sequencing, and PCR-based analyses have also found two euFULI and two euFULII genes. This suggests the loss of one paralog from each of the euFULI and euFULII clades following a whole-genome triplication or, alternatively one or more duplication events . For the purposes of this paper, we will refer to the euFULI and euFULII subclades by the name currently used for the tomato gene in each subclade . Thus, the two euFULI subclades will be referred to as the FUL1 and FUL2 clades, and the euFULII subclades will be referred to as the MPB10 and MBP20 subclades . In our gene tree, while the FUL2, MBP10, and MBP20 clades had high bootstrap support of 83, 99 and 89%, respectively, the FUL1clade had only 53% support .