These favorable alleles of volatiles can be pyramided to improve overall fruit flavor via marker assisted selection. Specific esters are shared with apple , certain lactones are shared with peach and various terpenes are shared with citrus . Syntenic regions and orthologous genes could be exploited for flavor improvement in those species. Additional insights were gained for the strawberry gene regulatory landscape, SV diversity, complex interplays among cis- and trans- regulatory elements, and sub genome dominance. Previously, Hardigan et al. and Pincot et al. showed a large genetic diversity existing in breeding populations of Fragaria × ananassa, challenging previous assumptions that cultivated strawberry lacked nucleotide variation owing to the nature of its interspecific origin and short history of domestication . Our work corroborated their findings and showed that even highly domesticated populations harbor substantial expression regulatory elements and structural variants. Over half of the expressed genes in fruit harbored at least one eQTL, and 22 731 eGenes had impactful cis-eQTL. The distribution of trans-eQTL is not random, but rather is concentrated at a few hotspots controlled by putative master regulators . The aggregation of trans-eQTL also was observed in plant species such as Lactuca sativa and Zea mays . Furthermore, grow bucket we observed a substantial number of trans-eQTL among homoeologous chromosomes, similar to observations in other allopolyploid plant species .
In cotton, physical interactions among chromatins from different sub genomes have been identified via Hi-C sequencing , supporting a potential regulatory mechanism among homoeologous chromosomes. However, owing to the high similarity among four sub genomes and limited length of Illumina reads, false alignment to incorrect homoeologous chromosomes could arise, leading to ‘ghost’ trans-eQTL signals. Future studies are needed to scrutinize the homoeologous trans-eQTL and investigate the mechanism behind this genome-wide phenomenon. Higher numbers of trans-eQTL in the Fragaria vesca-like sub genome are consistent with its dominance in octoploid strawberry . By contrast, the highly mixed Fragaria viridis- and Fragaria nipponica- like sub genomes contained much smaller numbers of trans-eQTL. The characterization of naturally-occurring allelic variants underlying volatile abundance has direct breeding applications. First, this will facilitate the selection of desirable alleles via DNA markers. Second, understanding the causal mutations in alleles can guide precision breeding approaches such as gene editing to modify the alleles themselves and/or their level of expression. From a broader perspective, multi-omics resources such as this one will have value for breeding a wide array of fruit traits. Enhancing consumer satisfaction in fruit ultimately will depend on the improvement of the many traits that together enhance the overall eating experience.Natural competence is a phenomenon that allows bacteria to take up DNA segments from the environment and incorporate them into the genome via homologous recombination . Natural competence was first demonstrated in Streptococcus pneumoniae in 1928 by Frederick Griffith . Griffith showed that virulence genes were transferred from donor to recipient cells, converting the nonvirulent recipients into virulent pathogens . Since then, 80 bacterial species in divergent phyla have been described as naturally competent .
Although the exact reasons for occurrence of natural competence in bacteria still remain unknown, studies showed that natural competence is induced under conditions of starvation and DNA damage , and it has been hypothesized that the incoming DNA serves as a food source and DNA repair material. Another proposition is that natural competence allows acquisition of new genes and alleles, providing the recipient cells with adaptive advantages. In fact, a previous study showed an increased rate of adaptation by natural competence in Helicobacter pylori . interestingly, natural competence has been demonstrated in some of the most highly diverse and successful human pathogens such as H. pylori , Neisseria meningitidis and Neisseria gonorrhoeae , and Porphyromonas gingivalis , which require rapid adaptation to evade the immune response. Furthermore, natural competence also was described in two plant pathogens, Ralstonia solanacearum andXylella fastidiosa , both of which have very broad plant host ranges. Xylella fastidiosa is a bacterial pathogen affecting many economically important crops, such as grape, citrus, coffee, peach, and almond . The disease process is not completely understood, but it is proposed that X. fastidiosa forms biofilm-like aggregates and blocks xylem vessels, the conduits for water and nutrient transport in the plants . This blockage hinders xylem sap flow and starves the upper aerial parts of water and mineral nutrients, producing symptoms that resemble those of water and nutrient deficits. X. fastidiosa is transmitted by a number of xylem sap-feeding insects, including sharpshooter leaf hoppers and spittle bugs in which X. fastidiosa forms biofilms in the foregut .
Taxonomically, X. fastidiosa is divided into five subspecies based on multilocus sequence typing . Even within the subspecies, host range and genotype diversity have been described , and recombination events among strains have been detected among field-collected samples . In fact, homologous recombination was shown to have a greater effect in generating genetic diversity in X. fastidiosa than point mutation . Recent outbreaks of X. fastidiosa diseases in Europe and Asia and also in new plant hosts such as olive , blueberry , and pear suggest the great adaptation potential of this pathogen. In a number of plant species, X. fastidiosa is believed to live as a harmless endophyte without inducing disease symptoms . Coexistence in the same xylem system of different strains for a long time without killing the host represents a fertile environment for exchange of DNA material. Several MLST-based studies detected inter subspecific recombination among strains of X. fastidiosa and proposed recombination as the mechanism of new allele acquisition, leading to plant host shift and disease emergence. Inter subspecific recombination was described to generate strains that infect citrus and coffee , mulberry , and blueberry and blackberry . A recent study also showed intersubspecific recombination between coffee-infecting strains in plants intercepted in France . Natural competence could be an explanation for the frequent recombination events detected in X. fastidiosa. Natural competence in X. fastidiosa was recently described in vitro , the rate of homologous recombination was shown to be higher when the cells were growing exponentially in solid agar plates than in batch culture tubes, and minimal medium was more conducive than rich medium . With a plasmid as a donor DNA, 96 bp of flanking homology was sufficient to initiate recombination . Moreover, some competence-related and type IV pili genes were shown to be involved in the process . Although some of those studies were performed using plasmids as donor DNA, two strains were also shown to recombine in coculture conditions , although the capacity of these strains to act either as a donor or a recipient for DNA exchange was not determined in those studies. The objective of this study was to test the hypothesis that natural competence in X. fastidiosa occurs under flow conditions . Associated with this objective was the aim of elucidating whether previous observations of high frequencies of X. fastidiosa natural competence in vitro were dependent on batch culture conditions , which allow cell-tocell contact for longer times without replenishing of nutrients or removal of secreted molecules. Although natural competence and recombination are assumed to occur in natural habitats based on field surveys and DNA sequence data, dutch bucket for tomatoes experimental indications of its occurrence in the plant or insect host are not yet available for X. fastidiosa. Therefore, to circumvent the limitation of X. fastidiosa recombination tests in the natural hosts that are affected by uneven bacterial distribution and low populations , we performed recombination experiments in a microfluidic chamber system that mimics the natural environment of xylem vessels and insect foreguts. The MC system allows continuous media flow conditions and formation of biofilms and has been previously used to study the behavior of X. fastidiosa . The biofilm fraction of the MC and the planktonic and detached cell fraction can be collected separately, and the behavior of cells in the two fractions can be determined. Two strains used in all of the previous publications on this topic were used in this current study to facilitate comparison with the literature and to further our understanding of natural competence in X. fastidiosa. The results presented here show that growth under flow conditions supports natural competence in X. fastidiosa, with recombination frequencies equivalent to that on solid media, previously described to be the most conducive environment for natural competence in vitro . These findings support the hypothesis that recombination occurs at high rates under flow conditions, representing the natural habitats of X. fastidiosa.Xylella fastidiosa subspecies fastidiosa mutants NS1-CmR and pglA-KmR were used in this study.
The mutants were cultured in periwinkle wilt agar medium , modified by omitting phenol red and adding 1.8 g liter 1 bovine serum albumin and supplemented with the respective antibiotics. PD3 medium and modified X. fastidiosa medium were used when stated. Pectin was added to a final concentration of 0.01% as previously described . Kanamycin was used at 30 g ml 1 and chloramphenicol at 10 g ml 1 . Inocula were prepared by streaking cultures from the 80°C freezer stocks on PW agar plates and incubating the plates for 5 to 7 days at 28°C. Cultures were then restreaked onto new plates and incubated for another 5 to 7 days before use.To select a medium to test the occurrence of natural competence in MCs, three media were first tested in solid agar plates. XFM and PW, used in previous studies , were selected as positive- and negative-control media, respectively, for recombination. Natural competence experiments were performed according to the method of Kung et al. with some modifications. Briefly, cells of the NS1-CmR and pglAKmR mutants were prepared in liquid media by scraping the cultures from PW-antibiotic plates. Ten microliters of each strain was spotted on top of each other on the agar plates of PW, XFM, and PD3 without antibiotics, and the spots were allowed to dry for 1 h. The plates were then incubated at 28°C for 3 days. Next, two spots from the same plate were scraped off and suspended in 1 ml of PD3 to make one replication, and 3 to 4 replicates were included for each media type per experiment. The experiments were repeated independently twice for XFM and at least three times for PD3 and PW. Single mutant strains were included as controls. The suspensions were then serially diluted, and100- l aliquots of appropriate dilutions were plated on PW agar plates in triplicate supplemented with both antibiotics to recover recombinants at the antibiotic-resistant site and with a single antibiotic to check for the growth of both parents in the mixture. Appropriate dilutions also were plated onto PW plates without antibiotics for enumeration of total viable cells. Plates were incubated at 28°C for at least 14 days before CFU were enumerated. The recombination frequency at the antibiotic-resistant site was calculated as the ratio of recombinant CFU to total CFU in equal volumes of suspension. After selection of the media that supported recombination in the agar plates, the media were tested in the MCs for cell attachment and biofilm formation.To test for the specific components that may influence natural competence, an initial screen was performed by removing or adding components to PW and PD3 in solid agar plates as described above. The components tested were sodium citrate dehydrate, succinic acid, and starch ; BSA and L-glutamine ; and pectin. The effect of BSA was further tested by supplementing PD3 and PD3 plus L-glutamine with BSA and removing BSA from PW and XFM. Experiments were repeated three times independently with three replicates each time, except for PD3 plus L-glutamine treatment that was performed once with three replicates. The twitching motilities of both mutants were determined in media with and without BSA, according to previous studies with few modifications. Briefly, for PD3 and PW with and without BSA, media plates solidified with agar or Gelrite were divided into two halves, 10 to 12 spots of each mutant strain were made using a sterile toothpick, and plates were incubated at 28°C for 4 to 5 days. For XFM with and without BSA, plates solidified with agar were used and incubated for 10 to 12 days before measurements were recorded. Colony peripheral fringes were observed under 10 magnification using a Nikon Eclipse Ti inverted microscope , and fringe widths were measured for six colonies per plate per strain, with at least seven measurements per colony using a Nikon DS-Q1 digital camera connected to a Nikon Eclipse Ti inverted microscope and controlled by NIS-Elements imaging software version 3.0.