Month: May 2025

Superior fruit quality is also associated with sugar levels. During fruit ripening, sugar levels of the endocarp increase by importing hexose symplastically and/or apoplastically. Sugar transporters , sucrose transporter, and tonoplast sugar transporter have been demonstrated to regulate intercellular sugar transport in phloem and fruit.To the best of our knowledge, we are the first to report on the potential role of these genes during blueberry fruit development. In addition, homologs of A. thaliana TST1 and watermelon ClTST1 and ClTST3 were expressed during fruit ripening in blueberry. Elevated expression of a ClTST1 homolog was observed throughout fruit development, but the ClTST3 homolog showed very low expression. Another gene that is highly expressed during fruit maturation is vacuolar invertase. As described in other systems, its upregulation during fruit ripening coincided with the breakdown of starch to sucrose or a mixture of glucose and fructose, suggesting that it may be involved in the regulation of sugar accumulation in blueberry fruit. It was previously reported that vacuolar invertase modulates the hexose to sucrose ratio in ripening fruit. In addition, there are also two sugar transport protein homologs that exhibited developmental specific expression. However, their function remains largely unknown, thus, their potential role in sugar accumulation in the developing berry requires further investigation.Tandemly duplicated genes arise as a result of unequal crossing over or template slippage during DNA repair, exhibit high birth-death rates, grow bucket and typically are in co-regulated clusters in the genome.

Smaller scale duplications, which include tandem duplicates, are highly biased toward certain gene families including those involved in specialized metabolism. Furthermore, tandem duplications often results in the increased dosage of gene products and may improve the metabolic flux of ratelimiting steps in certain bio-synthetic pathways. Most genes associated with the biosynthesis of antioxidants have at least one tandem duplicate present in the highbush blueberry genome, with tandem array sizes ranging from 2 to 10 gene copies . The largest tandem arrays were found for HQT and HCT genes, which are co-regulated and involved in the CGA pathway . Differences in tandem array sizes were also observed between homoeologous chromosomes for various genes. For example, the C3H gene, which is involved in CGA biosynthesis , was present on all four homoeologous chromosomes but with varying tandem array sizes. One of the homoeologous chromosomes had two copies of C3H, while the other three homoeologous chromosomes had four copies. This suggests that copy number differences of C3H among subgenomes may be due to either selection for gene duplication or loss or, in the case of allopolyploidy, may be due to preexisting gene content differences among the diploid progenitor species. Genes in the anthocyanin pathway with other unique duplication patterns include CHS, CHI, OMT, and UFGT. The gene CHS, involved in the conversion of 4-coumaryl-CoA to naringenin chalcone, has two copies, and both have tandem duplicates in at least three of the homoeologous chromosomes. Interestingly, the gene CHI has a single preserved tandem gene duplicate on only one of the homoeologous chromosomes. However, additional copies of CHI were also identified more distantly away from the syntenic ortholog on another homoeologous chromosome, likely involving a transposition event following tandem duplication.

The OMT and UFGT genes all have tandem duplicates on all of the homoeologous chromosomes, although with varying array sizes, while the ANR gene involved in the conversion of anthocyanidin to proanthocyanidin is single copy on all homoeologous chromosomes. DFR gene, which is involved inthe conversion of dihydroquercetin/dihyromyricetin to leucoanthocyanidin, has a single tandem duplicate on only one of the homoeologous chromosomes. These findings suggest that there may have been greater selective pressure to retain tandem duplicates for genes encoding enzymes involved in anthocyanin production than conversion to proanthocyanidins. The vast majority of tandem duplicates are eventually lost ; however, in rare instances, some may undergo functional diversification. Gene expression analysis revealed that 83.4% of the tandem duplicates were expressed in at least one transcriptome library with 73.5% expressed in at least one of the fruit developmental stages. This suggests that a subset of these duplicate genes have non-functionalized, subfunctionalized, or neofunctionalized. Future studies are needed to more thoroughly investigate the functions of these genes with more diverse libraries and additional transcriptome analyses.Despite the economic importance of blueberry, molecular breeding approaches to produce superior cultivars have been greatly hampered by inadequate genomic resources and a limited understanding of the underlying genetics encoding important traits.

This has resulted in breeders having to solely rely on traditional approaches to generate new cultivars, each with widely varying fruit quality characteristics. For example, our analysis of a diversity panel consisting of 84 cultivars and wild species revealed that ”Draper” has antioxidant levels that are up to 19x higher than other cultivars. Thus, the genome of ”Draper” should serve as a powerful resource to the blueberry community for guiding future breeding efforts aimed at improving antioxidant levels among other important fruit quality traits. Furthermore, to our knowledge, this is not only the first genome assembly of the cultivated highbush blueberry but is also the first chromosome-scale and haplotype-phased genome for any species in the order Ericales. Ericales includes several other high-value crops and wild species with unique life history traits . Thus, we anticipate that this reference genome, plus associated datasets, will be useful for a wide variety of evolutionary studies. Here, we also leveraged the genome to identify candidate genes and pathways that encode superior fruit quality in blueberry, including those associated with pigmentation, sugar, and antioxidant levels. Furthermore, we found that genes encoding key biosynthetic steps in various antioxidant pathways are enriched with tandem gene duplicates. For example, tandem gene duplications have expanded gene families that are involved in the biosynthesis of anthocyanins. This suggests that, in addition to a recent whole genome duplication, tandem duplications may have greatly contributed to the metabolic diversity observed in blueberry . These tandem duplicates may have evolved new functions , possibly involved in the biosynthesis of novel compounds, and/or were selected to improve the metabolic flux of specific biosynthetic steps that alter the dosage of certain endpoint metabolites. Future studies are needed to further investigate the possible role of tandem duplications in having modified metabolite levels and composition in wild and cultivated blueberry. Our analyses also revealed that highbush blueberry, a tetraploid, likely arose from the hybridization of two distinct parents, possibly allopolyploidy, based on the sequence divergence, unique transposable element insertions, and subgenome expression patterns. Our analyses revealed that the subgenomes in highbush blueberry may be controlling a distinct set of genetic programs . The dominantly expressed subgenome in most surveyed tissues becomes the lowest expressed during fruit development. This observation is similar to findings in allopolyploid wheat where developmental and adaptive traits were shown to be controlled by different subgenomes. For example, cell type- and stage dependent subgenome expression dominance was observed in the developing wheat grain. We argue that both highbush blueberry and hexaploid wheat, each now with high-quality reference genomes, make excellent systems to further investigate these underlying mechanisms of subgenome dominance. Subgenome dominance has far-reaching implications to numerous research areas including breeding efforts. For example, marker-assisted breeding needs to target the correct set of dominant homoeologs given the trait in polyploids that exhibit subgenome dominance. Thus, we anticipate that this genome, combined with improved insights into subgenome dominance, dutch bucket for tomatoes will greatly accelerate molecular breeding efforts in the cultivated highbush blueberry.The genome of ”Draper” was assembled using the DeNovoMAGIC software platform , which is a de Bruijn graph-based assembler designed for higher polyploid, heterozygous, and/or repetitive genomes.

The Chromium 10X data were utilized to phase, elongate, and validate haplotype scaffolds. Four Dovetail Hi-C libraries were prepared as described previously and sequenced on an Illumina HiSeq X system with paired-end 150 bp reads to a total of 90.7X physical coverage of the genome . The de novo genome assembly, raw genomic reads, and Dovetail Hi-C library reads were used as input data for HiRise, a software pipeline designed specifically for using proximity ligation data to scaffold genome assemblies. Illumina genomic and Dovetail Hi-C library sequences were aligned to the draft input assembly using a modified SNAP read mapper. The separations of Dovetail Hi-C read pairs mapped within draft scaffolds were analyzed by HiRise to produce a likelihood model for genomic distance between read pairs, and the model was used to identify and break putative misjoins and to make joins to close gaps between contigs.Plant tissue samples were collected from blueberry cv. Draper grown in the growth chamber . For the fruit developmental series, three biological replicates each of berries at seven developmental stages were collected from cv. Draper in a field at the Horticulture Teaching and Research Center, Michigan State University, in July 2017. All plant tissues were immediately flash frozen in liquid nitrogen, and total RNA isolation was performed using the KingFisher Pure RNA Plant kit . Isolated total RNA was quantified using a Qubit 3 fluorometer . All samples were submitted to the Michigan State University Research Technology Support Facility Genomics core and sequenced with paired-end 150 bp reads on an Illumina HiSeq 4000 system .The draft genome of V. corymbosum cv. Draper was annotated using the MAKER annotation pipeline. Transcript and protein evidence used in the annotation included protein sequences downloaded from A. thaliana and UniprotKB plant databases, V. corymbosum ESTs from NCBI, and transciptome data assembled with StringTie from different blueberry tissues . A custom repeat library and Repbase were used to mask repetitive regions in the genome using Repeatmasker. Ab initio gene prediction was performed using gene predictors SNAP and Augustus. The resulting MAKER Max gene set was filtered to select gene models with Pfam domain and annotation edit distance <1.0. The filtered gene set was further scanned for transposase coding regions. The amino acid sequence of predicted genes was searched against a transposase database. The alignment between the genes and the transposases was further filtered for those caused by the presence of sequences with low complexity. The total length of genes matching transposases was calculated based on the output from the search. If more than 30% of gene length aligned to the transposases, the gene was removed from the gene set. Furthermore, to assess the completeness of annotation, the V. corymbosum Maker standard gene set was searched against the BUSCO v.3 plant dataset . Genes were annotated with pfam domains using InterProScan v5.26–65.0.To identify and classify repetitive elements in the genome, LTR retrotransposon candidates were searched using LTRharvest and LTR finder and further identified and classified using LTR retriever . A nonredundant LTR library was also produced by LTR retriever. Miniature inverted transposable elements were identified using MITE-Hunter. MITEs were manually checked for target site duplications and terminal inverted repeats and classified into superfamilies . Those with ambiguous Target Site Duplication and Terminal Inverted Repeats were classified as ”unknowns.” Using the MITE and LTR libraries, the V. corymbosum genome was masked using Repeatmasker. The masked genome was further mined for repetitive elements using Repeatmodeler. The repeats were then categorized into two groups: sequences with and without identities. Those without identities were searched against the transposase database; if they had a match, they were considered a transposon. The repeats were then filtered to exclude gene fragments using ProtExcluder and summarized using the ‘fam coverage.pl’ script in the LTR retriever package. The assembly continuity of repeat space was assessed using the LLAI deployed in the LTR retriever package. LAI was calculated based on either 3 Mb sliding windows or the whole assembly using LAI = /Total LTR-RT length. For the sliding window estimation, a step of 300 Kb was used . To account for dynamics of LTR retrotransposons, LAI was adjusted by the mean identity of LTR sequences in the genome based on all-versus-all blastn search, which was also performed by the LAI program.Illumina adapters were removed from the raw reads using Trimmomatic/0.33, and trimmed reads were filtered using FASTX Toolkit. After quality assessment using FastQC , the filtered reads were then aligned to the V. corymbosum genome using STAR. For the samples that were used for annotation, transcript assembly was performed de novo using StringTie.

These findings also will help breeders to select for genomic variants underlying volatiles important to flavor. New markers can be designed from regulatory regions of key aroma volatiles, including multiple medium-chain volatiles shown to improve strawberry flavor and consumer liking , methyl thioacetate contributing to overripe flavor and methyl anthranilate imparting grape flavor . In the present study, a new functional HRM marker for mesifurane was developed and tested in multiple populations . These favorable alleles of volatiles can be pyramided to improve overall fruit flavor via marker assisted selection. Strawberry also shares common volatiles with a variety of fruit crops. 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 subgenome 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, hydroponic nft channel 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, 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 subgenomes have been identified via Hi-C sequencing , supporting a potential regulatory mechanism among homoeologous chromosomes. However, owing to the high similarity among four subgenomes 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 subgenome are consistent with its dominance in octoploid strawberry . By contrast, the highly mixed Fragaria viridis- and Fragaria nipponica- like subgenomes 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 spittlebugs 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 intersubspecific recombination among strains of X. fastidiosa and proposed recombination as the mechanism of new allele acquisition, leading to plant host shift and disease emergence. Intersubspecific 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 , nft growing system 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, 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.Bacterial strains, media, and culture conditions. 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. Media selection for natural competence and growth in microfluidic chambers. 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.