Zhanao Deng

Cloning and characterization of NBS-LRR class resistance-gene candidate sequences in citrus.

Abstract Numerous disease resistance gene-like DNA sequences were cloned from an intergeneric hybrid of Poncirus and Citrus, using a PCR approach with degenerate primers designed from conserved NBS (nucleotide-binding site) motifs found in a number of plant resistance genes. Most of the cloned genomic sequences could be translated into polypeptides without stop codons, and the sequences contained the characteristic motifs found in the NBS-LRR class of plant disease resistance genes. Pairwise comparisons of these polypeptide sequences indicated that they shared various degrees of amino-acid identity and could be grouped into ten classes (RGC1–RGC10). When the sequences of each class were compared with known resistance-gene sequences, the percentage of amino-acid identity ranged from 18.6% to 48%. To facilitate genetic mapping of these sequences and to assess their potential linkage relationship with disease resistance genes in Poncirus, we developed CAPS markers by designing specific primers based on the cloned DNA sequences and subsequently identifying restriction enzymes that revealed genetic polymorphisms. Three of the amplified DNA fragment markers (designated as 18P33a, Pt9a, and Pt8a) were associated with the citrus tristeza virus resistance gene (Ctv), and one fragment (Pt8a) was associated with the major gene responsible for the citrus nematode resistance (Tyr1); both genes are from Poncirus and of importance to citrus survival and production. These polymorphic fragments were located on two local genetic linkage maps of the chromosome region from Ctv to Tyr1. These results indicate that resistance-gene candidate sequences amplified with the NBS-derived degenerate primers are valuable sources for developing markers in disease resistance-gene tagging, mapping, and cloning.

Factors affecting Agrobacterium-mediated transformation and regeneration of sweet orange and citrange

Epicotyl explants of sweet orange and citrange were infected with Agrobacterium strain EHA101 harboring binary vector pGA482GG, and factors affecting the plant regeneration and transformation efficiency were evaluated. Increasing the wounded area of explants by cutting longitudinally into two halves, and optimization of inoculation density, dramatically enhanced both regeneration and transformation frequency. Inclusion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the explant pretreatment medium and the co-culture medium improved the transformation efficiency by decreasing the escape frequency. More than 90% rooting frequency of transformed citrange shoots was achieved by two-step culture: first on media supplemented with auxins, and then on media without hormones. Inclusion of 20 mg l−1 kanamycin in rooting medium efficiently discriminated transformed shoots from non-transgenic escaped shoots. Shoot grafting in vitro was used to regenerate transformed plants, due to the slow growth of most sweet orange shoots.

Inheritance of citrus nematode resistance and its linkage with molecular markers.

Abstract Eleven RAPD markers linked to a gene region conferring resistance to citrus nematodes in an intergen-eric backcross family were identified. Two sequence- characterized amplified region markers linked to a citrus tristeza virus resistance gene and one selected resistance gene candidate marker were evaluated for their association with citrus nematode resistance. A nematode-susceptible citrus hybrid, LB6-2 [Clementine mandarin (Citrus reticulata)×Hamlin orange (C. sinensis)], was crossed with the citrus nematode-resistant hybrid Swingle citrumelo (C. paradisi×Poncirus trifoliata) to produce 62 hybrids that were reproduced by rooted cuttings. The plants were grown in a greenhouse and inoculated with nematodes isolated from infected field trees. The hybrids segregated widely for this trait in a continuous distribution, suggesting possible polygenic control of the resistance. Bulked segregant analysis was used to identify markers associated with resistance by bulking DNA samples from individuals at the phenotypic distribution extremes. Linkage relationships were established by the inheritance of the markers in the entire population. A single major gene region that contributes to nematode resistance was identified. The resistance was inherited in this backcross family from the grandparent Poncirus trifoliata as a single dominant gene. QTL analysis revealed that 53.6% of the phenotypic variance was explained by this major gene region. The existence of other resistance-associated loci was suggested by the continuous phenotypic distribution and the fact that some moderately susceptible hybrids possessed the resistance-linked markers. The markers may be useful in citrus rootstock breeding programs if it can be demonstrated that they are valid in other genetic backgrounds.

Construction of a bacterial artificial chromosome (BAC) library for citrus and identification of BAC contigs containing resistance gene candidates

Abstract A BAC library was constructed from the genomic DNA of an intergeneric Citrus and Poncirus hybrid. The library consists of 24,576 clones with an average insert size of 115 kb, representing approximately seven haploid genome equivalents and is able to give a greater than 99% probability of isolating single-copy citrus DNA sequences from this library. High-density colony hybridization-based library screening was performed using DNA markers linked to the citrus tristeza virus (CTV) resistance gene and citrus disease resistance gene candidate (RGC) sequences. Between four and eight clones were isolated with each of the CTV resistance gene-linked markers, which agrees with the library’s predicted genome coverage. Three hundred and twenty-two clones were identified using 13 previously cloned citrus RGC sequences as probes in library screening. One to four fragments in each BAC were shown to hybridize with RGC sequences. One hundred and nine of the RGC BAC clones were fingerprinted using a sequencing gel-based procedure. From the fingerprints, 25 contigs were assembled, each having a size of 120–250 kb and consisting of 2–11 clones. These results indicate that the library is a useful resource for BAC contig construction and molecular isolation of disease resistance genes.

Comprehensive meta-analysis, co-expression, and miRNA nested network analysis identifies gene candidates in citrus against Huanglongbing disease

BackgroundHuanglongbing (HLB), the most devastating disease of citrus, is associated with infection by Candidatus Liberibacter asiaticus (CaLas) and is vectored by the Asian citrus psyllid (ACP). Recently, the molecular basis of citrus–HLB interactions has been examined using transcriptome analyses, and these analyses have identified many probe sets and pathways modulated by CaLas infection among different citrus cultivars. However, lack of consistency among reported findings indicates that an integrative approach is needed. This study was designed to identify the candidate probe sets in citrus–HLB interactions using meta-analysis and gene co-expression network modelling.ResultsTwenty-two publically available transcriptome studies on citrus–HLB interactions, comprising 18 susceptible (S) datasets and four resistant (R) datasets, were investigated using Limma and RankProd methods of meta-analysis. A combined list of 7,412 differentially expressed probe sets was generated using a Teradata in-house Structured Query Language (SQL) script. We identified the 65 most common probe sets modulated in HLB disease among different tissues from the S and R datasets. Gene ontology analysis of these probe sets suggested that carbohydrate metabolism, nutrient transport, and biotic stress were the core pathways that were modulated in citrus by CaLas infection and HLB development. We also identified R-specific probe sets, which encoded leucine-rich repeat proteins, chitinase, constitutive disease resistance (CDR), miraculins, and lectins. Weighted gene co-expression network analysis (WGCNA) was conducted on 3,499 probe sets, and 21 modules with major hub probe sets were identified. Further, a miRNA nested network was created to examine gene regulation of the 3,499 target probe sets. Results suggest that csi-miR167 and csi-miR396 could affect ion transporters and defence response pathways, respectively.ConclusionMost of the potential candidate hub probe sets were co-expressed with gibberellin pathway (GA)-related probe sets, implying the role of GA signalling in HLB resistance. Our findings contribute to the integration of existing citrus–HLB transcriptome data that will help to elucidate the holistic picture of the citrus–HLB interaction. The citrus probe sets identified in this analysis signify a robust set of HLB-responsive candidates that are useful for further validation.

Construction of citrus gene coexpression networks from microarray data using random matrix theory

After the sequencing of citrus genomes, gene function annotation is becoming a new challenge. Gene coexpression analysis can be employed for function annotation using publicly available microarray data sets. In this study, 230 sweet orange (Citrus sinensis) microarrays were used to construct seven coexpression networks, including one condition-independent and six condition-dependent (Citrus canker, Huanglongbing, leaves, flavedo, albedo, and flesh) networks. In total, these networks contain 37 633 edges among 6256 nodes (genes), which accounts for 52.11% measurable genes of the citrus microarray. Then, these networks were partitioned into functional modules using the Markov Cluster Algorithm. Significantly enriched Gene Ontology biological process terms and KEGG pathway terms were detected for 343 and 60 modules, respectively. Finally, independent verification of these networks was performed using another expression data of 371 genes. This study provides new targets for further functional analyses in citrus.

Evaluation of Silicon for Managing Powdery Mildew on Gerbera Daisy

ABSTRACT Powdery mildew, caused by Erysiphe cichoracearum or Podosphaera fusca, is a common disease of gerbera daisy (Gerbera jamesonii) grown in Florida. Previous studies demonstrated that silicon reduces powdery mildew in Arabidopsis, cucumber, grape, strawberry, and wheat. In this study, two silicon (Si) sources, calcium silicate and potassium silicate, were evaluated for their ability to reduce powdery mildew in gerbera ‘Snow White’. The effect of calcium silicate in flower quality and the silicon uptake was also determined. Calcium silicate was not effective in reducing powdery mildew or in improving flower quality. The silicon content of gerberas treated with potassium silicate was slightly greater than that in plants treated with calcium silicate. However, the severity of powdery mildew was not reduced by potassium silicate. The results suggest that silicon may not be useful for managing this disease of gerbera daisy, possibly due to the low accumulation of silicon by gerbera leaves.

De Novo Assembly, Annotation, and Characterization of Root Transcriptomes of Three Caladium Cultivars with a Focus on Necrotrophic Pathogen Resistance/Defense-Related Genes

Roots are vital to plant survival and crop yield, yet few efforts have been made to characterize the expressed genes in the roots of non-model plants (root transcriptomes). This study was conducted to sequence, assemble, annotate, and characterize the root transcriptomes of three caladium cultivars (Caladium × hortulanum) using RNA-Seq. The caladium cultivars used in this study have different levels of resistance to Pythium myriotylum, the most damaging necrotrophic pathogen to caladium roots. Forty-six to 61 million clean reads were obtained for each caladium root transcriptome. De novo assembly of the reads resulted in approximately 130,000 unigenes. Based on bioinformatic analysis, 71,825 (52.3%) caladium unigenes were annotated for putative functions, 48,417 (67.4%) and 31,417 (72.7%) were assigned to Gene Ontology (GO) and Clusters of Orthologous Groups (COG), respectively, and 46,406 (64.6%) unigenes were assigned to 128 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. A total of 4518 distinct unigenes were observed only in Pythium-resistant “Candidum” roots, of which 98 seemed to be involved in disease resistance and defense responses. In addition, 28,837 simple sequence repeat sites and 44,628 single nucleotide polymorphism sites were identified among the three caladium cultivars. These root transcriptome data will be valuable for further genetic improvement of caladium and related aroids.

Induction, Identification, and Characterization of Tetraploids in Japanese Privet (Ligustrum japonicum)

A number of privet species (Ligustrum spp.) that are important to the nursery and landscape industry have escaped cultivation and become invasive or weedy in the United States and other countries. Induced tetraploids in these species may produce new selections or cultivars with reduced or eliminated invasive potential. Applying drops of semisolid agar containing 0.1% to 0.3% colchicine and 0.2% dimethyl sulfoxide (DMSO) to newly emerged seedlings of japanese privet (Ligustrum japonicum Thunb.) resulted in 15.6% to 22.6% tetraploid induction. The nuclear DNA content of tetraploids was 5.31 pg/2C, 101.9% higher than that of diploids. Compared with diploid plants, tetraploids were more compact, with an average of 31.0% shorter plant height and 33.1% smaller canopy width. Tetraploids had 29.2% thicker internodes, and their leaves were 39.5% larger and 33.8% thicker, resulting in 42.1% to 24.1% greater fresh or dry leaf weights (per leaf) in tetraploids compared with diploids. Without indole-3-butyric acid (IBA) treatment, cuttings from tetraploids showed 28% lower rooting than diploids. IBA treatments improved the rooting of tetraploid cuttings, resulting in 65% rooting success. These results indicate that tetraploids can be readily induced in japanese privet and induced tetraploids show significant changes in plant growth and size, shoot growth, leaf morphology, and rooting of cuttings. The modified tetraploid induction method and the induced tetraploids are expected to be useful for producing new selections or cultivars with reduced invasive potential in japanese and other privets. Exotic plant invasions are considered one of the main causes of the degradation of ecosystems and the loss of biodiversity globally (Theoharides and Dukes, 2007). Ornamental horticulture has been recognized as the main source of plant invaders (Bell et al., 2003; Niemiera and Von Holle, 2009; Reichard andWhite, 2001; Rejm anek, 2014). The economic impacts of invasive plant species in the United States are estimated at nearly

A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants

35 billion (Pimentel et al., 2005). To help mitigate this huge impact and meet the nursery and landscape industry’s need for plant materials, horticulturists have been searching for cultivars with reduced invasive potential (Knox and Wilson, 2006; Trueblood et al., 2010; Wilson and Mecca, 2003; Wilson et al., 2004, 2012). Multiple ornamental breeding programs in the United States have initiated breeding projects to develop new cultivars with reduced or eliminated invasive potential in major invasive ornamental shrubs or trees (Anderson, 2007; Czarnecki et al., 2012; Freyre et al., 2012; Leonhardt and Shi, 2009; Olsen, 2007; Phillips et al., 2015; Ranney et al., 2007, 2010; Thammina et al., 2011; Vining et al., 2012). Developing genetic tools and generating germplasm resources that can be used for this objective have become essential and important for current and future ornamental plant breeding (Anderson, 2007; Li et al., 2004; Olsen, 2007; Thammina et al., 2011; Vining et al., 2012). Several species of Ligustrum L. (privet) are commonly used as ornamentals in many parts of the world (Dirr, 1998; Wilson et al., 2014). In the landscape, these plants are valued for their evergreen leaves, white flowers, adaptability to a range of landscape conditions, tolerance to pruning, resistance to diseases, and wide availability (Dirr, 1998). Some Ligustrum species have escaped cultivation and become naturalized in natural areas (Munger, 2003). For example, 16 countries report naturalization of chinese privet (Ligustrum sinense Lour.) (Morris et al.; 2002; Munger, 2003). In the United States, chinese privet has escaped in 20 states and is considered invasive in many states (FLEPPC, 2015; Munger, 2003). Glossy privet (Ligustrum lucidum W.T. Aiton) has escaped cultivation in 10 states in the United States (Munger, 2003; Wilson et al., 2014) and is also a weed in Australia (Panetta, 2000), New Zealand (Miller and Henzell, 2000), and Argentina (Hoyos et al., 2010). Japanese privet (L. japonicum Thunb.) is native to Japan and eastern Asia and was introduced to the United States from Japan and Korea in 1845 as an ornamental landscape plant (Munger, 2003; Wilson et al., 2014). Since then, this species has been widely used in the landscape in the southeastern, southern, and western United States. It has escaped cultivation and become naturalized in 12 southeastern states in the United States (Munger, 2003). Japanese privet commonly forms dense thickets in the field or forest understories, shading and displacing many native species. Once established, it is difficult to eradicate. The invasiveness of many ornamentals is attributed to a number of factors including prolific production of viable seeds. Thus, making plants sterile or seedless can reduce, even eliminate, their invasive potential (Anderson, 2007; Ranney, 2004, 2006). Several genetic tools have been used to reduce seed production, viability, and/or germination, including natural mutations, artificial mutagenesis, interspecific hybridization, ploidy manipulation (Czarnecki et al., 2012; Freyre et al., 2012; Trueblood et al., 2010), endosperm culture (Thammina et al., 2011), and transgenics (Li et al., 2004; Vining et al., 2012). So far, ploidy manipulation has resulted in considerable success and yielded multiple sterile, noninvasive cultivars or breeding lines in several important ornamental plants, such as HORTSCIENCE VOL. 51(11) NOVEMBER 2016 1371 Hypericum androsaemum L. (Trueblood et al., 2010), Lantana camara L. (Czarnecki et al., 2012), and Ruellia simplex Wright (Freyre et al., 2012). The key step in ploidy manipulation is chromosome doubling and induction of stable tetraploids, which are the gateway to obtain other ploidy levels (triploids, pentaploids, hexaploids, octoploids, etc.) through interploid crosses. Several antimitotic agents have been used to inhibit the separation of chromosomes at the anaphase of cell division and achieve chromosome doubling in ornamental plants (Contreras, 2012; Contreras et al., 2010; Nadler et al., 2012; Vining et al., 2012). Colchicine has been one of the widely used agents to induce tetraploids in numerous ornamental plants (Abdoli et al., 2013; Henny et al., 2009; Lehrer et al., 2008; Leonhardt and Shi, 2009). Several types of plant tissues or organs, such as seeds, seedlings, axillary buds or shoot tips, embryos, and cultured cells or tissues, have been used as the target for colchicine treatment with various rates of successful tetraploid induction and mixoploidy (Habbard et al., 2016; Henny et al., 2009; Jones et al., 2007; Lehrer et al., 2008; Leonhardt and Shi, 2009; Vining et al., 2012). The availability of an effective and efficient chromosome doubling and tetraploid induction protocol is critical for successful ploidy manipulation and development of sterile, noninvasive cultivars in invasive ornamental plants. Our literature searches and quick surveys of privet cultivars indicated a lack of tetraploids in glossy, chinese, and japanese privet. Preliminary work on treating axillary buds of privet plants with colchicine resulted in few solid tetraploids. Thus, the main objectives of this study were to use japanese privet as a model to evaluate the effectiveness and efficiency of the semisolid agar method for induction of stable tetraploids in privet and to characterize the effects of chromosome doubling and tetraploidy on shoot growth and leaf morphology. Materials and Methods Plant materials.Open-pollinated seedswere collected from mature shrubs of L. japonicum ‘Texanum’ grown at the University of Florida (UF) North Florida Research and Education Center, Quincy, FL, in Feb. 2010 and shipped to the UF Gulf Coast Research and Education Center (GCREC), Wimauma, FL. Dry seeds were sown onto a commercial potting mix (Metro Mix 200; Sun Gro Horticulture, Agawam, MA) in plastic containers (15 cm in diameter) in Feb. 2011. Seeds were covered with a layer of horticultural grade vermiculite ( 1 cm thick) and germinated in a growth room at 24 C with 16 h of light (120 to 150 mmol·m·s) and 8 h of dark. About 5weeks after sowing, seedlings emerged with fully expanded cotyledons. Induction, identification, and confirmation of tetraploids. The semisolid agar method of Jones et al. (2007) was used with some modifications. Colchicine was applied to the growing points of newly emerged seedlings at the cotyledonary stage to induce tetraploids. A 1% colchicine stock solution was made by dissolving colchicine power (Sigma-Aldrich, St. Louis, MO) in water. The colchicine stock solution was added to melted semisolid agar containing one-fourth-strength Murashige and Skoog (MS) basal salts, 0.55%agar, andDMSO (Sigma-Aldrich; final concentration 0.2%) to a final concentration. Three colchicine concentrations were used: 0.1%, 0.2%, and 0.3%. The colchicine-containing agar solution was kept at 40 C and then 30 mL were pipetted onto the growing point of each privet seedling. Treated seedlings were placed in a high humidity ( 100% relative humidity) growth chamber at 24 C in dark to minimize colchicine degradation by light. Control seedlings received semisolid agar without colchicine. All treatments were repeated during three consecutive days. After treatment, seedlings were grown in a greenhouse, and fertigated with 50 to 100 ppm nitrogen as needed. Seedlings were kept in community containers until they produced shoots with several true leaves and later transplanted individually into new containers (10 to 15 cm in diameter). Seedlings were grown in these containers for 1 year, and then transplanted into ground beds at the UF’s GCREC in Mar. 2013. All transplanted plants were grown in full sun, irrigated through drip tapes, and fertilized with 15 g of 15N–3.9P–12K controlled-release fertilizer (Osmocote; Scotts, Marysville, OH). To identify tetraploids, multiple fully expanded young leaveswere collected from each plant, and each leaf was analyzed separat