Cecilia McGregor

Transcriptome profiling of female alates and egg-laying queens of the Formosan subterranean termite.

Termites are known to have an extraordinary reproductive plasticity and capacity, but the underlying genetic patterns of termite reproductive biology are relatively understudied. The goal of this study was to identify genes for which expression levels differ between dealated precopulatory females (virgins) and egg-laying queens of the Formosan subterranean termite, Coptotermes formosanus Shiraki. We constructed a normalized polyphenic expressed sequence tag (EST) library that represents genomic material from most of the castes and life stages of the Formosan subterranean termite. Microarrays were designed using probes from this EST library and public genomic resources. Virgin females and queens were competitively hybridized to these microarrays and differentially expressed candidate genes were identified. Differential expression of eight genes was subsequently confirmed via reverse transcriptase quantitative PCR (RT-QPCR). When compared to virgins, queens had higher expression of genes coding for proteins related to immunity (gram negative binding protein), nutrition (e.g., termite-derived endo-beta-1,4-glucanase), protein storage, regulation of caste differentiation and reproduction (hexamerin, juvenile hormone binding protein). Queens also had higher transcript levels for genes involved in metabolism of xenobiotics, fat, and juvenile hormone (glutathione-S-transferase-like proteins, and cytochrome P450), among others. In particular, hexamerin, juvenile hormone binding protein, and a cytochrome P450 from the 4C subfamily are likely to be involved in initiating the inactive period during the reproductive cycle of the queen. Vice versa, virgins had higher expression than queens of genes related to respiration, probably due to recent flight activity, and several genes of unknown function.

Citrullus lanatus germplasm of southern Africa.

Citrullus lanatus germplasm from southern Africa is a rich source of diversity for cultivated watermelon. Wild, feral, and landrace populations of the species are found throughout the arid regions of southern Africa, where they serve as sources of water and food for humans and wildlife alike. Genetic resources from the region proved to be important sources of disease resistance for cultivated watermelon, contributing to the development of both Fusarium wilt- and anthracnose-resistant cultivars. Basic research, such as genomic mapping and the elucidation of drought tolerance, have also benefitted from the abundant genetic diversity. Currently, several ex situ collections in the region and the rest of the world house accessions originating from southern Africa. The USDA germplasm collection has been screened extensively for traits of interest in watermelon breeding, but full advantage has not been taken of some of the other collections. The C. lanatus germplasm from southern Africa is currently a largely un...

A Genetic Locus Associated with Resistance to Fusarium oxysporum f. sp. niveum Race 2 in Citrullus lanatus-type Watermelon

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (FON), is a devastating soil-borne disease limiting watermelon production across the world. Although many watermelon cultivars have been bred for resistance to FON races 0 and 1, the only released cultivars that are resistant to FON 2 are nonharvested pollenizers. The lack of FON 2–resistant edible cultivars is thought to be associated with linkage drag and/or preferential inheritance patterns observed when crossing the resistant, wild source (Citrullus amarus), with edible watermelon. A potential way to overcome these obstacles is to use a resistant C. lanatus as the source of resistance and to develop molecular markers to increase the efficiency of selection. Here we describe the identification of a quantitative trait locus (QTL) associated with FON 2 resistance in watermelon. The genotyping by sequencing (GBS) platform was used to generate single nucleotide polymorphisms (SNPs) in an F2 population (n = 178) developed from a cross between UGA147 (resistant) and ‘Charleston Gray’ (susceptible). Five hundred and one SNPs were placed on the watermelon physical map and used in the mapping of QTL. F3 lines were phenotyped for resistance to FON 2 in the greenhouse. An intermediate QTL associated with resistance to FON 2 was identified on chromosome 11 (Qfon11). This QTL is a potential target for marker-assisted selection (MAS) for FON 2 resistance in watermelon. Watermelon is a widely cultivated crop of the Cucurbitaceae family, popular for its sweet flesh and edible seeds (AchiganDako et al., 2008; Edelstein and Nerson, 2002; Robinson and Decker-Walters, 1997). The intense selection during watermelon domestication has led to low genetic diversity in the cultivated genotypes (Guo et al., 2013; Hawkins et al., 2001a; Levi et al., 2001). Genome sequencing of wild and cultivated Citrullus detected a loss of disease resistance genes in cultivated material (Guo et al., 2013). Therefore, watermelon crop wild relatives [CWR (Maxted et al., 2006)] are expected to be useful sources of pest and disease resistance for watermelon cultivar development. Indeed, numerous studies have identified resistance to watermelon pests and diseases in CWR (Boyhan et al., 1992, 1994; Davis et al., 2007; Gusmini et al., 2005; Netzer and Martyn, 1989; Sowell, 1975; Sowell et al., 1980; Strange et al., 2002; Tetteh et al., 2010; Thies and Levi, 2007; Wechter et al., 2012a). However, the incorporation of resistance loci from CWR is not without obstacles. The use of CWR in breeding is hampered by their poor agronomic traits, which can make the process of incorporating desired genes into elite material, without introgressing unwanted genes, long and tedious. For watermelon, there have been numerous efforts to incorporate disease resistance from C. amarus [previously C. lanatus var. citroides (Chomicki and Renner, 2015; Paris, 2015)] into elite material. The efforts associated with introgressing fusarium wilt resistance have been particularly successful on one hand (race 1), and frustrating on the other (race 2). Fusarium oxysporum f. sp. niveum is a soil-borne pathogen that causes vascular wilting in watermelon, often resulting in yield reduction and crop failure (Everts and Himmelstein, 2015; Martyn, 2014). Four FON races have been described (0, 1, 2, and 3) based on their virulence or the ability to overcome specific resistance in a set of differential cultivars (Bruton et al., 2007, 2010; Egel et al., 2005; Zhou et al., 2010). The ability of the pathogen’s chlamydospores to persist in the soil for a long time, combined with the rapid evolution of its races has rendered the management of fusarium wilt difficult. Until recently, soil fumigation with methyl bromide was a popular management practice for fusarium wilt; however, this chemical has been phased out due to environmental concerns. The use of resistant cultivars is the most preferred management method for fusarium wilt (Bruton et al., 2007; Martyn and Netzer, 1991; Zhou and Everts, 2001). Since William Orton developed the first fusarium wilt resistant cultivar, Conqueror, using a wild C. amarus as the resistance source (Orton, 1907, 1917), numerous cultivars with resistance to FON races 0 and 1 have been developed (Martyn, 2014 for review). In particular, the resistance to FON 1 in the cultivar Calhoun Gray has been extensively used in watermelon breeding and many modern cultivars contain this resistance (Everts and Himmelstein, 2015). Several recent molecular studies have shown that Fo-1 is associated with a major QTL (Qfon1.1; R = 23% to 60%) on chr. 1 (Lambel et al., 2014; Meru and McGregor, 2016; Ren et al., 2015) of the watermelon draft genome (Guo et al., 2013). High resistance to FON races 0, 1, and 2 was described in 1991 in PI 296341-FR (Martyn and Netzer, 1991; Zhang and Rhodes, 1993). Resistance to FON 1 in PI 296341-FR is reported to be associated with the same QTL region on chr. 1 (Qfon1.1) that confers resistance in ‘Calhoun Gray’ (Ren et al., Received for publication 1 Aug. 2016. Accepted for publication 18 Oct. 2016. This research was supported in part by a U.S. Department of Agriculture Research and Education grant (GEO-2009-04819) and a U.S. Department of Agriculture Specialty Crop Research Initiative grant (2014-51181-22471). Current address: Tropical Research and Education Center/Institute of Food and Agricultural Sciences, University of Florida, 18905 SW 280th Street, Homestead, FL 33031. Corresponding author. E-mail: cmcgre1@uga.edu. J. AMER. SOC. HORT. SCI. 141(6):617–622. 2016. 617 2015). Resistance to FON 2 in PI 296341-FR is thought to be polygenic and controlled by at least a pair of recessive genes in epistasis with other minor genes (Martyn and Netzer, 1991; Zhang and Rhodes, 1993). Two QTL, one on chr. 9 (R = 13.7%) and the other on chr. 10 (R = 12.5%) are associated with this resistance (Ren et al., 2015). Despite the identification of PI 296341-FR as a source of resistance to FON 2 more than 25 years ago, there are no sweet-fleshed watermelon cultivars with resistance to FON 2 currently available to growers, likely due to a combination of linkage drag, the genetic background of the donor, and the quantitative nature of the resistance. PI 296341-FR is a selection from aC. amarus accession with hard, nonsweet, white flesh (Martyn and Netzer, 1991). Severe segregation distortion, including preferential inheritance of C. amarus alleles (Hawkins et al., 2001b; Levi et al., 2011; McGregor and Waters, 2013; Sandlin et al., 2012) and low pollen fertility (McGregor and Waters, 2013) have been observed in crosses between C. lanatus and watermelon. The resistance from PI 296341-FR has been incorporated into the nonharvested Super Pollenizer series from Syngenta (Syngenta Seeds Inc., 2009), but efforts to introgress the resistance into sweet, edible cultivars have not been successful to date. The lack of success incorporating resistance from PI 296341-FR suggests that breeders face serious challenges when incorporating quantitative traits, not associated with major QTL, from C. amarus into elite germplasm. Additional sources of FON 2 resistance have been described in C. amarus (Boyhan et al., 2003; Dane et al., 1998; Martyn and Netzer, 1991; Wechter et al., 2012a, 2012b). Generally, the basis of these resistances are not well understood, but is thought to also be polygenic. It remains to be seen whether resistance introgression from these accessions will face the same challenges as PI 296341-FR. One way to circumvent the challenges encountered with resistance introgression fromC. amarus, is to introgress resistance from a source genetically closely related to the elite watermelon material. Boyhan et al. (2003) identified a number of watermelon accessions in the U.S. Department of Agriculture (USDA) germplasm collection with resistance to FON 2 with pink or red flesh (USDA, 2016). These accessions provide a potential path to FON 2 resistance introgression circumventing the problems associated with introgression from C. amarus. The efficiency of trait introgression can also be improved by the use of MAS. The first step toward the use ofMAS for quantitative traits is to identify loci associated with the trait of interest. The aim of the current study was therefore to identify QTL associated with FON 2 resistance in a watermelon accession of the C. lanatus type. Materials and Methods PLANT MATERIAL AND DNA EXTRACTION. The FON 2 resistance source used in this study is UGA147, a resistant selection originating from PI 169233 (Boyhan et al., 2003). UGA147 has variable pink and yellow flesh color and soluble solids concentration (SSC) between 7% and 8%. A cross was made in the greenhouse betweenUGA147 and ‘CharlestonGray’ (susceptible). A single F1 plant was selfed to yield F2 plants which were further selfed to generate 178 F2:3 families. DNA was extracted from leaf material of the parents, the F1 and each of the F2 plants using the E.N.Z.A 96-well format kit (Omega Bio-tek, Norcross, GA) according to themanufacturer’s instructions. The concentration and quality of the DNA was determined by absorbance measurements Fig. 1. Frequency distribution for disease severity at 26 d after inoculation for the (A) April, (B) July, and (C) joint screening of the ‘Charleston Gray’ (CG) · UGA147 F2:3 watermelon population with Fusarium oxysporum f. sp. niveum race 2. Disease severity was visually rated on a scale of 0 to 5 where, 0 = asymptomatic plants, 1 = initial wilting on one leaf, 2 = continued wilting in more than one leaf, 3 = all leaves wilted, 4 = all leaves wilted and stem collapsing, and 5 = dead plants (Meru and McGregor, 2016). 618 J. AMER. SOC. HORT. SCI. 141(6):617–622. 2016. (Infinite M200 PRO; Tecan Group, M€annedorf, Switzerland) and by agarose gel electrophoresis. To ensure that the quality of the extracted DNA was sufficient for digestion with restriction enzymes, samples of the