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Krista C. Shellie, Robert D. Meyer, and T. Erik Mirkov

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Soon O. Park, Kevin M. Crosby, Rongfeng Huang, and T. Erik Mirkov

Male sterility is an important trait of melon in F1 hybrid seed production. Molecular markers linked to a male-sterile gene would be useful in transferring male sterility into fertile melon cultivars and breeding lines. However, markers linked to the ms-3 gene for male sterility present in melon have not been reported. Our objectives were to identify randomly amplified polymorphic DNA (RAPD) markers linked to the ms-3 gene controlling male sterility using bulked segregant analysis in an F2 population from the melon cross of line ms-3 (male-sterile) × `TAM Dulce' (male-fertile), convert the most tightly linked RAPD marker to the ms-3 gene into a sequence characterized amplified region (SCAR) marker based on a specific forward and reverse 20-mer primer pair, and confirm the linkage of the RAPD and SCAR markers with the ms-3 gene in an F2 population from the cross of line ms-3 × `Mission' (male-fertile). A single recessive gene controlling male sterility was found in F2 individuals and confirmed in F3 families. Two RAPD markers that displayed an amplified DNA fragment in the male-sterile bulk were detected to be linked to the ms-3 gene in the F2 population from the cross of line ms-3 × `TAM Dulce'. RAPD marker OAM08.650 was closely linked to the ms-3 gene at 2.1 cM. SCAR marker SOAM08.644 was developed on the basis of the specific primer pair designed from the sequence of the RAPD marker OAM08.650. The linked RAPD and SCAR markers were confirmed in the F2 population from the cross of line ms-3 × `Mission' to be consistently linked to the ms-3 gene at 5.2 cM. These markers were also present in 22 heterozygous fertile F1 plants having the ms-3 gene. The RAPD and SCAR markers linked to the ms-3 gene identified, and confirmed here could be utilized for backcrossing of male sterility into elite melon cultivars and lines for use as parents for F1 hybrid seed production.

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Ed Stover, Richard R. Stange Jr., T. Gregory McCollum, Jesse Jaynes, Michael Irey, and Erik Mirkov

Huanglongbing {HLB [associated with Candidatus Liberibacter sp. (CLas)]} and asiatic citrus canker {ACC [causal organism Xanthomonas citri ssp. citri (XCC)]} are bacterial diseases that seriously threaten sustainability of the Florida citrus (Citrus sp.) industry. Sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) are highly susceptible to ACC and improvement through conventional breeding is a long-term process, making transgenic solutions attractive. No strong HLB resistance has been identified within cultivated citrus scion types: creation of transgenic citrus that would permit economic citrus production where HLB is endemic is a high priority. Little is known about the HLB pathosystem and thus broad-spectrum antimicrobial peptides (AMPs) have been the focus for current work, and identification of safe and effective transgenes is essential to our efforts. In vitro assessment of minimum inhibitory concentration (MIC) for 44 AMPs was conducted using Sinorhizobium meliloti and Agrobacterium tumefaciens as surrogates for the unculturable CLas because they are closely related alpha proteobacteria (class Alphaproteobacteria). XCC is also a gram-negative bacterium and was included in these analyses in anticipation that HLB and ACC resistance can be achieved with the same AMP transgene if expressed using non-tissue-specific promoters. Twenty AMPs from diverse sources were initially tested. AMPs with the lowest MICs included tachyplesin I from horseshoe crab (Tachypleus tridentatus), SMAP-29 from sheep (Ovis aries), D4E1 and D2A21 (which are synthetic AMPs derived through evaluation of critical amino acid residues in AMPs, overall peptide structure, and AMP effectiveness), the human (Homo sapiens) LL-37, and the honeybee (Apis mellifera) venom AMP melittin. These AMPs inhibited growth of all three test bacterial species at 1 μM or less. An additional 20 synthetic AMPs were designed based on structures of the most effective AMPs and seven of these showed effectiveness at 1 μM or less across all three test bacteria. Most AMPs were comparable in effectiveness across the three bacterial species, but some species × AMP interactions were observed. Hemolytic activity was assessed by exposure of porcine erythrocytes (from Sus scrofa) to the AMPs. Hemolysis from most AMPs was not significantly different from water, whereas melittin was highly hemolytic.