Kirk W. Pomper

Identification of random amplified polymorphic DNA (RAPD) markers for self-incompatibility alleles in Corylus avellana L.

Abstract Random amplified polymorphic DNA (RAPD) markers were identified for self-incompatibility (SI) alleles that will allow marker-assisted selection of desired S-alleles in hazelnut (Corylus avellana L.). DNA was extracted from young leaves collected from field-planted parents and 26 progeny of the cross OSU 23.017 (S1S12)×VR6-28 (S2S26) (OSU23×VR6). Screening of 10-base oligonucleotide RAPD primers was performed using bulked segregant analysis. DNA samples from 6 trees each were pooled into four ‘bulks’, one for each of the following: S1 S2, S1 S26, S2 S12, and S12 S26. ‘Super bulks’ of 12 trees each for S1, S2, S12, and S26 were then created for each allele by combining the appropriate bulks. The DNA from these four super bulks and from the parents was used as a template in the PCR assays. A total of 250 primers were screened, and one RAPD marker each was identified for alleles S2 (OPI07750) and S1 (OPJ141700). OPJ141700 was identified in 13 of 14 S1 individuals of the cross OSU23×VR6 used in bulking and yielded a false positive in 1 non-S1 individual. This same marker was not effective outside the original cross, identifying 4 of 5 S1 progeny in another cross, ‘Willamette’×VR6-28 (‘Will’×VR6), but yielded false positives in 4 of 9 non-S1 individuals from the cross ‘Casina’×VR6-28 (‘Cas’×VR6). OPI07750 served as an excellent marker for the S2 allele and was linked closely to this allele, identifying 12 of 13 S2 individuals in the OSU23×VR6 population with no false positives. OPI07750 was found in 4 of 4 S2 individuals from ‘Will’×VR and 7 of 7 S2 individuals of ‘Cas’×VR6 with no false positives, as well as 10 of 10 S2 individuals of the cross OSU 296.082 (S1S8)×VR8-32 (S2S26), with only 1 false positive individual out of 21 progeny. OPI07750 was also present in 5 of 5 cultivars carrying the S2 allele, with no false-positive bands in non-S2 cultivars, and correctly identified all but 2 S2 individuals in 57 additional selections in the breeding program. In the OSU23×VR6 population, the recombination rate between the marker OPJ141700 and the S1 allele was 7.6% and between the OPI07750 marker and the S2 allele was 3.8%. RAPD marker bands were excised from gels, cloned, and sequenced to enable the production of longer primers (18 or 24 bp) that were used to obtain sequence characterized amplified regions (SCARs). Both the S1 and S2 markers were successfully cloned and 18 bp primers yielded the sole OPJ141700 product, while 24-bp primers yielded OPI07750 as well as an additional smaller product (700 bp) that was not polymorphic but was present in all of the S-genotypes examined.

Emerging Fruit Crops

Hundreds of fruit species with commercial potential are currently in a status of low economic importance. Some, such as quince, pomegranate, and figs, have been cultivated for thousands of years. Others have only been locally collected and consumed from wild populations of the fruit. The development of these underappreciated crops depends on a range of factors including the cultivation limitations, yields, uses of the fruit, and marketing potential. Although initially many crops are developed using selections from the wild, as they are developed, breeding programs work toward improving the crop for both production and quality. This chapter examines nine emerging crops chosen among hundreds of potential crops which are currently showing much promise as commercial crops. These include five tree fruits, namely, pawpaw, quince, mayhaw, pomegranate, and fig, and four berry crops, namely, blue honeysuckle, elder, goji, and ‘ōhelo.

Nosema apis infection in honey bee (Apis mellifera) queens

Summary Queen honey bees were fed Nosema apis spores in sucrose solution, returned to their hives, and examined later for N. apis infection by a polymerase chain reaction test. Eggs, larvae and pupae from the hives were also examined for infection on three observation dates during a 39 day period following the inoculation of the queens. Six of seven surviving queens developed N. apis infections in their ventriculi, but none had detectable N. apis in their ovaries. No eggs, larvae or pupae taken from the hives of Nosema inoculated queens contained detectable N. apis. These results suggest that N. apis is not transmitted vertically, unlike many other Microsporidia in other invertebrate hosts. In an effort to determine if the stress of shipping increased the susceptibility of Nosema exposed bees, another set of mated, queen honey bees was fed N. apis spores in sucrose, and the queens were then either mailed in small shipping cages, from Kentucky to Indiana and back, or kept caged for the same period in the laboratory. These queens were then dissected and their ovaries and ventriculi examined for N. apis. Of the mailed queens, all developed infection, whilst 86% of the queens not mailed became infected, but this difference was not statistically significant. N. apis was not detected in any of the ovaries of mailed queens nor of those queens not mailed.

Loss of ripening capacity of pawpaw fruit with extended cold storage.

The fruit ripening traits of pawpaw [ Asimina triloba (L.) Dunal] were examined after harvest and after cold storage at -2, 2, 4, and 6 degrees C for up to 12 weeks. Generally, fruits stored at 2-4 degrees C for 4 weeks ripened normally, but those stored at -2 degrees C did not ripen normally, those stored at 6 degrees C were overripe, and by 6-8 weeks those stored at 2-4 degrees C had a lower respiration rate and ethylene production, lower firmness, and lower pH than fruit cold-stored for 4 weeks or less. These changes, and the occasional development of brown discoloration in the pulp once the fruits were moved back to room temperature, were evidence of chilling injury by 6 weeks. After harvest and through 4 weeks of cold storage, the main volatile compounds produced by fruit were methyl and ethyl octanoates and hexanoates. Volatile production significantly increased >5-fold in fruit ripened for 72 h after harvest or after removal from up to 4 weeks of cold storage. Fruit cold-stored for 6 weeks or more produced fewer total volatiles and esters but increased levels of such off-flavor compounds as ethyl acetate, ethyl propionate, and hexanoic and decanoic acids. Alcohol acyltransferase (AAT) activity declined in cold-stored fruit but was not correlated with either total volatile production or total ester production. Alcohol dehydrogenase activity did not change during ripening after harvest or cold storage. Lipoxygenase activity was highest just after harvest or after 2 weeks of cold storage, but was low by 4 weeks. Thus, ripening of pawpaw fruit seems to be limited to 4 weeks at 2-4 degrees C with loss of ability to continue ripening and chilling injury symptoms evident at colder temperatures and after longer periods of cold storage.

Progress in Studies on Hormonal Sex Reversal and Genetic Sex Control in Black Crappie

Sex control can solve the problem of stunted black crappie populations in small impoundments. The main objectives of the present study were (1) to identify sex-reversed males of black crappie from a previously obtained androgen-treated group using test crosses, and (2) to develop broodstock of sex-reversed males by masculinization of fish from those crosses. An additional objective of the study was to try to identify sex-specific RAPD markers, which might be used for identification of sex-reversed males. The progenies resulting from test crosses were divided into two groups: Group I (control) fish were raised without hormonal treatment, while Group II fish were subjected to androgen (MT) treatment. Seven progenies were obtained from the cross of preliminary androgen-treated males with normal females. Six of seven progenies had sex ratio in Groups I close to 1:1, and one progeny consisted of females only. This shows that only one male, which generated this progeny, was a sex-reversed homogametic fish (XX) while the other males were heterogametic normal (XY) fish. In Group II the androgen 17α-methyltestosterone (MT) was orally administered to crappie with an artificial diet (30 mg/kg) for 40 days beginning 35 days post hatching; androgen-treated groups consisted of 95–100% males. Sex-specific random-amplified polymorphic DNA markers were not identified in black crappie.

Clonality of Pawpaw (Asimina triloba) Patches in Kentucky

Abstract Pawpaw [Asimina triloba (L.) Dunal] is a tree-fruit (see overleaf, page 2) native to the southeastern region of the United States. Kentucky State University serves as the USDA-National Clonal Germplasm Repository for pawpaw, therefore assessing genetic diversity across the pawpaws native range is a high priority. Pawpaw is usually found in large patches as an understory tree and root suckering likely occurs. To determine if native pawpaw patches are clonal, DNA was extracted from leaf samples collected from trees in six native patches in three counties in central Kentucky. Two ISSR-PCR primers yielded three polymorphic and six monomorphic markers in the six patches. Three patches did not display any polymorphic markers in each patch, suggesting they were clonal. However, three other patches did show polymorphic markers within each patch, indicating these patches were not clonal and contained trees of at least two genotypes within each patch. This study suggests that to assess the genetic diversity of a pawpaw patch or local population, more intensive sampling strategies will be required.

Potential of Ripe Pawpaw Fruit Extract as an Insecticide and Feeding Deterrent for Striped Cucumber Beetle (Coleoptera: Chrysomelidae) on Squash

Laboratory experiments were performed to study the effects of pawpaw, Asimina triloba (L.) Dunal, fruit extract on mortality and feeding deterrence of striped cucumber beetle, Acalymma vittatum (F.). Recently, fruit tissues of pawpaw were found to contain phenolic and antioxidant compounds, as well as annonaceous acetogenin compounds having insecticidal activity. Ripe pawpaw fruit pulp from a range of pawpaw varieties was extracted with 100% ethyl alcohol to obtain acetogenin compounds. Pulp extracts of 0, 10, 100, 1,000, 10,000 and 50,000 ppm were then used to assess feeding deterrence and mortality of beetles. Buttercup squash leaf disks 3.5 cm in diameter were treated individually with each concentration and placed on water moistened filter paper in plastic Petri dishes (9 cm diam). Five striped cucumber beetles were placed on each leaf disk. All Petri dishes were then placed in an environmental growth chamber at 27°C and a 16:8 h light:dark photoperiod. Feeding activity was recorded 1, 4 and 24 h after beetle introduction. After 24 h the beetles were removed. Beetles did not feed on treated squash leaves at either 1 or 4 h of exposure. However, significant feeding occurred between 4 and 24 h after beetle introduction. Feeding was lowest and feeding damage least on 50,000 ppm pawpaw-treated leaf disks compared with leaf disks treated with < 10,000 ppm dilutions. Pawpaw fruit extract reduced feeding by 89% and 97% in the 10,000 and 50,000 ppm treatments, respectively. The calculated LC50 value was 50,538 ppm whereas the LCF10 (concentration at which only 10% of the leaves were consumed) was 2,033 ppm. At 10,000 ppm 10% of the beetles were killed; however, only 3% of the leaf tissue was consumed. Thus, pawpaw fruit extract may be an effective insect feeding deterrent. The duration of treatment effectiveness and susceptibility of other pest and beneficial insect species to the extracts also needs to be examined.

Lady Beetle Composition and Abundance in Sweet Corn Bordered by Pasture, Buckwheat or Sunflower Companion Plantings

ABSTRACT To determine the effect habitat management/border plantings have on beneficial insect diversity and abundance in agricultural crops, sweet corn, Zea mays L. ‘Garrison®’, was grown in replicated plots on Kentucky State Universitys Agricultural Research and Demonstration Farm. Each 25 m × 12 m plot was bordered on each length by a 2 m wide border of un-mowed pasture, buckwheat (Fagopyrum esculentum Moench), or dwarf sunflower (Heliathus annuus L. var. ‘Big Smile’). Yellow sticky traps 15 cm × 15 cm were used to capture lady beetles weekly through anthesis. Pink lady beetle, Coleomegilla maculata (DeGeer); Asian lady beetle, Harmonia axyridis (Pallas); spotless lady beetle, Cycloneda munda (Say); and seven spotted lady beetle, Coccinella septempunctata L. were caught in this study. The pink lady beetle was the most abundant species overall in all three borders and in the sweet corn plots with 79% and 94%, respectively. There were greater numbers of pink lady beetles in buckwheat (P < 0.00001) and s...

The Pawpaw Peduncle Borer, Talponia plummeriana Busck (Lepidoptera: Tortricidae): A Pest of Pawpaw Fruit

ABSTRACT The pawpaw peduncle borer, Talponia plummeriana Busck (Lepidoptera: Torticidae), is a pest of pawpaw flowers often boring into the peduncle and causing flower drop. Here we document the first occurrence of this insect infesting ripe pawpaw fruit. Infested fruit that had been collected at the Kentucky State University Research and Demonstration Farm in Franklin County, Kentucky were dissected and small tan colored larvae with brown head capsules were discovered in the fruit. Pawpaw peduncle borer adults were reared from fruit held at room temperature in the laboratory.

Pawpaw Patch Genetic Diversity, and Clonality, and its Impact on the Establishment of Invasive Species in the Forest Understory

ABSTRACT The pawpaw (Asimina triloba) is a native understory tree of 26 states of the Eastern and Midwestern United States. Pawpaws genetic diversity and clonality in relation to this trees ability to compete with local invasive species in Kentucky has not been examined. The objectives of this study were two-fold: to determine the genetic diversity and clonality displayed in seven native pawpaw patches located at the Kentucky State University Environmental Education Center (KSU-EEC), The Kentucky River, Cove Spring Park, and the KSU Research and Demonstration Farm in Franklin County using DNA microsatellite markers; and to determine if patches reduced the incidence of invasive species. Twenty-five trees from seven patches in the four different locations were sampled for genetic analysis. Leaf samples were extracted using the DNAmite Plant Extraction Kit and products from four microsatellite loci were analyzed using a 3130 Applied Biosystems Gene sequencer. String grids were created and invasive plants c...