James R. McFerson

Microsatellite (SSR) markers reveal genetic identities, genetic diversity and relationships in a Malus×domestica borkh. core subset collection

Abstract A collection of 66 Malus×domestica Borkh. accessions from the USDA-ARS Plant Genetic Resources Unit’s core collection was screened with a set of eight SSR (simple sequence repeat) primers developed at the PGRU in order to determine genetic identities, estimate genetic diversity, and to identify genetic relationships among these accessions. All eight primer pairs generated multiple fragments when used in amplification reactions with DNA from these accessions. High levels of variation were detected with a mean of 12.1 alleles per locus and a mean heterozygosity across all eight loci of 0.693. The eight primer pairs utilized in this study unambiguously differentiated all but seven pairs of accessions in this collection of 66 M.×domestica Borkh. genotypes. The probability of matching any two genotypes at all eight loci in this study was approximately 1 in 1 billion. The markers detected two misnamed accessions in the collection. Genetic-identity data produced a genetic-relatedness phenogram which was concordant with geographic origins and/or known pedigree information. These SSR markers show great promise as tools for managing Malus ex situ germplasm collections as well as for collection and preservation strategies concerning wild Malus populations in situ.

Abundance and characterization of simple-sequence repeats (SSRs) isolated from a size-fractionated genomic library of Brassica napus L. (rapeseed).

A size-fractionated library of Brassica napus L. (rapeseed), composed of 15000 clones, was screened for the presence of GA-, CA-, and GATA-simple-sequence repeats (SSRs). GA-SSRs were four- and five-fold more abundant than CA- and GATA-SSRs, respectively, and present at a frequency of approximately one SSR for every 100 kb of DNA. Following the sequencing of 124 positive clones, primer pairs were designed and evaluated for seven selected SSRs. Products were amplified in an array of individuals of B. napus, B. oleracea and B. rapa, demonstrating that the seven SSRs were conserved among species. Two SSRs were polymorphic. Among 11 accessions, the dinucleotide (GA)-repeat, B.n.9A, yielded 12 fragments, while the tetranucleotide-repeat (GATA), B.n.6A2, revealed two fragments. Automated, fluorescence-based detection of polyacrylamide gels has been employed to simultaneously increase throughput, reduce unit cost, improve analytical resolution, and expedite data acquisition of SSR analysis. Though initial financial investment and technical capabilities may prevent some from directly employing our documented approach, SSR analysis warrants further investigation as a tool in genetic studies for enhancing both the conservation and utilization of genetic resources.

Identification of polymorphic, conserved simple sequence repeats (SSRs) in cultivated Brassica species

The application of simple sequence repeat (SSR) genotyping for the characterization of genetic variation in crop plants has been hindered by ready access to useful primer pairs and potentially limited conservation of the repeat sequences among related species. In this phase of work, we report on the identification and characterization of SSRs that are conserved in Brassica napus L. (rapeseed) and its putative progenitors, B. oleracea L. (cabbage, and related vegetable types) and B. rapa (vegetable and oil types). Approximately 140 clones from a size-fractionated genomic library of B. napus were sequenced, and primer pairs were designed for 21 dinucleotide SSRs. Seventeen primer pairs amplified products in the three species and, among these, 13 detected variation between and within species. Unlike findings on SSR information content in human, no relationship could be established between the number of tandem repeats within the target sequence and heterozygosity. All primer pairs have been designed to work under identical amplification conditions; therefore, single-reaction, multiplex polymerase chain reaction (PCR) with these SSRs is possible. Once moderate numbers of primer pairs are accessible to the user community, SSR genotyping may provide a useful method for the characterization, conservation, and utilization of agricultural crop diversity.

Comparative virus resistance and fruit yield of transgenic squash with single and multiple coat protein genes

Five transgenic squash lines expressing coat protein (CP) genes from cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV 2) were analyzed in the field for their reaction to mixed infections by these three viruses and for fruit production. Test plants were exposed to natural inoculations via aphids in trials simulating the introduction of viruses by secondary spread from mechanically infected susceptible border row plants. Plants of transgenic line CZW-3 expressing the CP genes from CMV, ZYMV, and WMV 2 displayed the highest level of resistance with no systemic infection, although 64% exhibited localized chlorotic dots which were mainly confined to older leaves. CZW-3 plants had a 50-fold increase in marketable yield compared to controls and the highest predicted cash returns. Plants of transgenic line ZW-20 expressing the CP genes from ZYMV and WMV 2 displayed high levels of resistance to these two potyviruses, but 22% became infected by CMV. However, ZW-20 plants provided a 40-fold increase in marketable yield relative to controls and good estimated cash returns. Three transgenic lines expressing single CP genes from either ZYMV (line Z-33), WMV 2 (line W-164) or CMV (line C-14) developed systemic symptoms similar to those of controls but showed a delay of 2 to 4 weeks before the onset of disease. Plants of transgenic line Z-33 were highly resistant to ZYMV but not to WMV 2 and CMV. Interestingly, Z-33 plants had a 20-fold increase in marketable yield compared to controls and some predicted cash returns if market sale prices were high. This study indicates that virus-resistant transgenic lines are economically viable even if they are affected by viruses other than those to which they are resistant.

Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus, zucchini yellow mosaic virus, and watermelon mosaic virus-2 is resistant to these three viruses in the field

Cantaloupe line CZW-30 containing coat protein gene constructs of cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV-2) was investigated in the field over two consecutive years for resistance to infections by CMV, ZYMV, and/or WMV-2. Resistance was evaluated under high disease pressure achieved by mechanical inoculations and/or natural challenge inoculations by indigenous aphid vectors. Across five different trials, homozygous plants were highly resistant in that they never developed systemic symptoms as did the nontransformed plants but showed few symptomatic leaves confined close to the vine tips. Hemizygous plants exhibited a significant delay (2–3 weeks) in the onset of disease compared to control plants but had systemic symptoms 9–10 weeks after transplanting to the field. Importantly, ELISA data revealed that transgenic plants reduced the incidence of mixed infections. Only 8% of the homozygous and 33% of the hemizygous plants were infected by two or three viruses while 99% of the nontransformed plants were mixed infected. This performance is of epidemiological significance. In addition, control plants were severely stunted (44% reduction in shoot length) and had poor fruit yield (62% loss) compared to transgenic plants, and most of their fruits (60%) were unmarketable. Remarkably, hemizygous plants yielded 7.4 times more marketable fruits than control plants, thus suggesting a potential commercial performance. This is the first report on extensive field trials designed to assess the resistance to mixed infection by CMV, ZYMV, and WMV-2, and to evaluate the yield of commercial quality cantaloupes that are genetically engineered.

Transgenic Melon and Squash Expressing Coat Protein Genes of Aphid-borne Viruses do not Assist the Spread of an Aphid Non- transmissible Strain of Cucumber Mosaic Virus in the Field

Transgenic melon and squash containing the coat protein (CP) gene of the aphid transmissible strain WL of cucumber mosaic cucumovirus (CMV) were grown under field conditions to determine if they would assist the spread of the aphid non-transmissible strain C of CMV, possibly through heterologous encapsidation and recombination. Transgenic melon were susceptible to CMV strain C whereas transgenic squash were resistant although the latter occasionally developed chlorotic blotches on lower leaves. Transgenic squash line ZW-20, one of the parents of commercialized cultivar Freedom II, which expresses the CP genes of the aphid transmissible strains FL of zucchini yellow mosaic (ZYMV) and watermelon mosaic virus 2 (WMV 2) potyviruses was also tested. Line ZW-20 is resistant to ZYMV and WMV 2 but is susceptible to CMV. Field experiments conducted over two consecutive years showed that aphid-vectored spread of CMV strain C did not occur from any of the CMV strain C-challenge inoculated transgenic plants to any of the uninoculated CMV-susceptible non- transgenic plants. Although CMV was detected in 3% (22/764) of the uninoculated plants, several assays including ELISA, RT- PCR-RFLP, identification of CP amino acid at position 168, and aphid transmission tests demonstrated that these CMV isolates were distinct from strain C. Instead, they were non-targeted CMV isolates that came from outside the field plots. This is the first report on field experiments designed to determine the potential of transgenic plants expressing CP genes for triggering changes in virus-vector specificity. Our results indicate that transgenic plants expressing CP genes of aphid transmissible strains of CMV, ZYMV, and WMV 2 are unlikely to mediate the spread of aphid non-transmissible strains of CMV. This finding is of practical relevance because transgenic crops expressing the three CP genes are targeted for commercial release, and because CMV is economically important, has a wide host range, and is widespread worldwide.

Assessment and management of plant genetic diversity: considerations of intra- and interspecific variation

Abstract Effective management of plant genetic diversity incorporates agricultural development as a critical element of conservation. Advancement of renewable production of food and other plant-derived essentials of human culture must be compatible with and supportive of stewardship of those biological resources basic to agriculture. Balance must be struck between the accumulation of short-term dividends contributing to an enhanced quality of life and maintenance of insurance consisting of representative and well-characterized genetic diversity for the future. Plant genetic diversity is complex in theory and practice. To best understand plant genetic diversity, it is essential to consider variation as richness and distribution at both the intra- and interspecific levels. If a species is defined as a potentially interbreeding population, then the diversity existing in the gene pool is manifested as its resilience in response to selection, whether natural or artificial. Depending on the state of our genetic understanding of a taxon, genetic diversity may be considered at different organizational levels: gene pool, population, individual, genome, locus, and DNA base sequence. Concepts and techniques of classical genetics must be consolidated with recent progress in population, systematic and molecular biology. This synthesis will allow for a clearer characterization of both intra- and interspecific variation of wild and domesticated plants and greatly enhance conservation and utilization of plant genetic resources for the future.

Application of isozyme data to the management of the United States national Brassica oleracea L. genetic resources collection

SummaryManagement of a genetic resources collection is more effective if a curator can accurately identify genotypes and accessions as well as assess intraspecific genetic relationships and the genetic structure of species. Consequently, a study was conducted to determine whether data from starch gel electrophoresis of a specific set of isozymes from plants of Brassica oleracea (cole crops) would be useful in answering four questions the answers to which are essential for effective curatorial activities: Can individual plants be identified? Can specific accessions be identified? What are the genetic relationships among botanical varieties within B. oleracea? What is the genetic structure of the species B. oleracea? Six loci from 4 enzyme systems (LAP, PGD, PGI, and PGM) were analyzed. Individuals and accessions could not usefully be identified using these isozymes, but genetic relationships within and genetic structure of the species were easily determined, resulting in specific recommendations for improving collection management. As expected, highly selected commercial lines exhibited less diversity than average, and so may be of limited value when trying to maximize diversity with a minimum number of accessions. In contrast, landraces and weakly selected lines are more diverse than average, and thus are useful in maximizing per accession diversity. Not only was 93% of the genetic variability in B. oleracea found among accessions and a mere 7% among varieties, but also a cluster analysis showed that accessions of a single botanical variety are often more similar to those of a different variety than to each other. These results suggest that in order to create a genetic resources collection of B. oleracea that faithfully represents the diversity in the species, a curator should assemble a broad array of accessions originating from diverse agroecological niches, having different levels of improvement, and representing all botanical varieties.

U.S. Peach Producer Preference and Willingness to Pay for Fruit Attributes

This study investigates U.S. peach producers’ willingness to pay (WTP) for potential improvement of peach fruit attributes. Data were collected from 124 U.S. peach producers. The choice experiment and socioeconomic data were analyzed using mixed logit (ML) models to estimate the producer WTP and preferences for peach attributes. The results indicate that the WTP for attribute values vary across peach producers from different production regions (California and eastern United States), with different selling targets (fresh and processed) and different orchard sizes (smaller or larger than 15 acres). These results provide useful information for peach breeders in prioritizing traits in their breeding programs. Peach [Prunus persica (L.) Batsch] is an important crop for both fresh and processed markets in worldwide. It is increasingly challenging for peach producers to select the ideal scion cultivar that satisfies market requirements and maximizes their profits, especially considering the high capital investment for establishment and time lag to generating a positive cash flow (Day et al., 2009; Yue et al., 2014). Total farm gate value of U.S. peach production was

A Reflective, Processed-Kaolin Particle Film Affects Fruit Temperature, Radiation Reflection, and Solar Injury in Apple

606 million in 2015 and used production was 825,415 tons, including 467,680 tons for processing (USDA NASS, 2016). Although commercial production is distributed across 23 states, California is the largest producer, accounting for 73% of total U.S. production in 2015. Forty-eight percent of fresh peaches and 96% of processed peaches were produced in California, with South Carolina, Georgia, and New Jersey the second, third, and fourth top production states, respectively. Total peach production in 2015 dropped by 13% from 978,260 tons in 2012 (USDA NASS, 2013). Meanwhile, the average price of fresh peach in 2015 increased by 12% to