Genetic diversity and population structure of castor (Ricinus communis L.) germplasm within the US collection assessed with EST-SSR markers.

Castor is an important oilseed crop and although its oil is inedible, it has multiple industrial and pharmaceutical applications. The entire US castor germplasm collection was previously screened for oil content and fatty acid composition, but its genetic diversity and population structure has not been determined. Based on the screening results of oil content, fatty acid composition, and country origins, 574 accessions were selected and genotyped with 22 polymorphic EST-SSR markers. The results from cluster analysis, population structure, and principal component analysis were consistent, and partitioned accessions into four subpopulations. Although there were certain levels of admixtures among groups, these clusters and subpopulations aligned with geographic origins. Both divergent and redundant accessions were identified in this study. The US castor germplasm collection encompasses a moderately high level of genetic diversity (pairwise dissimilarity coefficient = 0.53). The results obtained here will be useful for choosing accessions as parents to make crosses in breeding programs and prioritizing accessions for regeneration to improve germplasm management. A subset of 230 accessions was selected and will be planted in the field for establishing a core collection of the US castor germplasm. Further evaluation of the US castor germplasm collection is also discussed.

The Potato Cryobank at The International Potato Center (Cip): A Model for Long Term Conservation of Clonal Plant Genetic Resources Collections of the Future.

BACKGROUND: Cryobanks are a secure, efficient and low cost method for the long-term conservation of plant genetic resources for theoretically centuries or millennia with minimal maintenance. OBJECTIVE: The present manuscript describes CIP's modified protocol for potato cryopreservation, its large-scale application, and the establishment of quality and operational standards, which included a viability reassessment of material entering the cryobank. MATERIALS AND METHODS: In 2013, CIP established stricter quality and operational standards under which 1,028 potato accessions were cryopreserved with an improved PVS2-droplet protocol. In 2014 the viability of 114 accessions cryopreserved in 2013 accessions were reassessed. RESULTS: The average recovery rate (full plant recovery after LN exposure) of 1028 cryopreserved Solanum species ranged from 34 to 59%, and 70% of the processed accessions showed a minimum recovery rate of ≥20% and were considered as successfully cryopreserved. CONCLUSION: CIP has established a new high quality management system for cryobanking. Periodic viability reassessment, strict and clear recovery criteria and the monitoring of the percent of successful accessions meeting the criteria as well as contamination rates are metrics that need to be considered in cryobanks.

Detection and Classification of SPLCV Isolates in the U.S. Sweetpotato Germplasm Collection via a Real-Time PCR Assay and Phylogenetic Analysis.

The United States Department of Agriculture-Agricultural Research Service sweetpotato (Ipomoea batatas) germplasm collection contains accessions that were initially collected from various countries worldwide. These materials have been maintained and distributed as in vitro plantlets since the mid-1980s. The status of viral infection by the emerging Sweet potato leaf curl virus (SPLCV) and other Begomovirus spp. in this germplasm has yet to be determined. In order to minimize the potential distribution of virus-infected clones, all accessions in the collection were tested for SPLCV using a real-time polymerase chain reaction assay. In total, 47 of 701 accessions of in vitro plantlets tested positive for SPLCV. The presence of SPLCV detected in these materials was confirmed via biological indexing using the indicator plants I. nil and I. muricata. Symptoms appeared more rapidly on I. muricata than on I. nil. Nucleotide polymorphisms among the isolates were evaluated by sequencing the AV1 coat protein gene from 24 SPLCV-infected accessions. The results revealed that the SPLCV isolates shared high sequence identity. Ten nucleotide substitutions were identified, most of which were synonymous changes. Phylogenetic analysis was conducted on those 24 SPLCV isolates in combination with six described SPLCV species and various SPLCV strains from GenBank to evaluate the relationships among viral species or strains. The results from this analysis indicated that most of the AV1 genes derived from previously classified SPLCV species clustered together, some of which formed well-supported monophyletic clades, further supporting the current taxonomy. Overall, identification of SPLCV-infected germplasm will allow approaches to be employed to eliminate the virus from the collection and limit the distribution of infected materials.

First Report of Tomato spotted wilt virus Infecting African Clover (Trifolium tembense) in Georgia.

Tomato spotted wilt virus (TSWV; family Bunyaviridae, genus Tospovirus), which is vectored by several species of thrips (order Thysanoptera, family Thripidae), causes a destructive disease that affects many economically important host plants such as tomatoes, peppers, and peanuts. Controlling the spread of this disease is challenging, and currently, only limited strategies are available to prevent and/or control its dissemination, including early diagnosis, destruction of infected material, and elimination of the vector. TSWV has been previously reported in subterranean clover (Trifolium subterraneum), white clover (T. repens), and various unidentified wild clovers (Trifolium spp.) in North America and Australia (1,3), but never before in an African species. T. tembense (Fresen.), an herbaceous annual African clover that is mainly used for grazing, is part of the national germplasm collection housed at the Plant Genetic Resources Conservation Unit in Griffin, GA. TSWV was found naturally infecting several accessions of this species being grown for regeneration in a greenhouse during 2008. Initial putative identification of the virus was done by visual inspection of host symptoms that included ringspots, necrotic and chlorotic local lesions, sometimes mild systemic wilting, and eventually an overall decline of healthy tissue in the infected plants. This was subsequently confirmed by double-antibody sandwich (DAS)-ELISA and reverse transcription (RT)-PCR. Primers (5'-ATGTCTAAGGTTAAGCTC-3' forward and 5'-TTAAGCAAGTTCTGTGAG-3' reverse) targeted the nucleocapsid gene of TSWV and amplified an expected product of approximately 800 bp (2). No product was amplified in any of the negative controls. Twenty-six individuals representing twelve plant accessions (PI 517788, 517790, 517792, 517793, 517809, 517832, 517842, 517845, 517851, 517871, 517876, and 517889) were screened for TSWV. Two to three individuals were targeted from each accession. Samples were chosen on the basis of the availability of leaf tissue to perform two diagnostic assays, ELISA and RT-PCR. Samples chosen for this study were all naturally infected by thrips. All but four individuals representing two plant accessions tested positive for the virus. The RT-PCR data substantiated the DAS-ELISA results and confirmed the suspected infection. More than 26% of the positive samples naturally infected by TSWV were further characterized by purifying and sequencing (bidirectionally) the RT-PCR product on an automated CEQ 8000 sequencer (Beckman Coulter, Fullerton, CA). The resulting sequences were aligned and edited using AlignIR (LI-COR, Lincoln, NE). More than 700 bp of sequence data (GenBank Accession No. FJ183743-FJ183746) was compiled and they displayed 98% identity with deposited TSWV nucleocapsid gene sequences in GenBank, with no similarity to any other targets. To our knowledge, this is the first report of TSWV infection in T. tembense. Accessions potentially resistant to TSWV within this species were identified and need to be further substantiated. T. tembense is a wild, native clover in Africa and could serve as a weed host for infection of nearby agronomically important crops. References: (1) I. Bitterlich and L. S. MacDonald. Can. Plant Dis. Surv. 73:137, 1993. (2) R. J. Holguín-Peña and E. O. Rueda-Puente. Plant Dis. 91:1682, 2007. (3) C. R. Wilson. Plant Pathol. 47:171, 1998.

Application of TILLING and EcoTILLING as Reverse Genetic Approaches to Elucidate the Function of Genes in Plants and Animals.

With the fairly recent advent of inexpensive, rapid sequencing technologies that continue to improve sequencing efficiency and accuracy, many species of animals, plants, and microbes have annotated genomic information publicly available. The focus on genomics has thus been shifting from the collection of whole sequenced genomes to the study of functional genomics. Reverse genetic approaches have been used for many years to advance from sequence data to the resulting phenotype in an effort to deduce the function of a gene in the species of interest. Many of the currently used approaches (RNAi, gene knockout, site-directed mutagenesis, transposon tagging) rely on the creation of transgenic material, the development of which is not always feasible for many plant or animal species. TILLING is a non-transgenic reverse genetics approach that is applicable to all animal and plant species which can be mutagenized, regardless of its mating / pollinating system, ploidy level, or genome size. This approach requires prior DNA sequence information and takes advantage of a mismatch endonuclease to locate and detect induced mutations. Ultimately, it can provide an allelic series of silent, missense, nonsense, and splice site mutations to examine the effect of various mutations in a gene. TILLING has proven to be a practical, efficient, and an effective approach for functional genomic studies in numerous plant and animal species. EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations and has been successfully utilized in animals and plants to discover SNPs including rare ones. In this review, TILLING and EcoTILLING techniques, beneficial applications and limitations from plant and animal studies are discussed.

Assessment of the genetic diversity and phylogenetic relationships of a temperate bamboo collection by using transferred EST-SSR markers.

Polymorphic expressed sequence tag - simple sequence repeat (EST-SSR) markers derived from major cereal crops were used to assess the genetic diversity of the USDA temperate bamboo collection consisting of 92 accessions classified in 11 separate genera and 44 species. A total of 211 bands were detected with a mean number of alleles per locus of 8.440. Phylogenetic relationships were determined by calculating genetic distances between all pairwise combinations and assessing differences in character data. The resulting dendrograms (unweighted pair group method with arithmetic means (UPGMA) and parsimony) clustered the accessions into 2 main clades, which corresponded to accessions characterized morphologically as either clumping (sympodial) or running (monopodial) bamboos. The majority of the accessions clustered according to their current taxonomic classification. These markers were also beneficial in identifying contaminated and (or) misidentified plots. Overall, these transferred markers were informative in differentiating the various bamboo accessions and determining the level of genetic variation within and among species and genera.