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Simple Sequence Repeat (SSR) markers developed in apple and pear were used to determine genetic relationships among heritage apple and pear cultivars from Portugal's Azore Islands, and to develop identity fingerprints for European and Asian pear accessions at the USDA–ARS National Clonal Germplasm Repository (NCGR). We used 11 SSR markers (six from apple and five from pear) to examine 18 heritage apple and 9 heritage pear cultivars from the Azores. Eight additional Portuguese and economically important cultivars of apple and eight of pear were used as standards. Cluster analysis separated the apple and pear accessions into two distinct groups. Among apple genotypes, 12 unique accessions and five groups of synonyms were identified, while, in pear, seven unique genotypes and three pairs of synonyms were found. None of the accessions obtained from the Azores corresponded to widely grown standard Portuguese apple or pear cultivars. To examine 144 NCGR pear accessions, we used nine polymorphic SSR loci that were developed from GenBank sequences. Cluster analysis identified five sets of synonyms (four in P. communis L. and one in P. ussuriensis Maxim.) and four pairs of homonyms (three in P. communis and one in P. pyrifolia Burm. f. Nakai), and confirmed three clonal sets. Morphological evaluations and additional SSR markers will be used to confirm these results, and to genetically document the identities of pear genotypes. SSR markers will greatly assist the management of ex situ pome fruit germplasm collections by helping to eliminate duplicate accessions and expanding the genetic diversity represented.
Sixty-nine accessions representing wild and domesticated highbush blueberry (Vaccinium corymbosum L.) germplasm were genotyped using 28 simple sequence repeats (SSRs). A total of 627 alleles was detected and unique fingerprints were generated for all accessions. Suspected duplicate accessions of `Coville' and `Ivanhoe' had DNA fingerprints that were identical to `Coville' and `Ivanhoe', respectively. Genetic similarity measures placed wild and cultivated blueberries in separate groups. Northern highbush blueberries grouped among ancestral clones that were used extensively in blueberry breeding such as `Rubel' and `Stanley'. Southern highbush blueberries formed a separate group from northern highbush blueberries. The microsatellite markers used here show excellent promise for further use in germplasm identification, in genetic studies of wild Vaccinium L. populations, and for constructing linkage maps.
Microsatellite markers for blueberry (Vaccinium L.) were created from a preexisting blueberry expressed sequence tag (EST) library of 1305 sequences and a microsatellite-enriched genomic library of 136 clones.
Microsatellite primers for 65 EST-containing simple sequence repeats (SSRs) and 29 genomic SSR were initially tested for amplification and polymorphism on agarose gels. Potential usefulness of these SSRs for estimating species relationships in the genus was assessed through cross-species transference of 45 SSR loci and cluster analysis using genetic distance values from five highly polymorphic EST-SSR loci. Cross-species amplification for 45 SSR loci ranged from 17% to 100%, and was 83% on average in nine sections. Cluster analysis of 59 Vaccinium species based on genetic distance measures obtained from 5 EST-SSR loci supported the concept of V. elliotii Chapm. as a genetically distinct diploid highbush species and indicated that V. ashei Reade is of hybrid origin. Twenty EST-SSR and 10 genomic microsatellite loci were used to determine genetic diversity in 72 tetraploid V. corymbosum L. accessions consisting mostly of common cultivars. Unique fingerprints were obtained for all accessions analyzed. Genetic relationships, based on microsatellites, corresponded well with known pedigree information. Most modern cultivars clustered closely together, but southern highbush and northern highbush cultivars were sufficiently differentiated to form distinct clusters. Future use of microsatellites in Vaccinium will help resolve species relationships in the genus, estimate genetic diversity in the National Clonal Germplasm Repository (NCGR) collection, and confirm the identity of clonal germplasm accessions.
Three microsatellite-enriched libraries of the european hazelnut (Corylus avellana L.) were constructed: library A for CA repeats, library B for GA repeats, and library C for GAA repeats. Twenty-five primer pairs amplified easy-to-score single loci and were used to investigate polymorphism among 20 C. avellana genotypes and to evaluate cross-species amplification in seven Corylus L. species. Microsatellite alleles were estimated by fluorescent capillary electrophoresis fragment sizing. The number of alleles per locus ranged from 2 to 12 (average = 7.16) in C. avellana and from 5 to 22 overall (average = 13.32). With the exception of CAC-B110, di-nucleotide SSRs were characterized by a relatively large number of alleles per locus (≥5), high average observed and expected heterozygosity (Ho and He > 0.6), and a high mean polymorphic information content (PIC ≥ 0.6) in C. avellana. In contrast, tri-nucleotide microsatellites were more homozygous (Ho = 0.4 on average) and less informative than di-nucleotide simple sequence repeats (SSRs) as indicated by a lower mean number of alleles per locus (4.5), He (0.59), and PIC (0.54). Cross-species amplification in Corylus was demonstrated. These microsatellite markers were highly heterozygous and polymorphic and differentiated among genotypes of C. avellana irrespective of geographical origin. They will aid in fingerprinting genotypes of the european hazelnut and other Corylus species, genome mapping, and genetic diversity assessments.
Propagation of Corylus avellana stem cuttings may be limited by either root initiation or bud abscission. We divided juvenile shoots of 3 varieties growing in layering beds in mid-July into 4 or 5 3-node cuttings with leaves at the upper two nodes, except that terminal cuttings had one expanded leaf. Cuttings were treated with 5 mM IBA in 50% EtOH, a mixture of A. rhizogenes strains A7 + 22 or left untreated. IBA and bacteria stimulated rooting of cuttings from all shoot positions. Rooting of the terminal cuttings (<50%) was less than that of the sub-terminal cuttings (>80%). Bud retention was <50% on terminal cuttings, nearly 100% on sub-terminal cuttings. Using juvenile stock plants of various varieties, sub-terminal cuttings treated with Agrobacterium or 5 mM IBA may yield 70-90% cuttings with both roots and buds, Agravitropic roots, characteristic of genetic transformation, were observed on Agrobacterium-treated cuttings. Dot blots probed for TL-DNA were negative, however.
Propagation of Corylus avellana stem cuttings may be limited by either root initiation or bud abscission. We divided juvenile shoots of 3 varieties growing in layering beds in mid-July into 4 or 5 3-node cuttings with leaves at the upper two nodes, except that terminal cuttings had one expanded leaf. Cuttings were treated with 5 mM IBA in 50% EtOH, a mixture of A. rhizogenes strains A7 + 22 or left untreated. IBA and bacteria stimulated rooting of cuttings from all shoot positions. Rooting of the terminal cuttings (<50%) was less than that of the sub-terminal cuttings (>80%). Bud retention was <50% on terminal cuttings, nearly 100% on sub-terminal cuttings. Using juvenile stock plants of various varieties, sub-terminal cuttings treated with Agrobacterium or 5 mM IBA may yield 70-90% cuttings with both roots and buds, Agravitropic roots, characteristic of genetic transformation, were observed on Agrobacterium-treated cuttings. Dot blots probed for TL-DNA were negative, however.
Edible European pears (Pyrus communis sp. communis L.) are thought to be derived from wild relatives native to the Caucasus Mountain region and eastern Europe. We collected genotype, phenotype, and geographic origin data for 145 P. communis individuals derived from seeds collected from wild relatives. These individuals are currently maintained in the USDA–ARS National Plant Germplasm System (NPGS) in Corvallis, Ore. Pear genotypes were obtained using 13 microsatellite markers. A Bayesian clustering method grouped the individual pear genotypes into 12 clusters. The subspecies of pears native to the Caucasus Mountains of Russia, Crimea, and Armenia could be genetically differentiated from the subspecies native to eastern European countries. Pears with large fruit clustered closely together and are most closely related to a group of genotypes that are intermediate to the other groups. Based on the high number of unique alleles and heterozygosity in each of the 12 clusters, we conclude that the genetic diversity of wild P. communis is not fully represented in the NPGS
Twelve microsatellites were isolated from simple sequence repeat (SSR)-enriched genomic libraries of ‘Meeker’ red raspberry (Rubus idaeus) and ‘Marion’ blackberry (Rubus hybrid). These primer pairs plus one developed from a GenBank red raspberry sequence were evaluated in 48 raspberry and 48 blackberry genotypes. Only RhM031 did not generate a product in raspberry, whereas RiG001 failed to amplify in blackberry and hybrid accessions. The number of polymerase chain reaction products per primer pair in the 12 SSRs that successfully amplified was higher in blackberry genotypes and their hybrids than in raspberry, ranging from three to 29 in blackberry (average, 14.4) and from one to 15 in red raspberry (average, 7.5). Diversity estimates were determined for 10 of 12 SSRs that amplified up to two products in 44 red raspberry genotypes. The best SSR loci based on high observed and expected heterozygosities, high polymorphism information content, and low inbreeding coefficient were RiM019, RhM003, and RhM011. They mapped to three different linkage groups (5, 2, and 7, respectively) in red raspberry and differentiated the unique genotypes identified with the 12 SSRs in each crop type.
Identifying and evaluating genetic diversity of culinary rhubarb (Rheum ×rhababarum) cultivars using morphological characteristics is challenging given the existence of synonyms and nomenclatural inconsistencies. Some cultivars with similar names are morphologically different, and seedlings may grow and become associated with the parental name. Morphological traits of one cultivar may vary when measured under different environmental conditions. Molecular markers are consistent for unique genotypes across environments and provide genetic fingerprints to assist in resolving identity issues. Microsatellite repeats, also called simple sequence repeats (SSRs), are commonly used for fingerprinting fruit and nut crops, but only 10 SSRs have previously been reported in rhubarb. The objectives of this study were to use short-read DNA sequences to develop new di-nucleotide-containing SSR markers for rhubarb and to determine if the markers were useful for cultivar identification. A total of 97 new SSR primer pairs were designed from the short-read DNA sequences. The amplification success rate of these SSRs was 77%, whereas polymorphism of those reached 76% in a test panel of four or eight rhubarb individuals. From the 57 potentially polymorphic primer pairs obtained, 25 SSRs were evaluated in 58 Rheum accessions preserved in the U.S. Department of Agriculture, National Plant Germplasm System. The primer pairs generated 314 fragments with an average of 12.6 fragments per pair. The clustering of many accessions in well-supported groups supported previous findings based on amplified fragment length polymorphisms (AFLPs). Cluster analysis, using the proportion of shared allele distance among the 25 SSRs, distinguished each of the 58 accessions including individuals that had similar names or the same name. Accessions that grouped in well-supported clusters previously belonged to similar clusters with high bootstrap support based on AFLP. In summary, our technique of mining short-read sequencing data was successful in identifying 97 di-nucleotide-containing SSR sequences. Of those tested, the 25 most polymorphic and easy-to-score primer pairs proved useful in fingerprinting rhubarb cultivars. We recommend the use of short-read sequencing for the development of SSR markers in the identification of horticultural crops.