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Carlos Miranda, Jorge Urrestarazu, Luis G. Santesteban, José B. Royo, and Valero Urbina

research program was launched to evaluate the genetic diversity of the collection through detailed morphological and agronomical description, and fingerprinting analysis based upon molecular markers was initiated in 2005. Microsatellite or simple sequence

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L.J. Grauke, Muhammad J. Iqbal, Avutu S. Reddy, and Tommy E. Thompson

A microsatellite-enriched library was developed from `Halbert', a native pecan [Carya illinoinensis (Wangenh.) K. Koch] selection from Coleman County, Texas. A genomic library enriched for simple sequence repeats (SSR) containing 6144 clones was archived in 384 well plates for screening. In total, 439 clones were identified after Southern hybridization using di- and tri-nucleotide repeats as probes. In total, 125 positive clones were sequenced and primers were designed for 24 repeats. The SSR markers chosen for analysis include di-(CT and GA) and tri-nucleotide repeats (CTT, GAA and GAT). Of the 24 primer pairs tested, 19 successfully amplified microsatellites from `Halbert'. DNA was isolated from 48 pecan and hickory accessions selected to strategically represent the genetic diversity of the National Clonal Germplasm Repository (NCGR) Carya collections. The 19 SSR primers that produced good amplification products in `Halbert' were used to evaluate the collection, with 11 revealing polymorphism. The number of fragments amplified with different primer combinations ranged from 4 to 32 in the 48 genotypes tested. Evaluation of the data confirms the utility of the microsatellites in delimiting known relationships.

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Yiqun Weng, Shanna Johnson, Jack E. Staub, and Sanwen Huang

and Staub (1999 ; used RAPD markers). Moreover, the microsatellite markers used here were developed from a whole genome sequence of cucumber inbred line 9930 through pre-selection of long repeat motifs ( Ren et al., 2009 ), which are generally more

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R.J. Schnell, J.S. Brown, C.T. Olano, E.J. Power, C.A. Krol, D.N. Kuhn, and J.C. Motamayor

Three horticultural races of avocado (Persea americana Mill.) are known: Guatemalan, Mexican, and West Indian. Each race has unique characteristics and current commercial varieties have been selected from within the races or from interracial hybrids. Using 14 microsatellite loci we investigated the genetic variation among 224 accessions (394 plants) maintained at the National Germplasm Repository (NGR) in Miami, Fla., and a set of 34 clones from the University of California South Coast Field Station (SCFS) located in Irvine, Calif. The 14 microsatellite loci had an average of 18.8 alleles per locus and average unbiased genetic diversity was 0.83. The total propagation error in the collection, i.e., plants that had been incorrectly labeled or grafted, was estimated to be 7.0%. Although many unique alleles did exist, no useful race-specific markers were found. A general concordance between the horticultural race and the clusters obtained from molecular data was observed. Principal Coordinate Analysis (PCA) grouped the Guatemalan and Mexican races into two distinct clusters. The West Indian also grouped into a unique major cluster but with an outlying group. Using the PCA a change in the racial designation or interracial hybrid status for 50 accessions (19.7%) is proposed. The unbiased gene diversity estimate was highest in the Mexican and Guatemalan races and lower in the West Indian group. This demonstrates the need to collect more of the West Indian germplasm to broaden the genetic diversity and to emphasize the identification of individuals conferring resistance to Phytophthora Root Rot (PRR).

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M.I. Buteler, D.R. LaBonte, R.L. Jarret, and R.E. Macchiavelli

Using codominant molecular markers (microsatellites) for paternity identification was investigated in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.]. Two experimental populations (CIP and LAES), each consisting of progeny of known parentage, were scored for the presence or absence of alleles segregating at IB-316 and IB-318 microsatellite loci. Paternity was assessed using paternity exclusion and the most-likely parent methods. In the former, paternity is assigned based on the identification of incompatible parent-progeny marker data. In contrast, the latter method incorporates paternity exclusion and a log-likelihood or LOD score that weighs progeny allelic patterns as to the likelihood that they could have come from a given paternal parent. The number of correctly allocated progeny differed for the methods. Paternity exclusion correctly allocated 7% and 25% of the progeny in the LAES and CIP populations, respectively. The most-likely parent method correctly allocated 23% and 88% of the progeny in the LAES and CIP populations, respectively. The greater misassignments in the LAES population were attributed to low allelic diversity at the LAES IB-318 locus and a larger parental population. This study demonstrates the feasibility of identifying paternity in sweetpotato using a minimal number of loci.

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LJ Grauke, Maria Azucena Mendoza-Herrera, Carol Loopstra, and Tommy E. Thompson

Microsatellite or Simple Sequence Repeat (SSR) markers are being developed in ongoing research in the USDA ARS Pecan Breeding Program. These co-dominant markers provide a powerful tool for the verification of parentage. To confirm their utility, SSR profiles were used to confirm the parentage of 19 of the 25 controlled crosses released by the breeding program. Questions were raised concerning the parentage of some crosses thought to be known. When the genotype of the maternal parent is known, the paternal genotype necessary to have produced the progeny can be determined. A SAS program was written to query a database that includes 288 pecan accessions to find appropriate paternal genotypes given a maternal pattern. If neither parent is known, all possible parental combinations can be derived based on the progeny. Putative parents can be qualified on the basis of genotype as well as other evidence, such as nut morphology, dates of origin, locations of origin, and dichogamy. Using these techniques, putative parents are suggested for the historic cultivars `Riverside' and `Western'. Although the probabilities for a particular genotypic pattern can be determined based on allele frequencies within the population, assigning numeric probabilities to other evidence is more challenging. Meticulous records are necessary to establish the linkage between an inventory of an accession and its historic origin, thereby placing putative parents in combination at the proper place and appropriate time. Records of USDA–ARS National Plant Germplasm System, as exemplified by logbooks and vouchers of the McKay Collection of the National Arboretum, provide evidence for confident molecular genetic verification of cultivar identity and parentage, increasing the value of the living accessions in the NPGS.

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Yu Zong, Ping Sun, Xiaoyan Yue, Qingfeng Niu, and Yuanwen Teng

needed. In this study, we analyzed the geographical distribution of polymorphisms in nuclear microsatellite loci from the 18 P . betulaefolia populations that were reported in our previous study ( Zong et al., 2014 ). The main purpose of this study was

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Riaz Ahmad, Darush Struss, and Stephen M. Southwick

We evaluated the potential of microsatellite markers for use in Citrus genome analysis. Microsatellite loci were identified by screening enriched and nonenriched libraries developed from `Washington Navel' Citrus. Microsatellite-containing clones were sequenced and 26 specific PCR primers were selected for cross-species amplification and identification of cultivars/clones in Citrus. After an enrichment procedure, on average 69.9% of clones contained dinucleotide repeats (CA)n and (CT)n, in contrast to <25% of the clones that were identified as positive in hybridization screening of a nonenriched library. A library enriched for trinucleotide (CTT)n contained <15% of the clones with (CTT)n repeats. Repeat length for most of the dinucleotide microsatellites was in the range of 10 to 30 units. We observed that enrichment procedure pulled out more of the (CA)n repeats than (CT)n repeats from the Citrus genome. All microsatellites were polymorphic except one. No correlation was observed between the number of alleles and the number of microsatellite repeats. In total, 118 putative alleles were detected using 26 primer pairs. The number of putative alleles per primer pair ranged from one to nine with an average of 4.5. Microsatellite markers discriminated sweet oranges [Citrus sinensis (L.) osb], mandarin (Citrus reticulata Blanco), grapefruit (Citrus paradisi Macf.), lemon [Citrus limon (L.) Burm.f.], and citrange (hybrids of trifoliate orange and sweet orange), at the species level, but individual cultivars/clones within sweet oranges, mandarins and grapefruit known to have evolved by somatic mutation remained undistinguishable. Since these microsatellite markers were conserved within different Citrus species, they could be used for linkage mapping, evolutionary and taxonomic study in Citrus.

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Josh A. Honig, Vincenzo Averello, Stacy A. Bonos, and William A. Meyer

Jung, 2004 ) between genetic diversity assessed by RAPD markers and the PTM kentucky bluegrass classification system. In the current study we used microsatellite, or SSR, markers to study the genetic relationships of 247 kentucky bluegrass cultivars and

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Nahla V. Bassil, R. Botta, and S.A. Mehlenbacher

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.