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Luís Goulão, Luisa Monte-Corvo, and Cristina M. Oliveira

Variability of commercial plum (Prunus L. sp.) cultivars is unknown since breeding often involves intercrossing hybrids with several species but has been based on a low number of parents. Molecular markers like amplified fragment length polymorphisms (AFLP) and inter-simple sequence repeats (ISSR), which sample multiple loci simultaneously, have become increasingly popular, and were used to characterize 24 diploid and four hexaploid cultivars of plum. Seven AFLP and six ISSR primers were used, and resulted in amplification of 379 and 270 products, respectively. Unweighted pair-group method with arithmetic averages (UPGMA) dendrograms, based on similarity coefficients, reflected a clear separation between diploid and hexaploid plums. Among diploid plums, two pairs of cultivars were relatively distinct from the rest, namely `Golden Japan' and `Methley' and `Ozark Premier' and `Songold'. Furthermore, several cultivars were grouped together both with AFLP and ISSR analysis: 1) `Ambra', `Red Beaut', and `Black Beaut', 2) `Black Diamond' and `Royal Diamond', 3) `June Rose', `Santa Rosa', and `Royal Red', and iv) `Freedom', `Larry Ann', and `Queen Rosa'. Although the phenetic classification obtained by the two methods were similar (r = 0.73, for the diploid group), ISSR had a higher reproducibility and percentage of polymorphisms (87.4% vs. 62.8%) than AFLP. Methodological aspects of both markers systems are discussed. Results obtained suggest that the AFLP and ISSR approaches are valuable tools for identification of specific genotypes and analysis of phenetic relationships in plum.

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Kimberly J Walters, George L. Hosfield, and James D. Kelly

30 POSTER SESSION 4 (Abstr. 460-484) Breeding/Genetics/Molecular Markers

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Mario Crespo, James Nienhuis, Jan Tivang, and Paul Skroch

Knowledge of relative genetic distance among genotypes is useful in a breeding program because it permits organization of germplasm resources. Genetic distance (GD) was estimated among 113 faba bean, Vicia faba L. genotypes, which included three botanical varieties from different geographical areas around the world. The genotypes included 87 accessions from Bolivia, 14 accessions from the Middle East and North Africa, five accessions from Australia, and seven commercial varieties from Europe. Twenty-three RAPD primers were scored yielding four to 13 polymorphic bands resulting in a total of 165 bands. Our objective was to determine genetic relationships among accessions and cultivars as measured by RAPD markers. The genetic relationships were estimated using the ratio of discordant to total bands scored. A multidimensional scaling (MDS) plot indicated four clusters corresponding to: i) European commercial cultivars; ii) the Middle East, North Africa, and Australian accessions; iii) the Bolivian highland landraces; and iv) the Bolivian collection maintained in a valley environment. A permutation test confirmed the four clusters (P < 0.01). Sampling variance results indicated that a CV of 10% could be obtained with as few as 148 bands between groups. Selection and drift appears the main cause of divergence of two populations in the Bolivian faba bean collection. The results of this study indicated that RAPDs are a powerful tool for evaluation of germplasm conservation methods in faba bean.

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Soon O. Park, Dermot P. Coyne, Nedim Mutlu, Geunhwa Jung, and James R. Steadman

Common bacterial blight, incited by Xanthomonas campestris pv. phaseoli (Xcp) is a serious disease of common bean (Phaseolus vulgaris L.). Randomly amplified polymorphic DNA (RAPD) markers and flower color (V gene) previously were reported to be associated with six quantitative trait loci (QTL) affecting leaf and pod resistance to Xcp. However, the markers for the QTL were not confirmed in different populations and environments to indicate their merit in breeding. The objective was to determine if the associations of RAPD markers and the V gene with QTL for leaf and pod resistance to Xcp in a recombinant inbred (RI) backcross population from the cross BC2F6 `PC-50' × XAN-159 and for leaf resistance to Xcp in an F2 population from a different cross pinto `Chase' × XAN-159 could be confirmed. One or two genes from XAN-159 controlled leaf and pod resistance to Xcp. Among six QTL previously detected, five in the RI backcross population and three in the F2 population were confirmed to be associated with resistance to Xcp. The V gene and RAPD marker BC437.1050 on linkage group 5 were most consistently associated with leaf and pod resistance to two to five Xcp strains in the RI backcross population and with leaf resistance to two Xcp strains in the F2 population. One to three QTL affecting leaf and pod resistance to Xcp accounted for 22% to 61% of the phenotypic variation. Gene number (one to two) estimations and number of QTL (one to three) detected for leaf and pod resistance to Xcp in the RI backcross population were generally in agreement. The marker BC437.1050 and V gene, along with other resistance genes from other germplasm, could be utilized to pyramid the different genes into a susceptible or partially resistant bean line or cultivar to enhance the level of resistance to Xcp.

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Yiping Zhang and John R. Stommel

The carotenoids have an important influence on tomato fruit quality and enhance the fruit contribution to human nutrition. Expression of the high pigment (hp) locus in tomato results in increased total carotenoids and increased efficiency of utilization of the polyenes. A similar mutant, dark green (dg), contains higher level of chlorophyll in immature fruit and results in darker red pigmentation, both externally and internally in ripe fruit. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses were performed using two pairs of near isogenic lines (NILs) designed to be isogenic at the hp and dg loci. Sixty-four AFLP primer pairs and more than 1000 RAPD 10-mer primers were screened for polymorphism between each pair of the NILs. One RAPD marker was identified to be linked to the hp gene, and two AFLP primer pairs showed polymorphic fragments which distinguished the dg NILs. The markers identified in this study will be converted to allele specific SCAR (sequence characterized amplified region) markers, which are more useful in marker-assisted selection breeding programs.

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Soon O. Park, Dermot P. Coyne, and James R. Steadman

Bean rust, caused by Uromyces appendiculatus, is a major disease of common bean (Phaseolus vulgaris). The objective was to identify RAPD markers linked to the gene (Ur-7) for specific resistance to rust race 59 using bulked segregant analysis in an F2 segregating population from the common bean cross GN1140 (resistant to rust) × Nebraska #1 (susceptible to rust). A single dominant gene controlling specific resistance to race 59 was found in the F2 and was confirmed in the F3. Seven RAPD markers were detected in a coupling-phase linkage with the Ur-7 gene. Coupling-phase RAPD markers OAA11.500, OAD12.550, and OAF17.900 with no recombination to the Ur-7 gene were found. Three RAPD markers were identified in a repulsion-phase linkage with the Ur-7 gene among the three markers at a distance of 8.2 cM. This is the first report on RAPD markers linked to the Ur-7 gene in common bean. The RAPD markers linked to the gene for specific rust resistance of Middle American origin detected here, along with other independent rust resistance genes from other germplasm, could be used to pyramid multiple genes into a bean cultivar for more-durable rust resistance.

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Soon O. Park, Dermot P. Coyne, Nedim Mutlu, James R. Steadman, and Geunhwa Jung

Common bacterial blight, incited by Xanthomonas campestris pv. phaseoli (Xcp), is a serious disease of common bean (Phaseolus vulgaris). RAPD markers and flower color (V gene) previously had been reported to be associated with six QTL affecting leaf and pod resistance to Xcp. However, the markers for the QTL were not confirmed in different populations and environments to indicate their merit in breeding. Our objective was to determine if the associations of RAPD markers and the V gene with QTL for leaf and pod resistance to Xcp in a RI backcross population from the cross BC2F6 `PC-50' × XAN-159 and for leaf resistance to Xcp in a F2 population from a different cross Pinto `Chase' × XAN-159 could be confirmed. Among six QTL previously detected, five in the RI backcross population and three in the F2 population were confirmed to be associated with resistance to Xcp. The V gene and RAPD marker BC437.1050 on linkage group 5 were most consistently associated with leaf and pod resistance to two to five XCP strains in the RI backcross population and with leaf resistance to two Xcp strains in the F2 population. The confirmed marker BC437.1050 and V gene on linkage group 5, along with other resistance genes from other germplasm, could be used to pyramid the different genes into a bean cultivar to enhance the resistance to Xcp.

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James M. Bradeen and Philipp Simon

The Y2 locus conditions α- and β-carotene accumulation in the xylem (core) of carrot roots. The dominant allele suppresses carotene, but not xanthophyll accumulation, resulting in yellow-cored roots. Individuals homozygous for the recessive allele are rich in carotenes and are therefore orange-cored. Increased consumer interest in high carotene produce requires improved understanding of carotene biosynthesis and color development and more-efficient breeding techniques. We examined 103 F2 individuals generated from inbred populations differing in core carotene content. Bulked segregant analysis identified AFLP bands putatively linked to Y2. Linkage was confirmed for some bands by mapping. Linked bands were excised from gels, re-amplified, cloned into pGEM vectors, and sequenced. Cloned fragments and sequence information were used to characterize larger genomic regions to identify codominant markers. Currently we are developing codominant, PCR-based markers that can be used to rapidly genotype individuals in breeding programs, to characterize wild, feral, and cultivated populations for diversity and evolution studies, and to examine the role of Y2 in carotene accumulation.

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Courtney A. Weber, Gloria A. Moore, Zhanao Deng, and Fred G. Gmitter Jr.

Mapping quantitative trait loci (QTL) associated with freeze tolerance was accomplished using a Citrus grandis (L.) Osb. × Poncirus trifoliata (L.) Raf. F1 pseudo-testcross population. A progeny population of 442 plants was acclimated and exposed to temperatures of -9 °C and -15 °C in two separate freeze tests. A subpopulation of 99 progeny was genotyped for random amplified polymorphic DNA (RAPD), cleaved amplified polymorphic sequence (CAPS), sequence characterized amplified region (SCAR), and sequence tagged site (STS) markers to produce a linkage map for each parent. Potential QTL were identified by interval mapping, and their validity was corroborated with results from means comparison (t test), one-way analysis of variance (F test), and bulked segregant analysis (BSA). Multiple analytical methods provided evidence supporting putative QTL and decreased the probability of missing significant QTL associated with freeze tolerance. QTL with a large effect on freeze tolerance were located on both the Citrus and Poncirus linkage maps. In addition, clusters of markers with significantly different means between marker present and absent classes indicating minor QTL that contribute smaller effects on the level of tolerance were found on the linkage maps of both species.

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Jack E. Staub, Fenny Dane, Kathleen Reitsma, Gennaro Fazio, and Anabel López-Sesé

Genetic relationships among 970 cucumber (Cucumis sativus L.) plant introductions (PIs) in the U.S. National Plant Germplasm System (NPGS) were assessed by observing variation at 15 isozyme loci. Allozyme frequency data for these PIs were compared to allozyme variation in heirloom and modern (H&M) cultivars released from 1846-1985 (H&M cultivars; 178 accessions), and experimental commercial (EC) germplasm (EC germplasm; 82 accessions) in use after 1985. Multivariate analysis defined four distinct groups of accessions (Groups A-D), where Group A consisted of PIs received by the NPGS before 1992, Group B contained PIs from India and China obtained by NPGS after 1992, Group C consisted of EC germplasm, and Group D contained H&M cultivars. Morphological, abiotic stress (water and heat stress tolerance) and disease resistance evaluation data from the Germplasm Resources Information Network (GRIN) for the PIs examined were used in conjunction with estimates of population variation and genetic distance estimates to construct test arrays and a core collection for cucumber. Disease resistance data included the evaluation of angular leafspot [Pseudomonas lachrymans (E.F. Smith) Holland], anthracnose [Colletotrichum lagenarium (Ross.) Ellis & Halst], downy mildew [Pseudoperonospora cubensis (Berk. & Curt) Rostow], rhizoctonia fruit rot (Rhizoctonia solani Kuhn), and target leafspot [Corynespora cassiicola (Berk. & Curt) Wei] pathogenicity. The test arrays for resistance-tolerance to angular leafspot, anthracnose, downy mildew, rhizoctonia fruit rot, target leafspot, and water and heat stress consisted of 17, 16, 17, 16, 17, 16, and 16 accessions, respectively. The core collection consisted of accessions in these test arrays (115) and additional 32 accessions that helped circumscribe the genetic diversity of the NPGS collection. The core collection of 147 accessions (115 + 32) represents ≈11% of the total collection's size (1352). Given estimates of genetic diversity and theoretical retention of diversity after sampling, this core collection could increase curatorial effectiveness and the efficiency of end-users as they attempt to identify potentially useful germplasm.