Somatic embryos derived from walnut (Juglans regia L.) ovule tissues were evaluated to determine whether they were of zygotic or maternal origin. Molecular markers were used to permit evaluation at an early stage, before whole plant development. Somatic embryos developed from potentially apomictic `Sunland' and `Cisco' ovule tissue isolated from bagged putatively unpollinated flowers. Phosphoglucomutase (PGM) isozyme analysis showed that all of these embryos, except one from each cultivar, carry the same zymotype as the maternal tissue. However, restriction fragment length polymorphism (RPLP) analysis combined with isozyme evaluation demonstrated that the tested embryos originated from zygotic rather than maternal tissues. This study demonstrates the application of molecular marker analyses, particularly RFLPs, evaluation of somatic embryo origin.
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.
Molecular markers were used to assess genetic diversity in basil (Ocimum L. spp., Lamiaceae). Using randomly amplified polymorphic DNA (RAPD) analysis, 11 primers generated 98 polymorphic bands, ranging from 300 to 2,000 base pairs, that discriminated among 37 accessions across nine Ocimum spp. Means of genetic similarities within Ocimum spp. showed that the domesticated species, O. minimum L. (0.887), O. basilicum L. (0.769), and O. ×citriodorum Vis. (0.711) had highest similarity indices within species, while the nondomesticated, O. americanum L. (0.580), O. gratissimum L. (0.408), and O. kilimandscharicum Guerke (0.559) showed the lowest similarity. RAPD results indicated that O. minimum should not be considered a distinct species but rather a variety of O. basilicum. Consistent clusters among all but one of the O. ×citriodorum spp., all containing citral as the major constituent, were identified using bootstrap analysis. RAPD analysis was useful in discriminating among Ocimum spp., although within species resolution will require a higher number of polymorphic bands.
106 POSTER SESSION (Abstr. 335–343) Breeding and Genetics–Vegetables II (Molecular Markers and Physiological Genetics)
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.
Sw-5 is a locus introgressed from Lycopersicon peruvianum to some L. esculentum lines conferring dominant resistance to TSWV. Restriction fragment length polymorphism (RFLP) analyses positions Sw-5 to the long arm of chromosome 9 in the sub-telomeric region between CT71 and CT220. RFLP analyses suggest the introgressed region begins distal to CT71, includes CT220, and may extend to the telomere. Randomly amplified polymorphic DNA (RAPD) analyses with >700 random 10-mer primers identified a single 2.2-kbp band with one primer (primer #72 GAGCACGGGA) that is tightly linked to Sw-5. However, we have also produced an equivalent 2.2-kbp band in analysis of other TSWV-susceptible tomato breeding lines. Thus, this band likely derives from L. esculentum DNA very near to Sw-5 and the introgressed region. Additional analyses have recently detected a potential co-dominant RAPD polymorphism linked to Sw-5.
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.
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.
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.
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.