N. F. Weeden

Linkage among isozyme, RFLP and RAPD markers in Vicia faba

SummarySegregating allozyme and DNA polymorphisms were used to construct a preliminary linkage map for faba bean. Two F2 populations were analyzed, the most informative of which was segregating for 66 markers. Eleven independently assorting linkage groups were identified in this population. One of the groups contained the 45s ribosomal array and could be assigned to the large metacentric chromosome I on which the nucleolar organizer region is located. This linkage group also contained two isozyme loci, Est and Tpi-p, suggesting that it may share some homology with chromosome 4 of garden pea on which three similar markers are syntenic. Additional aspects of the map and the extent of coverage of the total nuclear genome are discussed.

Inter simple sequence repeat analysis of genetic relationships in the genus Vigna

The utility of inter simple sequence repeat (ISSR) DNA polymorphisms to distinguish taxa within the genus Vignawas investigated. Nineteen primers, most containing either aGA or CA repeat, generated amplification products that differed among the taxa examined. The ISSR polymorphisms produced by 15 of these primers were very effective for distinguishing taxa at the species level or below. The Vigna unguiculataaccessions analyzed formed a cohesive group and appeared to be most closely related to V. triphylla and V. reticulata.In contrast, ISSR analysis was not able to clearly differentiate subgeneric divisions within Vigna. We attribute this loss of resolution at the subgeneric level to the high rate of evolution of the sequences we examined. Several probable instances of misclassification or hybrid origin of an accession were identified.

Large-scale, cost-effective screening of PCR products in marker-assisted selection applications

A simple, PCR-based method has been developed for the rapid genotyping of large numbers of samples. The method involves a alkaline extraction of DNA from plant tissue using a slight modification of the procedure of Wang et al. (Nucleic Acids Res 21:4153–4154, 1993). Template DNA is amplified using allelespecific associated primers (ASAPs) which, at stringent annealing temperatures, generate only a single DNA fragment and only in those individuals possessing the appropriate allele. This approach eliminates the need to separate amplified DNA fragments by electrophoresis. Instead, samples processing the appropriate allele are identified by direct staining of DNA with ethidium bromide. Total technician time required for extraction, amplification and detection of 96 samples is about 4 h, and this time requirement can be reduced by automation. Excluding labor, cost per sample is less than

Linkage analysis of er-1, a recessive Pisum sativum gene for resistance to powdery mildew fungus (Erysiphe pisi D.C.)

0.40. The method is tested using the codominant isozyme marker, alcohol dehydrogenase (Adh-1) gene in pea (Pisum sativum), and applied to the screening of photoperiod genes in common bean (Phaseolus vulgaris L.).

Molecular-marker analysis of quantitative traits for growth and development in juvenile apple trees

Linkage analysis was used to determine the genetic map location of er-1, a recessive gene conditioning resistance to powdery mildew, on the Pisum sativum genome. Genetic linkage was demonstrated between er-1 and linkage group 6 markers after analyzing the progeny of two crosses, an F2 population and a set of recombinant inbred lines. The classes of genetic markers surrounding er-1 include RFLP, RAPD and allozyme markers as well as the morphological marker Gty. A RAPD marker tightly linked to er-1 was identified by bulked segregant analysis. After DNA sequence characterization, specific PCR primers were designed to convert this RAPD marker into a sequence characterized amplified region (SCAR).

Distinguishing among white seeded bean cultivars by means of allozyme genotypes

Abstract Random amplified polymorphic DNAs (RAPDs) were used in combination with a double pseudo-testcross mapping strategy to estimate the position and effects of quantitative trait loci (QTLs) for traits influencing juvenile tree growth and development in two apple cultivars. The mapping population consisted of 172 F1 trees from a cross between the columnar mutant ‘Wijcik McIntosh’ and a standard form disease-resistant selection NY 75441-58. Significant associations were found between markers and height increment, internode number, internode length, base diameter increment, base diameter after 9 years of growth, branch number, and leaf break. The number of genomic regions associated with each trait varied from one to eight. The amount of variation explained by linear regression on individual marker loci (R2) ranged from 3.9 to 24.3%, with an average of 7%. Multiple regression using markers for each putative QTL explained from 6.6 to 41.6% of the phenotypic variation, with an average value of 24.3%. A large number of traits had significant variation associated with the map position of the dominant columnar gene, Co. QTL stability over years was estimated by comparing the locations of putative QTLs for traits measured in multiple years. The majority of genomic regions were associated with a trait in only a single year, although regions associated with a trait in more than 1 year were also detected. The limitations of dominant markers and an outbred mapping pedigree for QTL analysis are discussed.

Identification of co-dominant RAPD markers tightly linked to fruit skin color in apple

SummaryAllozyme genotypes were determined at 10 loci for 90 cultivars of white seeded snap beans. Within cultivars the loci were homozygous and usually monomorphic, permitting the characterization of most cultivars by a single set of allozymes. A total of 72 allozyme combinations were observed among the cultivars tested, and 52 (58%) of the cultivars could be uniquely distinguished by allozyme genotype alone. The remaining 38 lines could be separated into small groups of 2–5 cultivars each.

Linkage mapping of quantitative trait loci controlling seed weight in pea (Pisum sativum L.).

A simple genetic basis for the red/yellow skincolor polymorphism in apple was verified using DNA markers. Bulked segregant analysis identified one 10-base oligomer that generated different fragments in each of the bulks. After testing the primer in four populations, two fragments were found to be associated with red skin color and another two fragments associated with yellow skin color. Three of the fragments (1160, 1180, and 1230 bp) were partly sequenced and found to share high sequence homology, suggesting these were generated from the same locus. A pair of universal primers were designed to amplify the fragments. In the ‘Rome Beauty’ x ‘White Angel’ population, two fragments were associated with red skin color; one fragment designated as A1 (1160 bp) was from ‘Rome Beauty’ and another fragment (A2, 1180 bp) was from ‘White Angel’. Progeny possessing both fragments, or either one, had red fruit. Both parents displayed an alternate fragment, a1 (1230 bp), associated with yellowskinned fruit. In three other crosses tested, only fragment A1 co-segregated with red skin color; two fragments, a1 and a2 (1230 bp and 1320 bp), were associated with yellow skin color. Our results are consistent with the hypothesis that the red/yellow dimorphism is controlled by a monogenic system with the presence of the red anthocyanin pigmentation being dominant. There was no indication that other modifier genes could reverse the effect of the locus (Rf) linked to the markers. Examination of amplification products in 56 apple cultivars and advanced breeding selections demonstrated that the universal primers could be used to correctly predict fruit skin color in most cases.

Distribution, inheritance and linkage relationships of ribosomal DNA spacer length variants in pea

Quantitative trait loci (QTLs) affecting seed weight in pea (Pisum sativum L.) were mapped using two populations, a field-grown F2 progeny of a cross between two cultivated types (‘Primo’ and ‘OSU442-15’) and glasshouse-grown single-seed-descent recombinant inbred lines (RILs) from a wide cross between a P. sativum ssp. sativum line (‘Slow’) and a P. sativum ssp. humile accession (‘JI1794’). Linkage maps for these crosses consisted of 199 and 235 markers, respectively. QTLs for seed weight in the ‘Primo’ x ‘OSU442-15’ cross were identified by interval mapping, bulked segregant analysis, and selective genotyping. Four QTLs were identified in this cross, demonstrating linkage to four intervals on three linkage groups. QTLs for seed weight in the ‘JI1794’ x ‘Slow’ cross were identified by single-marker analyses. Linkage were demonstrated to four intervals on three linkage groups plus three unlinked loci. In the two crosses, only one common genomic region was identified as containing seed-weight QTLs. Seed-weight QTLs mapped to the same region of linkage group III in both crosses. Conserved linkage relationships were demonstrated for pea, mungbean (Vigna radiata L.), and cowpea (V. unguiculata L.) genomic regions containing seed-weight QTLs by mapping RFLP loci from the Vigna maps in the ‘Primo’ x ‘OSU442-15’ and ‘JI1794’ x ‘Slow’ crosses.

Linkage mapping of sbm-1, a gene conferring resistance to pea seed-borne mosaic virus, using molecular markers in Pisum sativum

SummaryDNA restriction endonuclease fragment analysis is used to examine the genetic organization, inheritance and linkage associations of the ribosomal DNA in pea. The substantial variation observed in the length of the intergenic spacer region is shown to segregate in Mendelian fashion involving two independent genetic loci, designated Rrn1 and Rrn2. Linkage between Rrn1 and two marker loci on chromosome 4 establishes the approximate location of this tandem array. Rrn2 shows linkage with a set of isozyme loci which assort independently of other markers on all seven chromosomes. Combining these observations with previous cytological data, we suggest that Rrn2 and the isozyme loci linked to it constitute a new linkage group on chromosome 7. The general absence of spacer length classes common to both rRNA loci in any of the lines we examined indicates that little or no genetic exchange occurs between the nonhomologous nucleolar organizer regions.