The use of the isoenzymic marker gene Got-1 in the recognition of incompatibility S alleles in apple

The S incompatibility system of apple was confirmed through the application of the gene Got-1 for glutamate oxaloacetate transaminase as a marker for the S locus. The 11S alleles proposed by Kobel et al. (1939) were confirmed through anomalous segregations for Got-1 observed in 14 semi-compatible crosses and regular segregations observed in 2 fully compatible crosses. The S allele genotypes of Idared (S ₃ S ₇), Cox (S ₅ S ₉) and Fiesta (S ₃ S ₅) were determined and found to fall within the original series. By associating parental incompatibility genotypes with the segregation of Got-1 alleles, we were able to deduce the coupling of S and Got-1 alleles in 9 varieties.     

Genetic fingerprinting of pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of pigeonpea [Cajanus cajan (L.) Millsp.] cultivars and their related wild species. The use of single primers of arbitrary nucleotide sequence resulted in the selective amplification of DNA fragments that were unique to individual accessions. The level of polymorphism among the wild species was extremely high, while little polymorphism was detected within Cajanus cajan accessions. All of the cultivars and wild species under study could be easily distinguished with the help of different primers, thereby indicating the immense potential of RAPD in the genetic fingerprinting of pigeonpea. On the basis of our data the genetic relationship between pigeonpea cultivars and its wild species could be established.NCL Communication No. 6062     

An integrated genetic linkage map for eucalypts using RFLP,RAPD and isozyme markers

An integrated genetic linkage map for E. nitens was constructed in an outbred three-generation pedigree. Analysis of 210 RFLP, 125 RAPD and 4 isozyme loci resulted in 330 markers linked in 12 linkage groups covering 1462 cM (n=11 in eucalypts). The 12th linkage group is comprised of only 5 markers and will probably coalesce with another linkage group when further linked loci are located. Co-dominant RFLP loci segregating in both parents were used to integrate linkages identified in the male and female parents. Differences in recombination frequencies in the two parents were observed for a number of pairs of loci, and duplication of sequences was identified both within and between linkage groups. The markers were distributed randomly across the genome except for the RFLPs in linkage group 10 and for some loci showing segregation distortion, which were clustered into three regions of the map. The use of a large number of co-dominant RFLP loci in this map enables it to be used in other pedigrees of E. nitens and forms a basis for the detection and location of QTL in E. nitens and other eucalypt species.     

Intra- and inter-specific variations in Lens revealed by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate intra- and interspecific variations in the genus Lens (lentil). Twenty cultivars of L. culinaris ssp. culinaris, including 11 microsperma (small-seeded) and nine macrosperma (large-seeded) types, and 16 wild relatives (four accessions each of L. culinaris ssp. orientalis, L. odemensis, L. nigricans and L. ervoides), were evaluated for genetic variability using a set of 40 random 10-mer primers. Fifty reproducibly scorable DNA bands were observed from ten of the primers, 90% of which were polymorphic. Genetic distances between each of the accessions were calculated from simple matching coefficients. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). This study revealed that (1) expect for L. ervoides, the level of intraspecific variation in cultivated lentil is lower than that in wild species, (2) L. culinaris ssp. orientalis is the most likely candidate for a progenitor of the cultivated species, and (3) microsperma and macrosperma cultivars were indistinguishable by the RAPD markers identified here.     

Conversion of a RAPD-generated PCR product,containing a novel dispersed repetitive element,into a fast and robust assay for the presence of rye chromatin in wheat

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.     

Characterization of quantitative trait loci (QTLs) in cultivated rice contributing to field resistance to sheath blight (Rhizoctonia solani)

Sheath blight, caused by Rhizoctonia solani, is one of the most important diseases of rice. Despite extensive searches of the rice germ plasm, the major gene(s) which give complete resistance to the fungus have not been identified. However, there is much variation in quantitatively inherited resistance to R. solani, and this type of resistance can offer adequate protection against the pathogen under field conditions. Using 255 F₄ bulked populations from a cross between the susceptible variety Lemont and the resistant variety Teqing, 2 years of field disease evaluation and 113 well-distributed RFLP markers, we identified six quantitative trait loci (QTLs) contributing to resistance to R. solani. These QTLs are located on 6 of the 12 rice chromosomes and collectively explain approximately 60% of the genotypic variation or 47% of the phenotypic variation in the LemontxTeqing cross. One of these resistance QTLs (QSbr4a), which accounted for 6% of the genotypic variation in resistance to R. solani, appeared to be independent of associated morphological traits. The remaining five putative resistance loci (QSbr2a, QSbr3a, QSbr8a, QSbr9a and QSbr12a) all mapped to chromosomal regions also associated with increased plant height, three of which were also associated with QTLs causing later heading. This was consistent with the observation that heading date and plant height accounted for 47% of the genotypic variation in resistance to R. solani in this population. There were also weak associations between resistance to R. solani and leaf width, which were likely due to linkage with a QTL for this trait rather than to a physiological relationship.     

Irregular meiosis in a somatic hybrid between S. bulbocastanum and S. tuberosum detected by species-specific PCR markers and cytological analysis

A system of randomly amplified polymorphic DNA (RAPD) markers was developed to facilitate the transfer of S. bulbocastanum (blb) genes into the S. tuberosum (tbr) genome by hybridization and backcrossing. DNA from tbr, blb and the hexaploid hybrid was used as a template for polymerase chain reaction (PCR) amplification. Polymorphic RAPD products, originating from 10-mer primers, specific for blb were cloned and sequenced at their ends to allow the synthesis of 18-mer primers. The 18-mer primers allowed a more reproducible assay than the corresponding RAPDs. Of eight 18-mer primer pairs, four amplified the expected products specific for blb. However, the stringency of the primer annealing conditions needed to be carefully optimized to avoid amplification of the homeologous tbr product, suggesting that the original RAPD polymorphisms were due to single base-pair changes rather than deletions or insertions. Two primers used for amplification of backcross 2 progeny segregated in a 11 (presence:absence) ratio; the other two were unexpectedly absent. The most likely explanation for the loss of these markers is irregular meiosis in the original hexaploid hybrid and subsequent elimination of chromosomes. Cytological analysis of the meiosis in the hybrid demonstrated widespread irregular pairing and the presence of lagging univalents. In addition, the first backcross individual used as the parent for the second backcross had 54 chromosomes instead of the predicted 60. In conclusion, our results demonstrate that PCR technology can be used for the efficient isolation of taxon-specific markers in Solanum. Furthermore, by the use of these markers we detected the loss of chromosomes that was subsequently shown by cytological analysis to be caused by irregular meiosis of the somatic hybrid.     

Nature of 2n gamete formation and mode of inheritance in interspecific hybrids of diploid Vaccinium darrowi and tetraploid V. corymbosum

RAPD markers were used to determine the level of heterozygosity transmitted via 2n gametes from V. darrowi selection Florida 4B (Fla 4B) to inter-specific hybrids with tetraploid V. corymbosum cv Bluecrop. The tetraploid hybrid US 75 was found to contain about 70% of Fla 4B's heterozygosity, a value attributed to a first-division restitution (FDR) mode of 2n gamete production. Crossovers during 2n gamete formation were evidenced by the absence of 16 dominant alleles of Fla 4B in US 75, and direct tests of segregation in a diploid population involving Fla 4B. RAPD markers that were present in both Fla 4B and US 75 were used to determine the mode of inheritance in a segregating population of US 75xV. corymbosum cv Bluetta. Thirty-one duplex loci were identified which segregated in a 51 ratio, indicating that US 75 undergoes tetrasomic inheritance.     

Combined genetic and physiological analysis of a locus contributing to quantitative variation

The natural variation of many traits is controlled by multiple genes, individually referred to as quantitative trait loci (QTL), that interact with the environment to determine the ultimate phenotype of any individual. A QTL has yet to be described molecularly, in part because strategies to systematically identify them are underdeveloped and because the subtle nature of QTLs prevents the application of standard methods of gene identification. Therefore, it will be necessary to develop a systematic approach(es) for the identification of QTLs based upon the numerous positional data now being accumulated through molecular marker analyses. We have characterized a QTL by the following three-step approach: (1) identification of a QTL in complex populations, (2) isolation and genetic mapping of this QTL in near-isogenic lines, and (3) identification of a candidate gene using map position and physiological criteria. Using this approach we have characterized a plant height QTL in maize that maps to chromosome 9 near the centromere. Both map position and physiological criteria suggest the gibberillin biosynthesis gene dwarf3 as a candidate gene for this QTL.     

An extended map of the sugar beet genome containing RFLP and RAPD loci

An updated map of sugar beet (Beta vulgaris L. ssp. vulgaris var altissima Doell) is presented. In this genetic map we have combined 248 RFLP and 50 RAPD loci. Including the loci for rhizomania resistance Rr1, hypocotyl colour R and the locus controlling the monogerm character M, 301 loci have now been mapped to the nine linkage groups covering 815 cM. In addition, the karyotype of some of the Beta vulgaris chromosomes has been correlated with existing RFLP and RAPD linkage maps.