Showing papers in "Theoretical and Applied Genetics in 1995"

Detection of genetic diversity in closely related bread wheat using microsatellite markers.

Abstract: Wheat microsatellites (WMS) were used to estimate the extent of genetic diversity among 40 wheat cultivars and lines, including mainly European elite material. The 23 WMS used were located on 15 different chromosomes, and revealed a total of 142 alleles. The number of alleles ranged from 3 to 16, with an average of 6.2 alleles per WMS. The average dinucleotide repeat number ranged from 13 to 41. The correlation coefficient between the number of alleles and the average number of repeats was only slight (r s = 0.55). Based on percentage difference a dendrogram is presented, calculated by the WMS-derived data. All but two of the wheat cultivars and lines could be distinguished. Some of the resulting groups are strongly related to the pedigrees of the appropriate cultivars. Values for co-ancestry (f) of 179 pairs of cultivars related by their pedigrees (f⩾0.1) averaged 0.29. Genetic similarity (GS) based on WMS of the same pairs averaged 0.44. The rank correlation for these pairs was slight, with r s = 0.55, but highly significant (P<0.001). The results suggest that a relatively small number of microsatellites can be used for the estimation of genetic diversity and cultivar identification in elite material of hexaploid bread wheat.

Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method

Abstract: The restriction patterns of two chloroplast fragments and one mitochondrial DNA fragment, amplified by PCR with universal primers, were studied to determine the mode of inheritance of these organelles in 143 progeny of five intraspecific crosses in pedunculate oak (Quercus robur L.). The results indicate that both genomes are maternally inherited, an observation which agrees with the commonly observed pattern of inheritance in angiosperms. They confirm that both chloroplast DNA and mitochondrial DNA can be used as a source of seed-specific markers for the study of the geographic structure of oaks. This is the first report of organelle inheritance within the Fagaceae, an important and widespread tree family.

Locating genes associated with root morphology and drought avoidance in rice via linkage to molecular markers.

Abstract: This research was undertaken to identify and map quantitative trait loci (QTLs) associated with five parameters of rice root morphology and to determine if these QTLs are located in the same chromosomal regions as QTLs associated with drought avoidance/tolerance. Root thickness, root:shoot ratio, root dry weight per tiller, deep root dry weight per tiller, and maximum root length were measured in three replicated experiments (runs) of 203 recombinant inbred lines grown in a greenhouse. The lines were from a cross between indica cultivar Co39 andjaponica cultivar Moroberekan. The 203 RI lines were also grown in three replicated field experiments where they were drought-stressed at the seedling, early vegetative, and late-vegetative growth stage and assigned a visual rating based on leaf rolling as to their degree of drought avoidance/tolerance. The QTL analysis of greenhouse and field data was done using single-marker analysis (ANOVA) and interval analysis (Mapmaker QTL). Most QTLs that were identified were associated with root thickness, root/shoot ratio, and root dry weight per tiller, and only a few with deep root weight. None were reliably associated with maximum root depth due to genotype-by-experiment interaction. Root thickness and root dry weight per tiller were the characters found to be the least influenced by environmental differences between greenhouse runs. Correlations of root parameters measured in greenhouse experiments with field drought avoidance/tolerance were significant but not highly predictive. Twelve of the fourteen chromosomal regions containing putative QTLs associated with field drought avoidance/tolerance also contained QTLs associated with root morphology. Thus, selecting for Moroberekan alleles at marker loci associated with the putative root QTLs identified in this study may be an effective strategy for altering the root phenotype of rice towards that commonly associated with drought-resistant cultivars.

The use of microsatellite DNA markers for soybean genotype identification

Abstract: Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT)n/(TA)n and (ATT)n/(TAA)n that are composed of tandemly repeated 2–5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, “n”, results in PCR product length differences. The SSR alleles present at three (AT)n/(TA)n and four (ATT)n/(TAA)n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.

Structural evolution of wheat chromosomes 4A, 5A, and 7B and its impact on recombination

Abstract: The construction of comparative genetic maps of chromosomes 4Am and 5Am of Triticum monococcum and chromosomes of homoeologous groups 4, 5 and 7 of T. aestivum has provided insight into the evolution of these chromosomes. The structures of chromosomes 4A, 5A and 7B of modern-day hexaploid bread wheat can be explained by a 4AL/5AL translocation that occurred at the diploid level and is present both in T. monococcum and T. aestivum. Three further rearrangements, a 4AL/7BS translocation, a pericentric inversion and a paracentric inversion, have taken place in the tetraploid progenitor of hexaploid wheat. These structural rearrangements and the evolution of chromosomes 4A, 5A and 7B of bread wheat are discussed. The presence of the 4AL/5AL translocation in several Triticeae genomes raises two questions — which state is the more primitive, and is the translocation of mono- or poly-phylogenetic origin?

RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat.

Abstract: A population of single chromosome recombinant lines was developed from the cross between a frost-sensitive, vernalization-insensitive substitution line, 'Chinese Spring' (Triticum spelta 5A) and a frost-tolerant, vernalization-sensitive line, 'Chinese Spring' ('Cheyenne' 5A), and used to map the genes Vrn1 and Fr1 controlling vernalization requirement and frost tolerance, respectively, relative to RFLP markers located on this chromosome. The Vrn1 and Fr1 loci were located closely linked on the distal portion of the long arm of 5AL, but contrary to previous observations, recombination between them was found. Three RFLP markers, Xpsr426, Xcdo504 and Xwg644 were tightly linked to both. The location of Vrn1 suggests that it is homoeologous to other spring habit genes in related species, particularly the Sh2 locus on chromosome 7 (5H) of barley and the Sp1 locus on chromosome 5R of rye.

Estimation, variance and optimal sampling of gene diversity

Abstract: An extension of Nei's analysis of diversity in a subdivided population is proposed for a haploid locus. The differentiation G STbecomes a natural extension of Wright's F STand generalizes Weir and Cockerham's parameter of co-ancestry by relaxing the assumption of identical correlation for all the alleles. Inter- and intrapopulation variances of the estimated diversities and differentiation are derived. Finally, the optimal sampling strategy for measuring G STwhen a fixed number of individuals can be analysed is considered. It is shown that, at a given locus, there is a unique sample size per population which yields the smallest variance of G ST,regardless of the number of populations studied. These theoretical developments are illustrated with an analysis of chloroplast DNA diversity in a forest tree. The results emphasize the necessity of sampling many populations, rather than many individuals per population, for an accurate measurement of the subdivision of gene diversity at a single locus.

Estimation, variance and optimal sampling of gene diversity II. Diploid locus.

Abstract: Nei's analysis of diversity at a diploid locus is extended to a population subdivided into a large number of subpopulations. The diversities and the heterozygotes frequency are defined with respect to the total population and unbiasedly estimated in a two-stage random cluster sampling. The fixation indices F IS, F IT andF ST are derived, then inter- and intra-population variances of the estimated parameters are studied. We show that there is a unique sample size per population which yields the best accuracy in estimatingF ST and F IS, respectively, at a given locus. These results are illustrated with an analysis of DNA diversity in a forest tree and compared to those obtained under the Hardy-Weinberg assumption.

Identification of quantitative trait loci (QTLs) for heading date and plant height in cultivated rice ( Oryza sativa L.)

Abstract: ‘Lemont’ and ‘Teqing’ are both semidwarf rice varieties that differ in heading date by only 6 days. However, when ‘Lemont’ and ‘Teqing’ are crossed there is transgressive segregation for both heading date (HD) and plant height (PH). By testing 2418 F4 lines with 113 well-distributed RFLP markers, we identified and mapped chromosomal regions that were largely responsible for this transgressive segregation. QHd3a, a QTL from ‘Lemont’ that gives 8 days earlier heading, was identified on chromosome 3 approximately 3 cM from the marker RG348. Another QTL with a large effect, QHd8a, which gives 7 days earlier heading, was identified on chromosome 8 of ‘Teqing’ between RG20 and RG1034. Along with a QTL, QHd9a with a phenotypic effect of 3.5 days, these genomic regions collectively explain 76.5% of the observed phenotypic variance in heading date. Four QTLs which altered plant height from 4 to 7 cm were also mapped; these collectively explain 48.8% of the observed phenotypic variation in plant height. None of the QTLs for plant height mapped to chromosome 1, the location of the semidwarf gene sd-1. All three of the HD loci mapped to approximately the same genomic locations as PH QTLs, and in all cases, there was a reduction in height of approximately 1 cm for every day of earlier heading. The correspondence between the HD and some of the PH loci suggests that genes at these chromosome locations may have pleiotropic effects on both HD and PH. The observed heterosis in the F1 plants for HD can be largely explained by the dominance for earliness of the identified HD loci and distribution of earlier heading alleles in the parents. However, overdominance observed at one of the PH QTL may, at least in part, be responsible for the observed heterosis in PH.

Genotype-by-environment interaction in genetic mapping of multiple quantitative trait loci.

Abstract: The interval mapping method is widely used for the genetic mapping of quantitative trait loci (QTLs), though true resolution of quantitative variation into QTLs is hampered with this method. Separation of QTLs is troublesome, because single-QTL is models are fitted. Further, genotype-by-environment interaction, which is of great importance in many quantitative traits, can only be approached by separately analyzing the data collected in multiple environments. Here, we demonstrate for the first time a novel analytic approach (MQM mapping) that accommodates both the mapping of multiple QTLs and genotype-by-environment interaction. MQM mapping is compared to interval mapping in the mapping of QTLs for flowering time in Arabidopsis thaliana under various photoperiod and vernalization conditions.

Mapping the genome of rapeseed (Brassica napus L.). I. Construction of an RFLP linkage map and localization of QTLs for seed glucosinolate content.

Abstract: A linkage map of the rapeseed genome comprising 204 RFLP markers, 2 RAPD markers, and 1 phenotypic marker was constructed using a F1 derived doubled haploid population obtained from a cross between the winter rapeseed varieties 'Mansholt's Hamburger Raps' and 'Samourai'. The mapped markers were distributed on 19 linkage groups covering 1441 cM. About 43% of these markers proved to be of dominant nature; 36% of the mapped marker loci were duplicated, and conserved linkage arrangements indicated duplicated regions in the rapeseed genome. Deviation from Mendelian segregation ratios was observed for 27.8% of the markers. Most of these markers were clustered in 7 large blocks on 7 linkage groups, indicating an equal number of effective factors responsible for the skewed segregations. Using cDNA probes for the genes of acyl-carrier-protein (ACP) and β-ketoacyl-ACP-synthase I (KASI) we were able to map three and two loci, respectively, for these genes. The linkage map was used to localize QTLs for seed glucosinolate content by interval mapping. Four QTLs could be mapped on four linkage groups, giving a minimum number of factors involved in the genetic control of this trait. The estimated effects of the mapped QTLs explain about 74% of the difference between both parental lines and about 61.7 % of the phenotypic variance observed in the doubled haploid mapping population.

Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew.

Abstract: Several Triticum aestivum L.-Haynaldia villosa disomic 6VS/6AL translocation lines with powdery mildew resistance were developed from the hybridization between common wheat cultivar Yangmai 5 and alien substitution line 6V(6A). Mitotic and meiotic C-banding analysis, aneuploid analysis with double ditelosomic stocks, in situ hybridization, as well as the phenotypic assessment of powdery mildew resistance, were used to characterize these lines. The same translocated chromosome, with breakpoints near the centromere, appears to be present in all the lines, despite variation among the lines in their morphology and agronomic characteristics. The resistance gene, conferred by H. villosa and designated as Pm21, is a new and promising source of powdery mildew resistance in wheat breeding.

Genetic diversity in European and Mediterranean faba bean germ plasm revealed by RAPD markers.

Abstract: Broadening of the genetic base and systematic exploitation of heterosis in faba bean requires reliable information on the genetic diversity in the germ plasm. Three groups of faba bean inbred lines were examined by means of RAPDs (random amplified polymorphic DNAs) assays: 13 European small-seeded lines, 6 European large-seeded lines, and 9 Mediterranean lines. Out of 59 primers, 35 were informative and yielded 365 bands, 289 of which were polymorphic with a mean of 8.3 bands per primer. Monomorphic bands were omitted from the analyses and genetic distances (GD) were estimated via the coefficient of Jaccard. The mean GD among the European small-seeded lines was significantly greater than those among the lines of the other two groups. Repeatability of GD estimates was high. Cluster (UPGMA) and principal coordinate analyses identified European small-seeded lines and Mediterranean lines as distinct groups with European large-seeded lines located in between. The results are in harmony with published archaeobotanical findings. We conclude that RAPDs are useful for classification of germ plasm and identification of divergent heterotic groups in faba bean.

Characterization of highly variable (GA/CT) n microsatellites in the bur oak, Quercus macrocarpa

Abstract: The objective of this study was to ascertain the usefulness of polymerase chain reaction (PCR)-based microsatellite analysis for studying pollination and parentage in a wind-pollinated temperate tree. A small insert genomic library of the bur oak (Quercus macrocarpa) was constructed and screened for the presence of (CA/GT) n and (GA/CT) n repeats. The proportion of positive clones yielded estimates of 3×105 such dinucleotide repeats per genome, roughly comparable to abundances reported in other eukaryotic genomes. Thirteen positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) n motif was more abundant than the (CA/GT) n motif in these clones. The (GA/CT) n repeats also showed longer average repeat length (mean n=16.2 versus 7.3), suggesting that they are better candidates for yielding polymorphic genetic markers in oak genomes. Indeed, a survey of adult bur oaks and offspring in a small stand in northern Illinois at 3 of these (GA/CT) n microsatellite loci revealed Mendelian inheritance and extremely high levels of polymorphism, with the number of alleles at each locus ranging from 11–20 and heterozygosity ranging from 0.66 to 0.75. These results, indicating that (GA/CT) n microsatellites are both abundant and highly polymorphic in the bur oak genome, suggest that such genetic markers have tremendous potential for applications for studies of parentage, pollination and dispersal in temperate trees.

Application of two microsatellite sequences in wheat storage proteins as molecular markers.

Abstract: In eukaryotes, tandem arrays of simple-sequence repeat sequences can find applications as highly variable and multi-allelic PCR-based genetic markers. In hexaploid bread wheat, a large-genome inbreeding species with low levels of RFLP, di- and trinucleotide tandem repeats were found in 22 published gene sequences, two of which were converted to PCR-based markers. These were shown to be genome-specific and displayed high levels of variation. These characteristics make them especially suitable for intervarietal breeding applications.

Maximum-likelihood models for mapping genetic markers showing segregation distortion. 2. F2 populations.

Abstract: A maximum-likelihood approach is used in order to estimate recombination fractions between markers showing segregation distortion in backcross populations. It is assumed that the distortions are induced by viability differences between gametes or zygotes due to one or more selected genes. We show that Bailey's (1949) estimate stays consistent and efficient under more general assumptions than those defined by its author. This estimate should therefore be used instead of the classical maximum-likelihood estimate. The question of detection of linkage is also discussed. We show that the order of markers on linkage groups may be affected by segregation distortion.

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

Abstract: 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 ‘Lemont’x‘Teqing’ 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.

Genetic mapping of soybean cyst nematode race-3 resistance loci in the soybean PI 437.654.

Abstract: Resistance to the soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is difficult to evaluate in soybean [Glycine max (L.) Merr.] breeding. PI 437.654 has resistance to more SCN race isolates than any other known soybean. We screened 298 F6∶7 recombinant-inbred lines from a cross between PI 437.654 and ‘BSR101’ for SCN race-3 resistance, genetically mapped 355 RFLP markers and the I locus, and tested these markers for association with resistance loci. The Rhg 4 resistance locus was within 1 cM of the I locus on linkage group A. Two additional QTLs associated with SCN resistance were located within 3cM of markers on groups G and M. These two loci were not independent because 91 of 96 lines that had a resistant-parent marker type on group G also had a resistant-parent marker type on group M. Rhg 4 and the QTL on G showed a significant interaction by together providing complete resistance to SCN race-3. Individually, the QTL on G had greater effect on resistance than did Rhg 4, but neither locus alone provided a degree of resistance much different from the susceptible parent. The nearest markers to the mapped QTLs on groups A and G had allele frequencies from the resistant parent indicating 52 resistant lines in this population, a number not significantly different from the 55 resistant lines found. Therefore, no QTLs from PI 437.654 other than those mapped here are expected to be required for resistance to SCN race-3. All 50 lines that had the PI 437.654 marker type at the nearest marker to each of the QTLs on groups A and G were resistant to SCN race-3. We believe markers near to these QTLs can be used effectively to select for SCN race-3 resistance, thereby improving the ability to breed SCN-resistant soybean varieties.

Genetic mapping of QTLs controlling vegetative propagation in Eucalyptus grandis and E. urophylla using a pseudo-testcross strategy and RAPD markers.

Abstract: We have extended the combined use of the “pseudo-testcross” mapping strategy and RAPD markers to map quantitative trait loci (QTLs) controlling traits related to vegetative propagation in Eucalyptus. QTL analyses were performed using two different interval mapping approaches, MAPMAKER-QTL (maximum likelihood) and QTL-STAT (non-linear least squares). A total of ten QTLs were detected for micropropagation response (measured as fresh weight of shoots, FWS), six for stump sprouting ability (measured as # stump sprout cuttings, #Cutt) and four for rooting ability (measured as % rooting of cuttings, %Root). With the exception of three QTLs, both interval-mapping methods yielded similar results in terms of QTL detection. Discrepancies in the most likely QTL location were observed between the two methods. In 75% of the cases the most likely position was in the same, or in an adjacent, interval. Standardized gene substitution effects for the QTLs detected were typically between 0.46 and 2.1 phenotypic standard deviations (σp), while differences between the family mean and the favorable QTL genotype were between 0.25 and 1.07 (σp). Multipoint estimates of the total genetic variation explained by the QTLs (89.0% for FWS, 67.1 % for#Cutt, 62.7% for %Root) indicate that a large proportion of the variation in these traits is controlled by a relatively small number of major-effect QTLs. In this cross, E. grandis is responsible for most of the inherited variation in the ability to form shoots, while E. urophylla contributes most of the ability in rooting. QTL mapping in the pseudo-testcross configuration relies on withinfamily linkage disequilibrium to establish marker/trait associations. With this approach QTL analysis is possible in any available full-sib family generated from undomesticated and highly heterozygous organisms such as forest trees. QTL mapping on two-generation pedigrees opens the possibility of using already existing families in retrospective QTL analyses to gather the quantitative data necessary for marker-assisted tree breeding.

Identification and characterisation of sugarcane intergeneric hybrids, Saccharum officinarum x Erianthus arundinaceus, with molecular markers and DNA in situ hybridisation.

Abstract: Molecular markers were used to characterise sugarcane intergeneric hybrids between S. officinarum and E. arundinaceus. Very simple diagnostic tools for hybrid identification among the progeny were derived from isozyme electrophoresis and a sequence-tagged PCR. Two enzyme systems (GOT and MDH B) and PCR amplification revealing spacer-size variation in the 5s-rDNA cluster were found most convenient. Specific characterisation of the two genomic components was possible using RFLP and in situ hybridisation. The strong molecular differentiation between S. officinarum and E. arundinaceus allows the identification of numerous Erianthus-specific RFLP bands in the hybrids. Genomic DNA in situ hybridisation allows for the differentiation of the chromosomes contributed by S. officinarum and E. arundinaceus in chromosome preparations of the hybrids. In situ hybridisation with the 18s-5.8s-25s rDNA probe highlights the basic chromosome numbers in the two parental species. The potential of these techniques to monitor the Erianthus genome during the introgression process is discussed.

Molecular phytogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes.

Abstract: We investigated the molecular phylogeny of conifers using restriction endonuclease fragment length polymorphism of six polymerase chain reaction-amplified chloroplast genes — frxC, rbcL, psbA, psbD, trnK, and 16S. We detected 227 total site changes among species, representing 23, 26, 38, 48, 67, and 25 site changes in frxC, psbA, psbD, rbcL, trnK and 16S, respectively. The mean nucleotide substitution was 10.75% (SD 0.573) among species in five families. Forty maximally parsimonious trees were obtained using the Wagner parsimony method, and a 50% majority-rule consensus tree was obtained from them. Data analysis produced similar basic patterns when both the Wagner parsimony and the neighbor-joining methods were applied, and the main lineages were clearly separated. Taxaceae and Cephalotaxaceae species were used as the out-groups when applying Wagner parsimony methods. With the Wagner method, the consistency index was 0.510, the retention index was 0.879, and tree length was 435 steps. Our results indicated that Cupressaceae and Taxodiaceae are closely related families and that Sciadopitys verticillata is the basal lineage of Cupressaceae and Taxodiaceae. The neighbor-joining tree is similar to the 50% majority-rule consensus of the 40 Wagner parsimony trees except for the position of Keteleeria daversifolia, the Picea and Cedrus group, and the divergence within Cupressaceae.

Abundance and characterization of simple-sequence repeats (SSRs) isolated from a size-fractionated genomic library of Brassica napus L. (rapeseed).

Abstract: A size-fractionated library of Brassica napus L. (rapeseed), composed of 15000 clones, was screened for the presence of GA-, CA-, and GATA-simple-sequence repeats (SSRs). GA-SSRs were four- and five-fold more abundant than CA- and GATA-SSRs, respectively, and present at a frequency of approximately one SSR for every 100 kb of DNA. Following the sequencing of 124 positive clones, primer pairs were designed and evaluated for seven selected SSRs. Products were amplified in an array of individuals of B. napus, B. oleracea and B. rapa, demonstrating that the seven SSRs were conserved among species. Two SSRs were polymorphic. Among 11 accessions, the dinucleotide (GA)-repeat, B.n.9A, yielded 12 fragments, while the tetranucleotide-repeat (GATA), B.n.6A2, revealed two fragments. Automated, fluorescence-based detection of polyacrylamide gels has been employed to simultaneously increase throughput, reduce unit cost, improve analytical resolution, and expedite data acquisition of SSR analysis. Though initial financial investment and technical capabilities may prevent some from directly employing our documented approach, SSR analysis warrants further investigation as a tool in genetic studies for enhancing both the conservation and utilization of genetic resources.

Identification of molecular markers linked to the Agropyron elongatum -derived leaf rust resistance gene Lr24 in wheat

Abstract: The objective of this study was to identify molecular markers linked to the wheat leaf rust resistance gene Lr24 derived from Agropyron elongatum (3DL/3Ag translocation). Two near isogenic lines (NILs), ‘Arina’ and Lr24/7* “Arina”, were screened for polymorphism at the DNA level with 115 RFLP probes. Twenty-one of these probes map to the homoeologous group 3. In addition, 360 RAPD primers were tested on the NILs. Six RFLP probes showed polymorphism between the NILs, and 11 RAPD primers detected one additional band in the resistant NIL. The genetic linkage of the polymorphic markers with Lr24 was tested on a segregating F2 population (150 plants) derived from a cross between the leaf rust resistant Lr24/7* “Arina” and the susceptible spelt (Triticum spelta) variety ‘Oberkulmer’. All 6 RFLP markers were completely linked to Lr24: one was inherited as a codominant marker (PSR1205), one was in coupling phase (PSR1203) and 4 were in repulsion phase (PSR388, PSR904, PSR931, PSR1067) with Lr24. The localization of these probes on chromosome 3D was confirmed by nulli-tetrasomic analysis. Distorted genotypic segregation was found for the Codominant RFLP marker PSR1205. This distortion can be explained by the occurrence of hemizygous plants. One of the 11 RAPD markers (OPJ-09) also showed complete linkage to theLr24 resistance gene. The polymorphic RAPD fragment was cloned and sequenced. Specific primers were synthesized, and they produced an amplification product only in the resistant plants. This specific marker allows a reliable and rapid screening of a large number of genotypes in practical breeding. Analysis of 6 additional lines containing Lr24 revealed that 3 lines have a smaller chromosomal segment of A. elongatum than lines derived from ‘Agent’, a commonly used gene donor for the Lr24 resistance gene.

Detection of quantitative trait loci for agronomic, yield, grain and disease characters in spring barley (Hordeum vulgare L.).

Abstract: Quantitative trait loci (QTLs) have been revealed for characters in a segregating population from a spring barley cross between genotypes adapted to North-West Europe. Transgressive segregation was found for all the characters, which was confirmed by the regular detection of positive and negative QTLs from both parents. A QTL for all the agronomic, yield and grain characters measured except thousand grain weight was found in the region of the denso dwarfing gene locus. There were considerable differences between the location of QTLs found in the present study and those found in previous studies of North American germ plasm, revealing the diversity between the two gene pools. Thirty-one QTLs were detected in more than one environment for the 13 characters studied, although many more were detected in just one environment. Whilst biometrical analyses suggested the presence of epistasis in the genetic control of some characters, there was little evidence of interactions between the QTLs apart from those associated with yield. QTLs of large effect sometimes masked the presence of QTLs of smaller effect.

Impact of scoring error and reproducibility RAPD data on RAPD based estimates of genetic distance.

Abstract: RAPD band reproducibility and scoring error were evaluated for RAPDs generated by 50 RAPD primers among ten snap bean (Phaseolus vulgaris L.) genotypes. Genetic distances based on different sets of RAPD bands were compared to evaluate the impact of scoring error, reproducibility, and differences in relative amplification strength on the reproducibility of RAPD based genetic distance estimates. The measured RAPD data scoring error was 2%. Reproducibility, expressed as the percentage of RAPD bands scored that are also scored in replicate data, was 76%. The results indicate that the probability of a scored RAPD band being scored in replicate data is strongly dependent on the uniformity of amplification conditions between experiments, as well as the relative amplification strength of the RAPD band. Significant improvement in the reproducibility of scored bands and some reduction in scoring error was achieved by reducing differences in reaction conditions between replicates. Observed primer variability for the reproducibility of scored RAPDs may also facilitate the selection of primers, resulting in dramatic improvements in the reproducibility of RAPD data used in germplasm studies. Variance of genetic distances across replicates due to sampling error was found to be more than six times greater than that due to scoring error for a set of 192 RAPD bands. Genetic distance matrices computed from the RAPD bands scored in replicated data and RAPD bands that failed to be scored in replicated data were not significantly different. Differences in the ethidium bromide staining intensity of RAPD bands were not associated with significant differences in resulting genetic distance matrices. The assumption of sampling error as the only source of error was sufficient to account for the observed variation in genetic distance estimates across independent sets of RAPD bands.

Development of a consensus linkage RFLP map of cultivated sunflower (Helianthus annuus L.).

Abstract: This paper provides the first description of a consensus map of the cultivated sunflower genome (Helianthus annuus L., n=17 chromosomes), based on RFLP. A total of 180 probe-enzyme combinations were mapped on at least one of five segregating progenies (three F2 and two BC1 populations), revealing 237 loci that did not show any distortion of segregation. The consensus linkage map obtained with these loci covers 1150 cM and consists of 16 linkage groups of more than 20 cM, 7 groups of less than 20 cM and 18 unlinked loci. The mean distance between loci is 7 cM, but in some regions intervals of 20 cM remain. Genotypic and gametic segregation distortions affect about 7% of loci. It was found that 25% of the probes mapped using several different restriction enzymes or that on different progenies they revealed 2 or more loci.

Comparison of rapeseed cultivars and resynthesized lines based on allozyme and RFLP markers.

Abstract: It has frequently been suggested to use the resynthesis of rapeseed (Brassica napus) from B. campestris and B. oleracea to broaden its genetic base. The objective of the present study is twofold: (1) to compare the genetic variation within resynthesized rapeseed with a world-wide collection of oilseed rape cultivars, and (2) to compare genetic distances estimated from RFLP markers with distances estimated from a relatively small number of allozyme markers. We investigated 17 resynthesized lines and 24 rapeseed cultivars. Genetic distances were estimated either based on the electrophoresis of seven allozymes, with a total of 38 different bands, or based on RFLP data of 51 probe/enzyme combinations, with a total of 355 different bands. The results of allozyme and RFLP analyses agreed reasonably well. Genetic distances, estimated from two independent sets of RFLP data with 25 and 26 probe/enzyme combinations respectively, were highly correlated; hence about 50 RFLP markers are sufficient to characterize rapeseed material with a large genetic diversity. The cultivars were clustered into three groups: (1) spring rapeseed of European and Northern American origin, (2) winter rapeseed of European and Northern American origin, and (3) rapeseed of Asian origin. Several of the resynthesized rapeseed lines were similar to European winter rapeseed cultivars, whereas others had quite unique patterns. It is concluded, that resynthesized rapeseed is a valuable source for broadening the genetic variation in present breeding material of Brassica napus. However, different lines differ widely in their suitability for this purpose.

Localization of quantitative trait loci (QTL) for agronomic important characters by the use of a RFLP map in barley (Hordeum vulgare L.).

Abstract: Two hundred and fifty doubled haploid lines were studied from a cross between two 2-row winter barley varieties. The lines were evaluated for several characters in a field experiment for 3 years on two locations with two replications. From a total of 431 RFLP probes 50 were found to be polymorphic and subsequently used to construct a linkage map. Quantitative trait loci (QTLs) were determined and localized for resistance against Rhynchosporium secalis and Erysiphe graminis, for lodging, stalk breaking and ear breaking tendency, for the physical state before harvest, plant height, heading date, several kernel parameters and kernel yield. The heritability of the traits ranged from 0.56 to 0.89. For each trait except for kernel thickness, QTLs have been localized that explain 5–52% of the genetic variance. Transgressive segregation occurred for all of the traits studied.

Centromeric repetitive DNA sequences in the genus Brassica.

Abstract: Representatives of two major repetitive DNA sequence families from the diploid Brassica species B campestris and B oleracea were isolated, sequenced and localized to chromosomes by in situ hybridization Both sequences were located near the centromeres of many chromosome pairs in both diploid species, but major sites of the two probes were all on different chromosome pairs Such chromosome specificity is unusual for plant paracentromeric repetitive DNA Reduction of stringency of hybridization gave centromeric hybridization sites on more chromosomes, indicating that there are divergent sequences present on other chromosomes In tetraploid species derived from the diploids, the number of hybridization sites was different from the sum of the diploid ancestors, and some chromosomes had both sequences, indicating relatively rapid homogenization and copy number evolution since the origin of the tetraploid species

Identification of genomic regions affecting plant height in sorghum and maize.

Abstract: The objective of this study was to use restriction fragment length polymorphisms (RFLPs) to determine the genetic location and effects of genomic regions controlling plant height in sorghum. F2 plants (152) from the cross CK60 x PI229828 were used. Genomic and cDNA clones (106) identified 111 loci distributed among ten linkage groups covering 1299 cM. Interval mapping identified four regions, each in a separate linkage group. These regions may correspond to loci (dw) previously identified by alleles with qualitative effects. Also, these regions identified in sorghum may be orthologous to those previously reported for plant height in maize. Gene effects and gene action varied among genomic regions. In each region, PI229828 alleles resulted in increased plant height. Each region accounted for 9.2–28.7% of the phenotypic variation. Positive, additive effects ranged from 15 to 32cm. Tallness was dominant or overdominant and conferred by alleles from PI229828 for three quantitative trait loci (QTL). At the fourth QTL, PI229828 contributed to increased plant height, but short stature was partially dominant. One digenic interaction was significant. The presence of a PI229828 allele at one region diminished the effects of the other region. A multiple model indicated that these four regions collectively accounted for 63.4% of the total phenotypic variation. The utility of this information for germplasm conversion through backcross breeding is discussed.