Comparing the distribution of RAPD and RFLP markers in a high density linkage map of sugar beet.

The distribution of RAPD markers was compared with that of RFLP markers in a high density linkage map of sugar beet. The same mapping population of 161 F2 individuals was used to generate all the marker data. The total map comprises 160 RAPD and 248 RFLP markers covering 508 cM. Both the RAPD and the RFLP markers show a high degree of clustering over the nine linkage groups. The pattern is compatible with a strong distal localization of recombination in the sugar beet. It leads generally to one major cluster of markers in the centre of each linkage group. In regions of high marker density, dominant RAPD markers present in either linkage phase and codominant RFLP markers are subclustered relative to each other. This phenomenon is shown to be attributable to: (i) effects of the mapping procedure when dominant and codominant data are combined, (ii) effects of the mapping procedure when dominant data in both linkage phases are combined, and (iii) genuine differences in the way RAPD and RFLP markers are recruited.     

The detection of somaclonal variants of beet using RAPD

Summary Plantlets were regenerated by adventitious shoot budding in tissue culture from leaf explants of a single genotype of sugar beet. DNA was extracted from the parental plant and from 120 regenerants. RAPD analysis was carried out using five decanucleotide primers; 4,557 RAPD marker bands were examined and two polymorphisms were observed. Thirty secondary regenerants were then derived, using the same tissue culture technique, from thirty of the primary regenerants. Again RAPD analysis was employed and a single band polymorphism was observed out of 1,050 bands examined. The overall frequency of detection of somaclonal polymorphisms using RAPD (3 in 5,607 = 0.05%) is similar to frequencies previously reported using isozyme and RFLP technologies.Abbreviations BAP 6-benzylaminopurine - IBA indole-butyric acid - MS Murashige and Skoog medium (Murashige and Skoog, 1962) - RAPD Random Amplification of Polymorphic DNA     

Analysis of Genetic Diversity in Napier Grass (Pennisetum purpureum Schum) as Detected by RAPD and ISSR Markers

Randomly Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers were used to detect the DNA polymorphism among thirty Napier grass collections of wide geographical distribution. A total of 20 RAPD and 10 ISSR primers were used in this study. RAPD analysis produced 222 fragments of which, 195 were polymorphic with an average of 9.75 polymorphic fragments per primer. The ten ISSR primers produced a total of 98 fragments out of which 88 were polymorphic accounting for 89.8%. The Mantel test between two similarity matrices of the markers revealed a low correlation (r = 0.33) indicating low correspondence between polymorphism brought out by the two marker systems. The UPGMA clustering of genotypes eventhough was not similar when RAPD and ISSR derived dendrograms were compared, but showed a greater correspondence with geographical identity in both the marker systems employed. This correspondence was also evident when data from both the RAPD and ISSR markers were combined. The implications on collection and breeding of this important forage grass had been discussed.     

Linkage among the R, Y and BI loci in table beet

The primary pigments in table beet are the betalains, which are comprised of the red-violet betacyanins and the yellow betaxanthins. The presence of dominant alleles at two linked loci (R and Y) condition the qualitative production of betalain pigment in the beet plant. Red-pigmented roots are observed only in the presence of dominant alleles at both the R and Y loci, while white roots are conditioned by recessive alleles at the Y locus, and yellow roots by the genotype rrY-. A newly described gene ’blotchy’ (bl) conditions a blotchy or irregular pigment patterning in either red or yellow roots. The objective of the present investigation was to characterize the linkage relationships between the R and Y lociand the bl gene by evaluating segregating progenies developed from a series of matings of colored and white table beets. Due to epistatic interactions among the R, Y, and bl loci, algorithms for estimating linkage were developed using maximum-likelihood estimators for each cross. The two-point linkage estimate between the R and Y loci pooled over eight crosses was 7.4±1.7 cM. Segregation data indicated the bl gene is linked to the R and Y loci.The recombination fraction between R and bl was estimated from a pooled sample of four crosses at 16.7±10.8 cM. The most-likely gene order was R-Y-bl. These data demonstrate that the bl gene is a third locus conditioning betalain pigment production in table beet. The R-Y-bl genomic region is therefore important in the genetic control of betalain biosynthesis in table beet. Received: 18 May 1999 / Accepted: 29 August 1999     

RAPD-based genetic linkage maps of Tribolium castaneum.

A genetic map of the red flour beetle (Tribolium castaneum) integrating molecular with morphological markers was constructed using a backcross population of 147 siblings. The map defines 10 linkage groups (LGs), presumably corresponding to the 10 chromosomes, and consists of 122 randomly amplified polymorphic DNA (RAPD) markers, six molecular markers representing identified genes, and five morphological markers. The total map length is 570 cM, giving an average marker resolution of 4.3 cM. The average physical distance per genetic distance was estimated at 350 kb/cM. A cluster of loci showing distorted segregation was detected on LG9. The process of converting RAPD markers to sequence-tagged site markers was initiated: 18 RAPD markers were cloned and sequenced, and single-strand conformational polymorphisms were identified for 4 of the 18. The map positions of all 4 coincided with those of the parent RAPD markers.     

Populations composed entirely of hybrid colonies: bidirectional hybridization and polyandry in harvester ants

In eusocial Hymenoptera, haplodiploid life cycles, obligate sterile castes, and polyandry may facilitate selection for hybridization. We analyzed a broad hybrid zone between the ecologically distinct seed‐harvester ants Pogonomyrmex occidentalis (Cresson) and Pogonomyrmex maricopa (Wheeler) using mitochondrial (mt)DNA sequence data, eight morphological markers, and 14 random amplified polymorphic DNA (RAPD) markers. Average mtDNA sequence divergence among parental species was 11.34%, indicating secondary contact. RAPD markers were significantly correlated with morphological variation, confirming the interspecific hybrid origin of all morphologically putative hybrid colonies. A morphological hybrid index indicates an abundance of both F₁ hybrids and parental morphotypes within colonies. Individual character frequencies plotted against distance show coincident and concordant clines, suggesting little to no introgression. The structure of the hybrid zone is two‐fold. Within the western region, stark reversals in character frequencies coincide with overt soil differences, indicating a mosaic hybrid zone structure. The eastern region is a riparian habitat where four adjacent populations were composed entirely of hybrid colonies. These habitat associations suggest that hybrid worker genomes permit dispersal into intermediate environments that select against one or both parental species. The present study suggests that, in addition to retaining reproductive compatibility, ecologically distinct species of ants may generate hybrid colonies maintained by environmental selection. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 320–336.     

DNA markers linked to a T10 loose smut resistance gene in wheat (Triticum aestivum L.).

Screening for loose smut resistance in wheat is difficult. Selecting lines with DNA markers linked to loose smut resistance would be more reliable and less costly. Molecular markers linked to a race T10 loose smut resistance gene were identified using a F6 single seed descent segregating population. A RAPD marker and a RFLP marker were located on opposite flanks of the resistance gene and were shown to be loosely linked. The RAPD marker was converted to a user friendly polymorphic SCAR marker that represented a single genetically defined locus in hexaploid wheat. Using these two bracketing markers simultaneously, the error rate for T10 resistance selection due to crossing-over was reduced to 4%. These markers can be used for a faster and more reliable selection of T10 resistant plants than previous conventional loose smut ratings.     

In situ and Ex situ adsorption and recovery of betalains from hairy root cultures of Beta vulgaris

Various adsorbents were screened for in situ recovery of betalain pigments effluxed from hairy root cultures of red beet, Beta vulgaris. Alumina/silica (1:1) appeared ideal, showing in situ adsorption of 97% in a unit time of 30 min accounting for in situ recovery of 71.39% of the total betalaine effluxed. Other adsorbents such as Amberlite series (XAD-2 and -4), cyclodextrin, maltodextrin, dextrin white, and starches such as wheat starch and corn starch exhibited very poor in situ adsorption properties. Pretreatment of adsorbents with methanol significantly improved the adsorption capacities of some of the adsorbents, with a highest adsorption of 97.2% for alumina followed by alumina/silica (1:1) and higher adsorption by XAD-2 and -4. Complete in situ adsorption equilibrium was reached in 20 min for a solution containing 2.5 mg mL(-)(1) of betalain in adsorbents alumina, silica, and a mixture of alumina and silica. In situ betalain adsorption parameters for alumina/silica were determined using the Langmuir isotherm model where the adsorption capacity was found to be 0.174 mg g(-)(1) and the adsorption energy was 0.9 at pH 5.5 and 25 degrees C. Desorption of pigments from the adsorbents was invariably highest in poor adsorbents, indicating their poor adsorption energy for betalaines. Similarly, recovery by desorption was low in those adsorbents having high adsorption capacity, indicating that adsorbents such as activated ones with highest adsorption capacity with zero desorption property were unsuitable for the recovery of effluxed pigments. Ex situ recovery of betalain done using various combinations of alumina/silica and processed sand and different column geometries indicated that alumina with processed sand at a 2:1 ratio (w/w) and a minimum column material of 2 cm height and 2 cm diameter was good enough to cause 97% pigment adsorption from a solution containing 1.6 mg mL(-)(1). Desorption and recovery of pigments ex situ from columns were affected by various elution mixtures, where a gradient elution with ascending levels of HCl/ethanol in water resulted in 100% recovery of adsorbed pigments in a significantly lesser volume of eluent in a short period of 1 h. Different pigment flow rates of 0.2, 0.3, and 3.1 mL s(-)(1) through a column of alumina/processed sand indicated that a pigment equilibrium concentration of 0.18 mg mL(-)(1) at flow rates of 0.02 and 0.3 mL s(-)(1) resulted in a breakthrough at 110 and 14 min adsorbing 16.9 and 16.91 mg g(-)(1) betalain, respectively. From the breakthrough curves, the column capacities for respective flow rates were calculated as 8.86 and 9.6 mg g(-)(1), and the higher flow rates resulted in earlier breakthrough with lower capacity. Observations made in the present study are useful to develop a process for the on-line recovery of betalains effluxed from hairy roots.     

Development of coupling-repulsion-phase SCAR markers diagnostic for the sugar beet Rr1 allele conferring resistance to rhizomania

In sugar beet genotypes with the Holly type of resistance to rhizomania, a disease due to infection of the beet necrotic yellow vein virus (BNYVV), the major gene rrl is responsible for resistance. Twelve RAPD markers linked to rrl were selected by BSA and mapped on linkage group IV using a segregating population previously analysed by the same group. Markers F61050 and N9600 were tightly linked, respectively in coupling and repulsion, to the Rrl allele (recombination values of 1.4 cM for both markers). After sequencing the products amplified by F61050 and N9600, new PCR primers were used to generate the two SCAR markers F6 and N9. The simultaneous use of these markers in a PCR reaction allows the correct fingerprinting of rrl rrl, Rrl rrl and Rrl Rrl sugar beet plants in populations segregating for the Holly resistance. In a group of sugar beet elite lines containing the Holly type of rhizomania resistance, SCAR F6 is always present whereas the SCAR N9 fragment is absent. Thus, in marker-assisted selection with coupling-repulsion-phase markers, SCAR F6 can be used in combination with N9, or together with any other RAPD marker linked in repulsion to the Rrl allele.     

Genetic validation and characterization of RAPD markers differentiating black and red spruces: molecular certification of spruce trees and hybrids

 Picea mariana (black spruce) and P. rubens (red spruce) are closely related species which are difficult to differentiate morphologically. RAPD markers differentiating black and red spruces have been previously identified. In the present study, genetic validity of these markers was determined using samples representing range–wide provenances. Their applicability for certifying genetic identity of individual black, red trees and their hybrids from several sympatric and allopatric locations was demonstrated. These diagnostic fragments of both red and black spruce were present at a frequency of over 0.95 in allopatric provenances, but at a lower frequency in some sympatric provenances (0.43–1.00). Natural populations of red spruce exhibiting typical red spruce phenotype contained black spruce diagnostic RAPD fragments and black spruces growing in bogs with typical bog black spruce morphology, contained red spruce-specific RAPD markers. Some major RAPD markers were cloned and sequenced. The results reveal an extremely high degree of identity between the random primer and the primer binding sites on the genome. Amplification of black and red spruce genomic DNA with designed primers flanking the species-diagnostic RAPD markers indicates that most of RAPD markers used to differentiate black spruce from red spruce are not species specific since these sequences were detected in several spruce species using a more sensitive detection method. Received June 17, 2002; accepted August 5, 2002 Published online: February 4, 2003     

Changes in DNA-marker frequencies associated with response to contrasting selection methods in Arabidopsis

 Recurrent selection for specific combining ability (RS-SCA) and S1 family performance (RS-S1) were compared in replicated selection programs initiated from a common C0 base population of Arabidopsis. Three cycles of selection for aerial biomass were completed in each of two replicate programs of each selection method. Response to selection was measured on the basis of per se, S1 progeny, and testcross performance with a common tester. All selection programs improved testcross performance. Testcross gain per cycle in RS-S1 (7.15% cycle^-1) and RS-SCA (5.31% cycle^-1) were not significantly different. Performance of S1 progeny and populations per se significantly improved over cycles of selection using RS-S1 but were unchanged by RS-SCA. Codominant molecular marker-allele frequencies were recorded for each population at 22 polymorphic loci. Trends in marker-allele frequencies were tested by linear regression. Significant trends in marker-allele frequencies pooled over replicate programs were detected at 8 and 7 loci in the RS-S1 and RS-SCA programs, respectively. Marker alleles at 2 loci significantly changed frequency in response to both RS-S1 and RS-SCA programs. Marker alleles at 6 loci significantly changed frequency only in response to RS-SCA. Marker alleles at 6 other loci showed significant linear trends pooled over replicates only in RS-S1. No markers revealed increases in the frequency of different marker alleles within loci using the two selection methods. Possible genetic causes of marker frequency changes are discussed, as well as breeding implications.     

Identification,cloning and sequence analysis of a dwarf genome-specific RAPD marker in rubber tree [<Emphasis Type="Italic">Hevea brasiliensis</Emphasis> (Muell.) Arg.]

High-yielding dwarf clones of Hevea brasiliensis are tolerant to wind damage and therefore useful for high-density planting. The identification of molecular markers for the dwarf character is very important for isolating true-to-type high-yielding dwarf hybrid lines in the early stage of plant breeding programs. We have identified a dwarf genome-specific random amplified polymorphic DNA (RAPD) marker in rubber tree. A total of 115 random oligonucleotide 10-mer primers were used to amplify genomic DNA by PCR, of which 19 primers produced clear and detectable bands. The primer OPB-12 generated a 1.4-kb DNA marker from both natural and controlled F₁ hybrid progenies (dwarf stature) derived from a cross between a dwarf parent and a normal cultivated clone as well as from the dwarf parent; it was absent in other parent (RRII 118). To validate this DNA marker, we analyzed 22 F₁ hybrids (13 with a dwarf stature and nine with a normal stature); the dwarf genome-specific 1.4-kb RAPD marker was present in all dwarf-stature hybrids and absent in all normal-stature hybrids. This DNA marker was cloned and characterized. DNA marker locus specificity was further confirmed by Southern blot hybridization. Our results indicate that Southern blot hybridization of RAPD using probes made from cloned DNA fragments allows a more accurate analysis of the RAPD pattern based on the presence/absence of specific DNA markers than dye-stained gels or Southern blot analysis of RAPD blots using probes made from purified PCR products. Detection of RAPD markers in the hybrid progenies indicates that RAPD is a powerful tool for identifying inherited genome segments following different hybridization methods in perennial tree crops.     

DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.)

The technique of random amplified polymorphic DNA (RAPD) offers a broad range of applications in the investigation of plant genomes. A promising prospect is the use of RAPD products as genetic markers. We have investigated a possible organellar source of fragments in RAPD patterns of total DNA. Two nearly-isogenic lines of cytoplasmic male-sterile and male-fertile sugar beet (Beta vulgaris L.) were subjected to RAPD analysis with six different primers. Total, nuclear, mitochondrial (mt), and chloroplast (cp), DNA from each line were investigated. Reproducible DNA fingerprints could be obtained from both organellar DNAs. Differences in band patterns of mtDNA between cytoplasmic male-sterile and -fertile lines were observed with five out of six primers, whereas different cpDNA patterns were generated by one of the primers. Consequently, the RAPD technique can be used to discriminate between different cytoplasms. Clear evidence is provided for the organellar origin of fragments in genomic (total DNA) RAPD patterns. The consequences of these results for the interpretation of RAPD analyses are discussed.     

Random amplified polymorphic DNA (RAPD) and quantitative trait analyses across a major phylogeographical break in the Mediterranean ragwort Senecio gallicus Vill. (Asteraceae)

Random amplified polymorphic DNA (RAPD) and quantitative trait variation of the widespread and ephemeral Senecio gallicus were surveyed in 11 populations sampled from the Iberian Peninsula and southern France. The aim of the study was to compare population relationships and levels of geographical differentiation with chloroplast (cp) DNA and allozyme variation assessed previously in the same populations. Employing multivariate statistics, a moderate level of intraspecific differentiation was observed among populations from Iberian coastal and inland regions for both RAPDs and quantitative traits. However, RAPDs provided greater resolution in identifying additional population structure within the hypothesized, Pleistocene refugial source area of the species in coastal Iberia. A major part of the geographical subdivision in RAPD and quantitative traits was concordant with the coastal vs. inland divergence as previously inferred from cpDNA haplotype frequencies, but strongly contrasted with the geographical uniformity of the species for allozymes. This concordance across various nuclear and cytoplasmic markers (RAPDs/quantitative traits, cpDNA) suggests that geographical uniformity for allozymes is more attributable to low rates of evolution and/or small genome sampling rather than high rates of pollen dispersal, slow rates of nuclear lineage sorting, or indirect balancing selection. The present study underscores the value of using additional classes of nuclear markers for narrowing the numbers of competing causal hypotheses about intraspecific cpDNA-allozyme discrepancies and their underlying evolutionary processes.     

Isolation and characterization of RAPD-based markers linked to the beet cyst nematode resistance locus (Hs1 pat-1) on chromosome 1 of B. patellaris

A beet cyst nematode (BCN)-resistant telosomic addition of B. patellaris chromosome 1 in B. vulgaris was used to isolate 6 RAPD markers linked to the BCN resistance locus Hs1 ^pat-1. Southern analysis showed that the analyzed RAPD products contain either low-, middle or high-repetitive DNA. The relative positions of the random amplified polymorphic DNA (RAPD) markers and of the restriction fragment length polymorphism (RFLP) loci corresponding to the low-repetitive RAPD products were determined by deletion mapping using a panel of seven nematode-resistant B. patellaris chromosome-1 fragment additions. One RAPD marker, OPB11₈₀₀, was found to be present in two copies on the long arm telosome of B. patellaris chromosome 1. These copies are closely linked to the BCN resistance gene and flank the gene on both sides. On the basis of the nucleotide sequence of OPB11₈₀₀, sequence-tagged site (STS) primers were developed that amplify specific fragments derived from the two OPB11₈₀₀ loci. These STS markers can be used in the map-based cloning of the BCN gene, as they define start and finishing points of a chromosomal walk towards the Hs1 ^pat-1 locus. Two copies of the middle-repetitive OPX2₁₁₀₀ marker were mapped in the same interval of the deletion mapping panel as the resistance gene locus and thereby belong to the nearest markers as yet found for the BCN gene in B. patellaris.     

Identification of SCAR marker linking to longer frond length of <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Laminariales,Phaeophyta) using bulked-segregant analysis

To construct a molecular-marker-assisted selection (MAS) system, research was done on identifying molecular markers linking to longer frond length, a crucial selection index in the breeding of the commercially important seaweed Saccharina japonica. An F₂-segregant population of 92 individuals was obtained by crossing two prominent S. japonica strains. Genomic DNA from ten individuals with the longest frond and ten individuals with the shortest frond in the F₂-segregant population were mixed to create two DNA pools for screening polymorphic markers. In bulked-segregant analysis (BSA), out of 100 random amplified polymorphic DNA (RAPD) primers only two produced three polymorphic RAPD markers between the two DNA pools. In conversion of the three RAPD markers into sequence-characterized amplified region (SCAR) markers, only one was successfully converted into a SCAR marker FL-569 linking to the trait of longer frond. Test of the marker FL-569 showed that 80% of the individuals with longest fronds in a wild population and 87.5% of individuals with the longest fronds in an inbred line “Zhongke No. 2” could be detected by FL-569. Additionally, genetic linkage analysis showed that the SCAR marker could be integrated into the reported genetic map and QTL mapping showed that FL-569 linking to qL1-1. The obtained marker FL-569 will be beneficial to MAS in S. japonica breeding.     

Identification of genetic markers for Korean native cattle (Hanwoo) by RAPD analysis

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of 85.3%. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed 83.0% of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short microsatellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, AAC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found     

山羊经济性状标记辅助选择的遗传效应分析

本研究以辽宁绒山羊、柴达木绒山羊和柴达木山羊3个群体共147只山羊为研究对象,运用PAGE和RAPD技术对山羊的体重、绒产量和绒细度3个性状进行了与标记基因关系的遗传分析,结果表明：EsD2-2型、LAPBB型和PA-32-2型分别为体重、绒产量和绒细度性状的优势标记基因型;可利用标记辅助预测的方法充分利用多基因座标记基因间的互作效应;在体重上,寻找到有显著选择效应的RAPD条带11个,在绒产量和绒细度上分别为9和6个;在多目标性状选择中,CY0818/A0型和OPW19/C1型为体重和绒产量的双重优势RAPD标记,CY0818/G1型为体重和绒细度的双重优势RAPD标记。Abstract: The genetic relationships between economic traits and genetic markers were studied in 147 goats including Chaidamu goat (CS), Chaidamu Cashmere goat (CRS) and Liaoning Cashmere goat (LRS) in Qinghai province, China. CRS was the population of CS×LRS crossbred. The results showed as follows: the selection reaction of these blood protein polymorphisic loci were great, such as EsD, LAP and PA-3; and EsD2-2, LAPBB and PA-32-2 were the superior marker genotypes on body weight ,Cashmere yield and Cashmere fineness respectively by Least Square method. The interaction between marker genotypes at double loci was found frequently, and their ratio between interaction variance component and genetic variance was higher. With the method of marker assisted prediction( MAP), some interaction effect could be used effectively in the crossbreed population. On the aspect of random amplified polymorphic DNA (RAPD), the number of the superior RAPD marker bands were 11 on body weight trait, 9 and 6 RAPD marker bands on Cashmere yield and Cashmere fineness. For multi-goal traits, CY0818/A0 type and OPW19/C1 type were superior RAPD markers of body weight and Cashmere yield, CY0818/G1 type was superior one of body weight and Cashmere fineness.     

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.     

Genetic Linkage Maps of Eucalyptus Grandis and Eucalyptus Urophylla Using a Pseudo-Testcross: Mapping Strategy and Rapd Markers

We have used a ``two-way pseudo-testcross' mapping strategy in combination with the random amplified polymorhic DNA (RAPD) assay to construct two moderate density genetic linkage maps for species of Eucalyptus. In the cross between two heterozygous individuals many single-dose RAPD markers will be heterozygous in one parent, null in the other and therefore segregate 1:1 in their F(1) progeny following a testcross configuration. Meiosis and gametic segregation in each individual can be directly and efficiently analyzed using RAPD markers. We screened 305 primers of arbitrary sequence, and selected 151 to amplify a total of 558 markers. These markers were grouped at LOD 5.0, θ = 0.25, resulting in the maternal Eucalyptus grandis map having a total of 240 markers into 14 linkage groups (1552 cM) and the paternal Eucalyptus urophylla map with 251 markers in 11 linkage groups (1101 cM) (n = 11 in Eucalyptus). Framework maps ordered with a likelihood support >/=1000:1 were assembled covering 95% of the estimated genome size in both individuals. Characterization of genome complexity of a sample of 48 mapped random amplified polymorphic DNA (RAPD) markers indicate that 53% amplify from low copy regions. These are the first reported high coverage linkage maps for any species of Eucalyptus and among the first for any hardwood tree species. We propose the combined use of RAPD markers and the pseudo-testcross configuration as a general strategy for the construction of single individual genetic linkage maps in outbred forest trees as well as in any highly heterozygous sexually reproducing living organism. A survey of the occurrence of RAPD markers in different individuals suggests that the pseudo-testcross/RAPD mapping strategy should also be efficient at the intraspecific level and increasingly so with crosses of genetically divergent individuals. The ability to quickly construct single-tree genetic linkage maps in any forest species opens the way for a shift from the paradigm of a species index map to the heterodox proposal of constructing several maps for individual trees of a population, therefore mitigating the problem of linkage equilibrium between marker and trait loci for the application of marker assisted strategies in tree breeding.