Screening for scab resistance by RAPD markers in cultivars of apple (Malus spp.)

Genetic markers are a much faster and more practical alternative to classical methods for the identification of genes for scab resistance present in different apple cultivars. In our study, 28 scab-resistant cultivars, four wild sources of the resistance genes and 10 susceptible cultivars were screened for the presence of the RAPD fragments OPM18/900, OPD20/600 and OPA15/900, which are reported to be linked to the Vf gene. All three marker fragments were successfully amplified with different protocols in Vf-resistant cultivars including ‘M. floribunda 821’. No marker fragments were amplified in susceptible cultivars, three out of four Va-resistant cultivars, three out of four Vm-resistant cultivars, two Vr-resistant cultivars, ‘Antonovka PI 172612’ and ‘M. pumila R 12740-7A’. All three markers were found in the cv. ‘Nova Easygro’, reported to possess the Vr gene, and the cv. ‘Reglindis’, reported to be Va-resistant. M. atrosanguinea of unknown origin showed the presence of OPD20/600 and OPA 15/900 marker bands. The cvs. ‘Nova Easygro’, ‘Reglindis’ and M. atrosanguinea are probably carriers of the VF gene.     

The V f gene for scab resistance in apple is linked to sub-lethal genes

Summary V _f is the most widely used resistance gene in the breeding for scab resistant apple cultivars. Distorted segregation ratios for V _f -resistance have frequently been reported. Here we revealed that sub-lethal genes caused the distorted segregation. The inheritance of V _f was examined in six progenies by testing linked molecular markers. Three progenies showed distorted segregations that could be explained by three sub-lethal genes (sl1, sl2 and sl3), of which sl1, sl2 were closely linked to V _f . The s11 gene was located at about 14 cM from V _f and expressed itself only in the presence of another independently segregating sub-lethal gene sl3. Only the double homozygous recessive genotypes (sl1sl1 sl3sl3) were lethal, which occurred at first as dwarf and poor vigour plants during the first three months after germination. The sl2 gene was also linked to V _f and its lethality was expressed prior to seed germination and also required the homozygous recessive presence of sl3. The map position of sl3 has not yet been identified. The linkage of V _f to sub-lethal genes usually results in a shortage of V _f -resistant progenies. But in some exceptional crosses, it will lead to abundance of resistant seedling.An erratum to this article can be found at     

The use of a modified bulk segregant analysis to identify a molecular marker linked to a scab resistance gene in apple

Almost two-hundred random sequence decamer-primers were used to screen a pair of bulked samples and the donor parent Malus floribunda clone 821 for markers linked to the V_f gene conferring resistance to apple scab (Venturia inaequalis (Cke.) Wint.). A single primer was identified which generated a PCR fragment, OPK16/1300, from the donor parent M. floribunda clone 821 and the scab-resistant selections/cultivars bulk, but not from the scab-susceptible recurrent parent bulk. Co-segregation analysis using a segregating apple progeny and polymorphism analysis of individual scab-resistant Coop selections/cultivars confirmed that this marker was linked to the scab-resistance gene V_f with a recombination frequency of 4.3%. OPK16/1300 was then cloned and sequenced. Sequence-specific primers of 25 oligonucleotides based on the marker were synthesized, and used in turn to screen M. floribunda clone 821, scab-susceptible apple cultivars, scab-resistant apple cultivars, and scab-resistant Coop selections. A pair of sequence-specific primers of clone OPK16/1300 amplified a distinct single band of the same size as the RAPD clone. Thus, a sequence characterized amplified region (SCAR) marker was developed which can be used to identify polymorphisms of OPK16/1300 based on the presence or absence of a single band. This revised version was published online in July 2006 with corrections to the Cover Date.     

Early determination of genotypes for apple scab resistance by forced flowering of test cross progenies

Summary Apple selections with different major genes for resistance to apple scab (Venturia inaequalis) derived from Malus floribunda and M. pumila were crossed with each other. The progenies were screened as young seedlings for their reaction to V. inaequalis race 1. A gene for resistance from M. pumila, causing stellate necrotic (SN) lesions, was epistatic to a second gene for resistance from M. floribunda, causing irregular chlorotic (Chl) lesions. Although in most cases SN, Chl and susceptible phenotypes were clearly distinct, occasionally reactions were difficult to characterize or varied from one inoculation to another. Selected seedlings showing resistant or susceptible reactions were forced to flower in 16–20 months in the greenhouse and test crossed with susceptible cultivars. Test cross seedlings were screened for scab reaction. The presence of both genes for resistance in a resistant plant was indicated by presence of both Chl and SN resistant phenotypes in the test cross progeny. Chi-square analysis of four large progenies produced a good fit to the expected ratio. The use of the forced flowering technique to determine scab resistance genotypes in 28 months demonstrated its value in breeding apples with multiple disease resistance.     

Mapping Co, a gene controlling the columnar phenotype of apple, with molecular markers

The columnar phenotype is a very valuable genetic resource for apple breeding because of its compact growth form determined by the dominant gene Co. Using bulked segregant analysis combined with several DNA molecular marker techniques to screen the F₁ progeny of Spur Fuji × Telamon (heterozygous for Co), 9 new DNA markers (6 RAPD, 1 AFLP and 2 SSRs) linked to the Co gene were identified. A total of 500 10-mer random primers, 56 pairs of selective AFLP primers and 8 SSR primer pairs were screened. One RAPD marker S1142₆₈₂, and the AFLP marker, E-ACT/M-CTA₃₄₆, were converted into SCAR markers designated SCAR₆₈₂ and SCAR₂₁₆, respectively. These markers will enable early selection in progenies where Co is difficult to identify. The Co gene was located between the SSR markers CH03d11 and COL on linkage group 10 of the apple genetic linkage map. Finally, a local genetic map of the region around the Co gene was constructed by linkage analysis of the nine new markers and three markers developed earlier.     

Stability and potential use of RAPD markers in a sugarcane genealogy

Summary A complete ancestral history of the recently developed and closely related South African commercial sugarcane varieties N11 and NCo376, which differ markedly in their response to sugarcane mosaic virus (SCMV), was elucidated from archival records. The genealogy spans seven generations, starting with early intraspecific crosses between varieties of Saccharum officinarum and interspecific crosses between S. officinarum and either S. spontaneum or S. barberi. In total, the genealogy comprises 38 different varieties. Genomic DNA samples from N11 and NCo376 respectively were screened for polymorphisms using the PCR-RAPD technique. Ten polymorphic fragments ranging in molecular size from 317 to 1263bp were identified from a total of 1159 loci amplified with 100 random decamer primers. Two of the 10 polymorphic fragments were shown to be consistently present in N11 (resistant) and absent in NCo376 (susceptible), while 8 showed the reverse occurrence. The primers producing the polymorphisms were used to screen genomic DNA samples from all 19 varieties representing the genealogy. Results have indicated that (1) specific PCR-RAPD generated polymorphic fragments can indeed be identified across the seven generations; (2) certain fragments are sufficiently definitive to be used as markers to trace parentage; (3) the validity of documented crosses and/or the authenticity of germplasm material may be questioned using this technique, and (4) there is the potential to subject the markers to linkage analysis once a full and accurate assessment of the SCMV resistance phenotype is obtained.     

Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F₁ and F₂ progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stability of RAPD markers for determining cultivar specific DNA profiles in rye (Secale cereale L.)

Summary Cultivar specific DNA profiles in rye were revealed by polymerase chain reaction (PCR) using randomly amplified polymorphic DNA (RAPD) sequences. Ten base primers were used for the amplification of genomic DNA of rye cultivars by PCR. RAPD analysis was found to be reproducible among samples between PCR runs. When amplification profiles of different rye cultivars were compared using various primers, the overall profiles were cultivar specific. However, not all primers revealed polymorphisms. These primers appear to amplify conserved sequences in all rye cultivars. Intracultivar studies were conducted on two of the cultivars. In the cultivar Imperial, no polymorphisms were observed among ten plants analyzed with five primers. In the cultivar Balboa, polymorphisms were observed among fifty plants with four of the ten primers analyzed. Despite the small amount of intracultivar variability, RAPD analysis has the potential to be a rapid and reliable method of cultivar identification in this outcrossing species.     

Analysis of genetic diversity in Agave tequilana var. Azul using RAPD markers

By federal law in Mexico, A. tequilana Weber var. Azul is the only variety of agave permitted for the production of any tequila. Our objective was to assay levels of genetic variation in field populations of A. tequilana var. Azul using randomly amplified polymorphic DNA (RAPD) markers. Ten plants were collected from each of four different fields, with two fields being located in each of two principal regions of Mexico for the cultivation of A. tequilana var. Azul. The two regions are separated geographically by approximately 100km. Genetic relationships between A. tequilana var. Azul and two other varieties of A. tequilana Weber, ‘Chato’ and ‘Siguin’, were also investigated using RAPDs. Among the three varieties, 19 decamer primers produced 130 markers, of which 20 (15.4%) were polymorphic betweenA. tequilana var. Chato and A. tequilana var. Siguin. The results of RAPD analysis suggest that A. tequilana var. Siguin is more closely related to A. tequilana var. Azul than is A. tequilana var. Chato. Among the 40 field selections of A. tequilana var. Azul, only 1 of124 RAPD products (0.8%) was polymorphic and 39 of 40 plants were completely isogenic. This is one of the lowest levels of polymorphism detected to date for the analysis of a crop species, and is proposed to be the result of the promotion of a single conserved genotype over many years due to an exclusive reliance on vegetative propagation for the production of new planting materials. The significance of these results is discussed in relation to breeding programs focused on the improvement of A. tequilana var. Azul. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity and relationships among Lablab purpureus genotypes evaluated using RAPD as markers

Summary Genetic variation in 40 accessions of Lablab purpureus was evaluated using random amplified polymorphic DNA as markers. A high level of genetic variation in this species was detected but this was mainly restricted to the difference between cultivated and wild forms. Of the cultivated genotypes, genetic variation among Asian collections was significantly higher than that among African collections. The three most divergent cultivated genotypes were all from Asia. Four of the five wild accessions, two from Zimbabwe and the other two from Zambia, were closely related. The other one, CPI 31113 collected from Uganda, was highly divergent. The two commercial forage varieties used in Australia, Rongai from Kenya and Highworth from India, were not very different.     

Screening for resistance to Nectria galligena Bres. in cut shoots of apple

Summary To find a fast and reliable test to assess resistance to Nectria galligena in apple, different methods of inoculation were compared using macroconidia of N. galligena and one-year-old cut shoots from mature trees of Cox's Orange Pippin, IVT 69078-19, James Grieve and Jonathan.With the best inoculation method 11 genotypes were screened for resistance. Elstar, Golden Delicious, Jonathan and Lombart's Calville were highly resistant and the level of resistance of Ingrid marie, Gloster, Melrose, IVT 69078-19, Cox's Orange Pippin, James Grieve and Idared decreased in this order.The best inoculation method proved to be simple, giving results within four to nine weeks after inoculation.     

Dwarfing and lethal genes in apple progenies

Summary Three dwarf types are described, early dwarf, determined by two recessive genes d ₁ and d ₃, in the double homozygous state, and an additional recessive gene d ₄ and crinkle dwarf and sturdy dwarf each also determined by a recessive gene.The gene l, for pale green lethal was found to be closely linked to the gene V _f , for scab resistance.There was evidence of a regrowth promoting gene, G, in some seedlings homozygous for d ₁ and d ₃.     

Microsatellite (SSR) variation in a collection of Malus (apple) species and hybrids

A collection of 142 accessions of 23 Malus species, derived hybrids and cultivar accessions from the USDA-ARS Plant Genetic Resources Unit's core collection, which represents an extensive range of Malus species, was screened with a set of previously described SSR (simple sequence repeat) markers. The markers were used to determine genetic identities, estimate genetic diversity, identify genetic relationships among the accessions, and determine the utility of SSR primers developed from Malus ×domestica for making genetic assessments across the whole Malus genus. All eight primer pairs amplified multiple fragments when used in polymerase chain reactions with DNA from these accessions. High levels of variation were detected with a mean of 26.4 alleles per locus and a mean direct count heterozygosity across all eight loci equal to 0.623. The eight primer pairs used in this study unambiguously differentiated all but five pairs of accessions in this collection of 142 accessions of 23 Malus species, derived hybrids and cultivars. These SSR data were not useful in identifying genetic relationships among this diverse collection of accessions, with the majority of the accessions not clustering in ways concordant with taxonomic information and/or geographic origin. The resulting phenogram resolved only two meaningful clusters, for the taxonomically isolated Section Chloromeles and for M. fusca accessions, reflecting genetic relationships arising from geographic origin. The detection of identical accessions in the collection, which were previously considered to be unique, highlights the critical need to further bolster collections of certain Malus species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phylogenetic relationships as in Ocimum revealed by RAPD markers

Genetic relationships were examined among thirty germplasm accessions belonging to five Ocimum species using RAPD markers. A very high degree of polymorphism (98.20%) was observed. UPGMA cluster analysis of genetic similarity indices grouped all the accessions into two major clusters corresponding to previously reported botanical sections. Intra-clustering within the two clusters precisely grouped the accessions belonging to one species in one sub-cluster as expected from their genetic background. Our results show that RAPD technique is a sensitive, precise and efficient tool for genomic analysis in Ocimum species, that may be useful in future studies, by assigning new unclassified germplasm accessions to specific taxonomic groups and reclassifying previously classified accessions of other Ocimum species by traditional criteria on a more objective basis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of sex in Carica papaya L. using RAPD markers

Brazil is currently the worlds largest producer of papaya (Carica papayaL.), producing fruits for both the domestic market and export. Only fruits from hermaphrodite plants are marketed because they have the necessary commercial characteristics, i.e. they are pear-shaped and have thicker flesh and a smaller internal cavity. Increased papaya yield has been limited mainly by the ratio of female to hermaphrodite (1: 2) plants normally occurring in orchards. This ratio causes great losses to papaya producers and the identification of the sex of seedlings during the nursery stage would be an important advance. In our study random amplified polymorphic DNA (RAPD) analysis was used to differentiate between the sexual forms of three commercial C. papaya cultivars belonging to the Solo group. RAPD assays using the BC210 primer were able to detect hermaphrodites in all of the cultivars tested. The BC210₄₃₈molecular marker was much better at papaya sex differentiation than other markers described in the literature. This revised version was published online in August 2006 with corrections to the Cover Date.     

RAPD markers linked to a rust resistance gene in cultivated groundnut (Arachis hypogaea L.)

Groundnut rust (Puccinia arachidis Speg.) is an important air borne pathogen, which causes substantial losses in groundnut yield and quality. Although large numbers of accessions were identified as rust resistant in wild, interspecific derivative and cultivated groundnut species, transfer of resistance to well-adapted cultivars is limited due to linkage drag, which worsens yield potential and market acceptance. A F₂ mapping population comprising 117 individuals was developed from a cross between the rust resistant parent VG 9514 and rust susceptible parent TAG 24. Rust resistance was governed by single dominant gene in this cross. We identified 11 (out of 160) RAPD primers that exhibited polymorphism between these two parents. Using a modified bulk segregant analysis, primer J7 (5′CCTCTCGACA3′) produced a single coupling phase marker (J7₁₃₅₀) and a repulsion phase marker (J7₁₃₀₀) linked to rust resistance. Screening of the entire F₂ population using primer J7 revealed that the coupling phase marker J7₁₃₅₀ was linked with the rust resistance gene at a distance of 18.5 cM. On the other hand, the repulsion phase marker J7₁₃₀₀ was completely linked with rust resistance. Additionally, both J7₁₃₀₀ (P = 0.00075) and J7₁₃₅₀ (P < 0.00001) were significantly associated with the rust resistance. Marker J7₁₃₀₀ identified all homozygous rust resistant genotypes in the F₂ population and was present in all the eight susceptible genotypes tested for validation. Thus, J7₁₃₀₀ should be applicable for marker-assisted selection (MAS) in the groundnut rust resistance breeding programme in India. To the best of our knowledge this is the first report on the identification of RAPD markers linked to rust resistance in groundnut.     

Development of reliable PCR markers for the selection of the Vf gene conferring scab resistance in apple

Early selection of scab-resistant apple seedlings can be enhanced by the use of markers tightly linked to the Vf resistance gene. Two sequence characterized amplified regions (SCAR) markers have been obtained from previously described random amplified polymorphic DNA (RAPD) markers. AM19-SCAR is a codominant marker, while AM19-SCAR is dominant, as is the RAPD from which it was derived. A highly detailed map in the vicinity of the Vf gene was built through the cumulative analysis of about 600 seedlings from six different controlled crosses. The usefulness of these and other SCAR markers will be discussed in relation to combining the traditional phenotypic selection with MAS. The availability of two codominant, tightly linked markers flanking both sides of the resistance gene (AL07-SCAR and M18-CAPS) also makes it easy to identify the seedlings homozygous for the resistance gene.     

Inheritance and selection of some fruit characters of apple. I. Inheritance of low and high acidity

Summary Fruit acidity as such appears to be gouverned by a single major gene, low acidity being a recessive character. The additional genetic variation is explainable by the additive gene action model. This mode of inheritance causes the pH distribution of seedlings in progenies to shift with a higher mid-parent pH towards more low-acid cum sweet (pH 3.8) and fewer high-acid cum sour (pH 2.9) seedlings or vice versa with a lower midparent pH. As a result, in crosses between heterozygotes (Mama) the total proportion of undesirable seedlings -low plus high-acid ones-, remains similar, irrespective of the mid-parent pH. This proportion constituted about one third of the seedlings. A same proportion, but of mainly sour plants, was found in crosses between relatively acid homozygotes (Mama) or between these and similar heterozygotes. It was less than one quarter if the heterozygous partner was medium-acid and a few percentages only when both the homozygous and heterozygous partner were medium-acid. Choosing parents in the absence of information, chances are that the very large majority of the medium-acid types are heterozygotes, that among the relatively acid ones one in two is homozygous and that the odds to find homozygotes among the sour types are probably best. Attention was drawn to the importance of a high sugar content — which is quantitatively inherited — in parents with a relatively low pH to increase the chances of selecting seedlings with a good sugar/acid ratio.     

Fingerprinting of alfalfa meiotic mutants using RAPD markers

Summary A calendar of female sporogenesis and gametogenesis was made for both apomictic tetraploid (2n=4x=36) Brachiaria brizantha and Brachiaria decumbens and their apomictic F₁ hybrids with sexual tetraploid (2n=4x=36) Brachiaria ruziziensis. Microgametogenesis was used as a reference. Apospory was facultative in both species and hybrids. Environmental conditions had variable effects on the level of apomixis according to each genotype. Ratios of segregation into sexuals and apomicts in the interspecific hybrids suggest an oligogenic determinism with dominant apomixis in the genus Brachiaria. Highly apomictic and partially male fertile hybrids were identified and will be used in an improvement program to transfer genes for apomixis into the sexual species B. ruziziensis.     

DNA-RAPDs detect genetic variation and paternity in Malus

Summary DNA amplification fingerprinting using arbitrary primer(s) was applied to the identification of Malus species. Highly variable DNA fragment patterns were clearly detected by polyacrylamide gel electrophoresis of the amplified extension products, although three sports of Delicious exhibited the same fingerprint as the original cultivar. The fingerprinting of two triploid apple cultivars suggested that the parent contributing the 2n gamete was the female. We applied this fingerprinting to paternity analysis of an apple cultivar of which the pollen parent was unknown. By using 5 arbitrary primers and RFLP analysis of the amplified products, one cultivar was singled out for paternity among six putative candidates.