Molecular mapping of a novel blast resistance gene Pi38 in rice using SSLP and AFLP markers

Blast, caused by Magnaporthe grisea, is the most devastating disease of rice worldwide. In this study, the main objective was to identify and map a new gene for blast resistance, in an indica rice cultivar ‘Tadukan’ against blast fungal isolate B157, using molecular tools. F₂ segregating population was derived from ‘CO39’ (susceptible) and ‘Tadukan’ (resistant), and molecular mapping of the blast resistance gene was carried out using simple sequence length polymorphism (SSLP) and amplified fragment length polymorphism (AFLP) methods. Two SSLP markers, RM206 and RM21 and three AFLP markers (AF1: E‐aca/M‐ctt; AF2: E‐aca/M‐cat and AF3: E‐acc/M‐cac2) were identified to be linked to the resistance gene. The co‐segregation analysis using SSLP markers implied that the blast resistance gene designated Pi38 resides on rice chromosome 11.     

Identification of a molecular marker linked to the closed capsule mutant trait in sesame using AFLP

The identification of an amplified fragment length polymorphism (AFLP) marker linked to an agronomically useful trait in sesame is reported. A bulked segregant analysis (BSA) approach was adopted on segregating progenies of a cross between the closed capsule mutant line ‘cc3’, and the Turkish variety ‘Muganli‐57′. A total of 72 primer combinations were screened for linkage to the trait, but only one closely linked amplified fragment length polymorphism (AFLP) marker was identified. The linkage was confirmed by analysing the AFLP profile from single plants. The marker has the potential to accelerate breeding programmes aimed at modifying unwanted side‐effects of the closed capsule mutation by marker‐assisted selection.     

Molecular mapping of a new gene for resistance to frogeye leaf spot of soya bean in 'Peking'

Amplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeat, SSR) techniques were used to map the _RGSp_eking gene, which is resistant to most isolates of Cercospora sojina in the soya bean cultivar ‘Peking’. The mapping was conducted using a defined F₂ population derived from the cross of ‘Peking’(resistant) בLee’(susceptible). Of 64 EcoRI and MseI primer combinations, 30 produced polymorphisms between the two parents. The F₂ population, consisting of 116 individuals, was screened with the 30 AFLP primer pairs and three mapped SSR markers to detect markers possibly linked to Rcs_Peking. One AFLP marker amplified by primer pair E‐AAC/M‐CTA and one SSR marker Satt244 were identified to be linked to Res_Peking. The gene was located within a 2.1‐cM interval between markers AACCTA178 and Satt244, 1.1 cM from Satt244 and 1.0 cM from AACCTA178. Since the SSR markers Satt244 and Satt431 have been mapped to molecular linkage group (LG) J of soya bean, the Res_Peking resistance gene was putatively located on the LG J. This will provide soya bean breeders an opportunity to use these markers for marker‐assisted selection for frogeye leaf spot resistance in soya bean.     

A specific PCR assay for resistance to biotypes 1 and 2 of the rosy leaf curling aphid in apple based on an RFLP marker closely linked to the Sd1 gene

This report describes the conversion of a restriction fragment length polymorphism (RFLP) marker (the 2B12a locus). linked to the Sd₁ aphid resistance gene, to a polymerase chain reaction (PCR) based marker. A section of the 2BI2 probe was sequenced and two primers were designed lo amplify this sequence in the cultivars‘Prima’and‘Fiesta’: all the amplification products were the same size. After sequencing. two specific 24-mer oligonueleotides were synthesized (DdARM-5¹ and DdAR.M-3²) to exploit a single base-pair difference. These primers were used to screen 44 plants from the‘Prima’x‘Fiesta’family and generated a single amplification product (196bp). in approximately half of the seedlings, which was linked to the resistance gene Sd₁,. The DdARM primer combination was used to evaluate a range of apple cultivars and selections, including some varieties derived from‘Cox’and alternative sources of resistance reported in the literature. In parallel with this work, the phenotypic response of the same genotypes was either confirmed or determined in replicated glasshouse tests. The sequence characterized amplified regions (.SCAR) marker was amplified in all the resistant plants, with the exception of‘Northern Spy’and 3760 (the sources of Sd₂ and Sd₃ resistance, respectively), but never in the susceptible plants. The possible role of this marker in a marker-assisted breeding strategy, and its compatibility with a SCAR marker linked to the I, gene for resistance to apple scab. is discussed.     

Genetic diversity and relationships among pea cultivars revealed by RAPDs and AFLPs

In order to obtain an overview of the genetic diversity present within the set of pea cultivars released in Germany, 21 cultivars were analysed at the DNA level by random amplified polymorphic DNAs (RAPDs) and amplified fragment length polymorphisms (AFLPs), as well as for agronomic traits. Yield of grain cultivars ranged from 2.95 to 3.87 t/ha. Based on the screening of 60 RAPD primers and 32 Eco RI + 3/Mse I+3 AFLP primer combinations, 20 RAPD primers and 11 Eco RI + 3/MseI+ 3 primer combinations generating polymorphic and distinct fragments were chosen for estimation of genetic diversity. Twenty RAPD primers amplified a total of 314 scorable bands ranging from about 262 bp to 1996 bp. Of these, 175 fragments (55.7%) were polymorphic. Based on these data, genetic similarity (GS) was estimated between 0.80 (‘Lisa’ vs.‘Grapis’) and 0.94 (‘Bohatyr’ vs. ‘Sponsor’; mean GS = 0.88). Eleven AFLP primer combinations led to the amplification of 949 scorable fragments ranging from 43 to 805 bp and of these, 462 (48.7%) were polymorphic. Genetic similarity based on AFLPs was calculated between 0.85 (‘Lisa’ vs.‘Laser’) and 0.94 (‘Bohatyr’ vs. ‘Sponsor’, mean GS = 0.90). Correlation of genetic similarity estimated on RAPDs and AFLPs was estimated at r = 0.79** using Spearman's rank correlation coefficient and at r = 0.84 by the Mantel test, respectively. UPGMA cluster analysis carried out on these data separately for RAPDs and AFLPs and on the combined data reflected, to some extent, pedigree relationships and cophenetic correlations (r = 0.89 for RAPDs, r = 0.88 for AFLPs, and r = 0.93 RAPDs + AFLPs) indicate a good fit of respective clusters to genetic similarity data. The correlation of cluster analyses to pedigree information and the impact on parental genotype selection is discussed.     

Identification of commercial chicory cultivars for hydroponic forcing and their phenetic relationships revealed by random amplified polymorphic DNAs and amplified fragment length polymorphisms

One‐hundred and twenty‐four amplified fragment length polymorphism (AFLP) and 49 random amplified polymorphic DNA (RAPD) markers have been used to distinguish between 20 and 23 commercial chicory cultivars, respectively. These were all Cichorium intybus var. foliosum F₁ hybrids, currently used in hydroponic forcing. Five‐hundred and twenty RAPD primers (OPERON) were tested, of which 156 resulted in reproducible patterns and 26 yielded polymorphisms. Two‐hundred and fifty‐six AFLP primer‐combinations were tested and six combinations were selected for identification purposes. Similarity indices were measured and clustering has been done using pairwise comparison. Both types of marker provide similar conclusions. Two major clusters are formed, representing late and early cultivars. All cultivars were identified using 10 informative RAPD primers or three AFLP primer combinations. A low degree of polymorphism was detected between some early cultivars, suggesting a narrow genetic base in their breeding strategy.     

Identification of AFLP and SSR markers linked with the male fertility restorer gene of CMS 06J45 in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

In this study, AFLP and SSR techniques were combined with the bulk segregant analysis (BSA) method to map the restorer gene BrRfp using an F₂‐segregating population comprising 258 individuals developed by crossing the polima (pol)‐like cytoplasmic male sterility (CMS) line 06J45 and the restorer line 01S325 of heading Chinese cabbage. A survey of 2048 AFLP primer pairs identified 21 polymorphic fragments, approximately half of which exhibited high similarity with the A09 chromosome sequence of Brassica rapa in the Brassica database (BRAD). Based on the genome sequence, three specific AFLP fragments linked with BrRfp were successfully converted into sequence‐characterized amplified region (SCAR) markers, named SC1233, SC2673 and SC2141. Subsequently, 178 pairs of SSR primers were redesigned for further screening, with five producing polymorphic amplification patterns. Linkage analysis showed that these markers were distributed along both sides of the BrRfp gene, with two markers, SSR03 and SSR2528, co‐segregating with the BrRfp locus in the F₂ population. These results may be valuable for marker‐assisted selection and map‐based cloning in heading Chinese cabbage.     

Development of SCAR markers linked to the Ns locus in potato

A random amplified polymorphic DNA marker OPG17₄₅₀ linked to the Ns gene that confers resistance of potato to potato virus S (PVS), was used to develop sequence‐characterized amplified region (SCAR) markers. After cloning and sequencing of OPG17₄₅₀ new polymerase chain reaction (PCR) primers were designed to generate dominant (SCG17₃₂₁) and codominant (SCG17₄₄₈) markers. For SCG17₄₄₈, polymorphism between susceptible and resistant genotypes was recovered after digestion of the marker with the restriction enzyme Muni. In addition to the band corresponding to ‘susceptible’ allele that does not contain the Muni cleavage site, two bands of approximately 251 bp and 197 bp were observed in the resistant genotypes. The usefulness of these SCAR markers was verified in diploid potatoes possessing the Ns locus from clone G‐LKS 678147/60, and in tetraploid potatoes derived from G‐LKS 678147/60 and from clone MPI 65118/3.     

Evaluation of amplified fragment length polymorphism markers in tef, Eragrostis tef (Zucc.) Trotter, and related species

Tef is an important cereal crop in Ethiopia. This study was conducted to investigate (1) genetic diversity within and among three Eragrostis species (E. tef, E. pilosa and E. curvula), and (2) the relationship between E. tef, E. pilosa and E. curvula. A total of 529 AFLP markers were obtained, out of which 58% (368) were polymorphic, using 10 primer, combinations. The three species were separated distinctly using amplified fragment length polymorphism (AFLP), However, diversity revealed at the morphological trait level was not commensurate with that observed for AFLP. This was due to the small number of morphological traits available and their interaction with the environment. Within tef, ‘Rubicunda’ and DZ‐01‐1093 were found to be distantly related to the rest of the tef accessions. The diversity within species was such that E. pilosa was the most diverse followed by E. curvula and E. tef. Moreover, E. pilosa was more closely related to E. tef than E. curvula. Therefore, further study is needed of E. pilosa accessions and of ‘Rubicunda’ and DZ‐01‐1093 in a crossing programme to generate a population for selection and/or genetic mapping. A total of 19 cultivars or accessions had one or more unique fragments using one or more AFLP primers, indicating the potential of the technology in fingerprinting tef in a breeding or seed multiplication programme.     

Amplified fragment length polymorphism-derived microsatellite sequence linked to the Pch1 and Ep-D1 loci in common wheat

Amplified fragment length polymorphism (AFLP) markers linked to the Aegilops ventricosa‐derived chromosome segment in ‘VPM1’ on which the eyespot resistance gene, Pch1, and the endopeptidase gene, Ep‐D1b, occur were identified. One marker was isolated from the gel, cloned and sequenced. Sequence analysis revealed a microsatellite repeat motif. Sequence‐specific primers were designed to amplify a product containing the repeat motif, and the microsatellite marker was tested for cosegregation with the Ep‐D1b allele. Distinct alleles were produced by the Pch1 sources, normal wheat and wheat containing the Lr19 translocation. A recombination frequency of 0.02 was calculated between the microsatellite marker and Ep‐D1.     

AFLP–SSR maps of maize × teosinte and maize × maize: comparison of map length and segregation distortion

Two genetic linkage maps of Zea mays were constructed: one population comprised 94 F₂ individuals of a dent ‘B64’ × teosinte (Z. mays ssp. huehuetenangensis) cross while the second consisted of 94 F₂ individuals of a ‘B64’ × Caribbean flint ‘Na4’ cross. The level of polymorphism was higher in the ‘B64’ × teosinte combination than the ‘B64’ × ‘Na4’ combination. In the ‘B64’ × teosinte cross, a total of 338 amplified fragment length polymorphism (AFLP) and 75 simple sequence repeat (SSR) markers were mapped to 10 chromosomes, which covered 1402.4 cM. In the ‘B64’ × ‘Na4’ cross, a total of 340 AFLP and 97 SSR markers were mapped to 10 chromosomes, covering 1662.8 cM. Segregation distortion regions were found on chromosomes 4, 5 and 8 in the ‘B64’ × teosinte cross and on chromosome 9 in the ‘B64’ × ‘Na4’ cross. Comparison of the two maps revealed that the maize × teosinte map was 11.5% shorter than the maize × maize map. The maps generated in this study may be useful to identify genes controlling flooding tolerance.     

Integration of AFLP markers into an RFLP-based map of durum wheat

Amplified fragment length polymorphism (AFLP) is a powerful technique which can readily be applied to a wide range of species for mapping purposes. AFLPs were added to a linkage map of durum wheat constructed using restriction fragment length polymorphisms (RFLPs). The mapping population included 65 recombinant inbred lines derived from a cross between the durum wheat cultivar ‘Messapia’ and accession ‘MG4343’ of the wild Triticum turgidum ssp. dicoccoides (Körn.). Genomic DNA was digested with MseI (4‐cutter) and Sse83871 (8‐cutter). Using a silver‐staining protocol, 14 primer combinations revealed 421 clearly scorable amplicons including 100 polymorphisms. The presence of nine pairs of bands linked in repulsion phase with each pair generated by one primer combination suggested the presence of codominant alleles; sequence analysis of four band pairs confirmed their codominant nature. The integration of 80 AFLP loci extended the map in several telomeric regions, reduced the size of four large gaps present in the previous map, and eliminated one gap. The new map obtained after the inclusion of the 80 AFLP loci and eight additional RFLP loci spans 2063cM which represent a 52.6% increment compared with the previous map. Compared with the distribution of RFLPs, no significant clustering of AFLP markers was observed.     

Amplified fragment length polymorphism- and simple sequence repeat-based molecular tagging and mapping of greenbug resistance gene Gb3 in wheat

The greenbug, Schizaphis graminum (Rondani), is the most economically damaging aphid pest of wheat in the southern Great Plains of the USA. In this study, the single, dominant greenbug resistance gene, Gb3, was molecularly tagged and genetically mapped using amplified fragment length polymorphism (AFLP) and simple sequence repeat(SSR) markers. Three AFLP loci were associated with the Gb3 locus in linkage analysis with 75 F_2:3 families from the cross between two near‐isogenic lines (NILs) for Gb3,‘TXGBE273’ and ‘TXGBE281′. Two of these loci, XMgcc Pagg and Xmagg Patg cosegregate with Gb3 in the population analysed. Further analysis indicated that XMgcc Pagg and Xmagg Patg are specific for the Gb3 locus in diverse genetic backgrounds. Two SSR markers, Xgwm111 and Xgwm428 previously mapped in wheat chromosome 7D, were shown to be linked with Gb3, 22.5 cM and 33.1 cM from Gb3, respectively, in an F₂ population of ‘Largo’בTAM 107’, suggesting that Gb3 is located in the long arm of chromosome 7D. The two AFLP markers cosegregating with Gb3 are valuable tools in developing molecular markers for marker‐assisted selection of greenbug resistance in wheat breeding.     

Identification of a major QTL in cocoa (Theobroma cacao L.) associated with resistance to witches' broom disease

The witches’ broom disease caused by the fungus Crinipellis perniciosa is the main limiting factor for cocoa production in South America and the Caribbean. In Brazil, this disease affects almost all cocoa‐growing regions, causing serious economic, social and ecological damage. The aim of this study was to map genomic regions associated with resistance to C. perniciosa using an F₂ population derived from a cross between ‘Scavina‐6’(resistant) and ‘ICS‐1’(susceptible). The phenotypic index was determined as the average number of vegetative witches’ brooms per canopy area of each plant, the witches’ brooms were counted and eliminated during six field evaluations between May 1998 and August 1999. A total of 124 random amplified polymorphic DNA (RAPD) and 69 amplified fragment length polymorphism (AFLP) markers were mapped along 25 linkage groups covering 1713 cM of cocoa genome. After employing single factor and composite interval mapping analyses, a major quantitative trait loci (QTL) flanked by the marker AV14.940 was identified in the linkage group 11, explaining almost 35% of the resistance to witches’ broom. The present result suggests that this QTL acts as a major dominant component of resistance to this pathogen, with great potential for use in marker‐assisted selection procedures in cocoa breeding programmes.     

High-resolution mapping of the nud locus controlling the naked caryopsis in barley

The hulled or naked caryopsis character of barley is an important agronomic trait because of the direct link to its use. A single recessive gene, nud, located on the long arm of chromosome 7H, controls the naked caryopsis character. Previously, linked amplified fragment length polymorphism (AFLP) bands from bulked segregant analysis were screened, and the nud gene was mapped in a population of 151 F₂ plants. In the present study, the aim was to construct a high‐resolution map of the nud gene towards its positional cloning. Two AFLP bands were converted into sequence‐characterized amplified region (SCAR) markers (sKT5 and sKT9), and a previously reported SCAR marker sKT3 was improved for more reliable detection of polymorphism. A total of 2380 segregants derived from five cross‐combinations were analysed, and the nud gene was flanked by sKT3 and sKT9, at the 0.6‐cM proximal and the 0.06‐cM distal side, respectively. The SCAR markers developed in this study should be useful for marker‐assisted selection in naked barley breeding employing crosses between naked and hulled accessions.     

An STS marker distinguishing the rye-derived powdery mildew resistance alleles at the Pm8/Pm17 locus of common wheat

A sequence‐tagged site marker has been developed from restriction fragment length polymorphism marker probe IAG95 for the rye‐derived powdery mildew resistance Pm8/Pm17 locus of common wheat. This polymerase chain reaction marker enables the amplification of DNA fragments with different sizes from T1AL.1RS and T1BL.1RS wheat‐rye translocation cultivars with chromatin from ‘Insave’ and ‘Petkus’ rye, respectively, and therefore will be very useful in distinguishing Pm8‐carrying cultivars from Pm17‐carrying cultivars. Results obtained with that marker were compared with resistance tests performed on detached primary leaves of 29 wheat lines from two populations derived from doubled haploid production. The molecular assay corresponded well with the resistance tests in all the lines, and therefore will be helpful for the identification of Pm17 in lines in which other Pm genes or quantitative trait loci are present.     

Molecular analysis of the barley cv. 'Valticky' and its X-ray-derived semidwarf-mutant 'Diamant'

The outstanding semidwarf spring barley cultivar ‘Diamant’ was derived from the cv.‘Valticky’ by X‐ray mutagenesis. More than 120 European spring barley varieties trace back to ‘Diamant’. This variety and nine German spring barley cultivars having ‘Diamant’ in their pedigree, as well as four cultivars not related to ‘Diamant’, were analysed by amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs) in order to identify genomic regions associated with traits derived from ‘Diamant’. The total number of SSRs tested was 122, of which 88 were polymorphic taking all varieties into account, with 51 for ‘Valticky’ and ‘Diamant’, i.e. 42.8%. A varying level of polymorphism was detected on individual chromosomes (11.1–78.6%). In addition, AFLP revealed a high level of polymorphism. Out of the total number of 1591 bands, 670 were polymorphic for all varieties studied with 182 for ‘Valticky’ and ‘Diamant’, i.e. 11.4%. Based on SSR and AFLP data UPGMA cluster analysis was carried out, but no grouping according to the relatedness with ‘Diamant’ or ‘Valticky’, respectively, was found. It is concluded from these results that the mutagenic effect leading to the development of ‘Diamant’ out of ‘Valticky’ affected many genetic loci and is thus larger than expected.     

Mapping of a gene for leaf scald resistance in barley line 'B87/14' and validation of microsatellite and RFLP markers for marker-assisted selection

Seedlings of the barley line ‘B87/14’ were resistant to 22 out of 23 Australian isolates of Rhynchosporium secalis, the causal agent of leaf scald.‘B87/14’‐based populations were developed to determine the location of the resistance locus. Scald resistance segregated as a single dominant trait in BC₁F₂ and BC₁F₃ populations. Bulked segregant analysis identified amplified fragment length polymorphisms (AFLPs) with close linkage to the resistance locus. Fully mapped populations not segregating for scald resistance located these AFLP markers on chromosome 3H, possibly within the complex Rrs1 scald locus. Microsatellite and restriction fragment length polymorphism markers adjacent to the AFLP markers were identified and validated for their linkage to scald resistance in a second segregating population, with the closest marker 2.2 cM from the resistance locus. These markers can be used for selection of the Rrs.B87 scald‐resistance locus, and other genes at the chromosome 3H Rrs1 locus.     

Single nucleotide polymorphism genotyping of the barley waxy gene by polymerase chain reaction with confronting two-pair primers

A high‐throughput single nucleotide polymorphism (SNP) genotyping procedure was developed to select amylose‐free barley mutants whose waxy genes had a C‐ to T‐base substitution in exon 5, which converted Gln‐89 of the wild‐type gene into a termination codon. An F₂ population carrying an amylose‐free waxy gene was checked for segregation. Polymerase chain reaction with confronting two‐pair primers (PCR‐CTPP) produced allele‐specific PCR products that have different sizes and are inherited in a co‐dominant manner. Two alleles of the barley waxy gene with SNP were correctly identified in parental strains using the PCR‐CTPP procedure. Segregation of the SNP as detected by PCR‐CTPP in an F₂ population fitted the expected 1:2:1 ratio. The PCR‐CTPP procedure can provide a time saving and cost‐effective alternative to derived cleaved amplified polymorphic sequence in marker‐assisted selection.     

AFLP analysis of Solanum phureja DNA introgressed into potato dihaploids

Introgression of Solanum phureja DNA into S. tuberosum dihaploids was analysed by the use of amplified fragment length polymorphism (AFLP) markers. Five dihaploids, derived from crosses between S. tuberosum cv. Pentland Crown and two different S. phureja pollinators (IVP48 and EC90) were investigated by use of 17 AFLP primer pairs. Also 30 dihaploids, derived from pollination of five different S. tuberosum seed parents with S. phureja IVP101, were investigated for the presence of S. phureja‐specific markers. In total approximately 680 and 850 AFLP products were detected in the diploid S. phureja clones and in the tetraploid S. tuberosum genotypes, respectively. A total of 68 S. phureja IVP48‐specific markers were detected, while the total number of S. phureja IVP101‐specific markers was in the range of 72‐96, depending on the S. tuberosum seed parent. Introgression of DNA in the S. tuberosum cv. Pentland Crown dihaploids, after pollination with S. phureja IVP48 and S. phureja EC90, was demonstrated by the detection of 14 of 68 IPV48‐specific markers in the dihaploids. However, no DNA introgression was found in any of the 30 S. tuberosum dihaploids derived from S. phureja IVP101. Hence, S. phureja IVP101 is regarded as an excellent pollinator in the production of S. tuberosum dihaploids in potato breeding programmes because of the high yield of dihaploids per 100 berries, and because no introgression of DNA into the S. tuberosum dihaploids was evidenced.