Combined mapping of AFLP and RFLP markers in barley

AFLP marker technology allows efficient DNA fingerprinting and the analysis of large numbers of polymorphic restriction fragments on polyacrylamide gels. Using the doubled haploids from the F₁ of the cross Proctor × Nudinka, 118 AFLP markers were mapped onto a barley (Hordeum vulgare L.) RFLP map, also including five microsatellite and four protein marker loci. The AFLP markers mapped to all parts of the barley chromosomes and filled in the gaps on barley chromosomes 2L, 4L and 6 in which no RFLP loci had been mapped. Interestingly, the AFLP markers seldom interrupted RFLP clusters, but grouped next to them. The combined map covers 1873 cM, with a total of 282 markers. The merging of AFLP and RFLP markers increased the total map length; 402 cM were added to the map at the tips of chromosomes or in regions corresponding to earlier gaps. Another 375 cM resulted from mapping AFLP markers near to RFLP clusters or in between non-clustered RFLP markers.     

Chromosome landing at the Mla locus in barley (Hordeum vulgare L.) by means of high-resolution mapping with AFLP markers

 The complex Mla locus of barley determines resistance to the powdery mildew pathogen Erysiphe graminis f. sp. hordei. With a view towards gene isolation, a population consisting of 950 F₂ individuals derived from a cross between the near-isogenic lines ‘P01’ (Mla1) and ‘P10’ (Mla12) was used to construct a high-resolution map of the Mla region. A fluorescence-based AFLP technique and bulked segregant analysis were applied to screen for polymorphic, tightly linked AFLP markers. Three AFLP markers were selected as suitable for a chromosome-landing strategy. One of these AFLP markers and a closely linked RFLP marker were converted into sequence-specific PCR markers. PCR-based screening of approximately 70 000 yeast artificial chromosome (YAC) clones revealed three identical YACs harbouring the Mla locus. Terminal insert sequences were obtained using inverse PCR. The derived STS marker from the right YAC end-clone was mapped distal to the Mla locus. Received: 17 July 1998 / Accepted: 9 August 1998     

Application of fluorescence-based semi-automated AFLP analysis in barley and wheat

Genetic mapping and the selection of closely linked molecular markers for important agronomic traits require efficient, large-scale genotyping methods. A semi-automated multifluorophore technique was applied for genotyping AFLP marker loci in barley and wheat. In comparison to conventional ³³P-based AFLP analysis the technique showed a higher resolution of amplicons, thus increasing the number of distinguishable fragments. Automated sizing of the same fragment in different lanes or different gels showed high conformity, allowing subsequent unambigous allele-typing. Simultaneous electrophoresis of different AFLP samples in one lane (multimixing), as well as simultaneous amplification of AFLP fragments with different primer combinations in one reaction (multiplexing), displayed consistent results with respect to fragment number, polymorphic peaks and correct size-calling. The accuracy of semi-automated co-dominant analysis for hemizygous AFLP markers in an F₂ population was too low, proposing the use of dominant allele-typing defaults. Nevertheless, the efficiency of genetic mapping, especially of complex plant genomes, will be accelerated by combining the presented genotyping procedures. Received: 10 April 1999 / Accepted: 11 May 1999     

Comparative AFLP mapping in two hexaploid oat populations

Amplified fragment length polymorphisms (AFLPs) can be used to quickly develop linkage maps in plant species and are especially useful for crops with large genomes like oat (Avena sativa L., 2n=6x=42). High reproducibility and consistency are crucial if AFLP linkage maps are employed for comparative mapping. We mapped AFLP markers in combination with restriction fragment length polymorphism (RFLP) markers in two recombinant inbred populations of hexaploid oat in two laboratories to test the consistency of AFLP markers in a polyploid crop. Eight primer combinations produced 102 and 121 scoreable AFLP markers in the respective populations. In a population from the cross Kanota×Ogle, AFLP markers were placed onto a RFLP reference map consisting of 32 linkage groups. Nineteen linkage groups from another population from the cross Kanota×Marion were assigned to the reference map using AFLP and RFLP markers homologous to those used in the Kanota× Ogle cross. Reproducibility of AFLP assays was high in both laboratories and between laboratories. The AFLP markers were well-distributed across the genome in both populations. Many AFLP markers tended to extend the distance between adjacent RFLP markers in linkage analysis. Of the 27 polymorphic AFLPs common in both populations, 20 mapped to homologous linkage groups, 4 were unlinked in at least one population, and 3 mapped to different linkage groups in the two crosses. We believe that 1 of the 3 markers that mapped to a different linkage group in the two populations mapped to homoeologous linkage groups. The linkage map of hexaploid oat is not yet complete, and genomic rearrangements such as translocations exist among cultivars and are likely to account for the remaining two non-syntenous mapping results. AFLPs provide not only a fast and powerful tool for mapping but could be useful in characterizing genomic structural variations among germplasms in hexaploid oat. Received: 17 December 1999 / Accepted: 28 July 2000     

Deletion-based physical mapping of barley chromosome 7H

Chromosomal mutations in barley (Hordeum vulgare, 2n=2x=14, HH) chromosome 7H added to the common wheat (Triticum aestivum, 2n=6x=42, AABBDD) cultivar Chinese Spring were induced genetically by the gametocidal activity of certain alien chromosomes derived from wild species of the genus Aegilops. The rearranged barley chromosomes were characterized by C-banding, FISH and GISH. Twenty two deletion or translocation chromosomes in a hemizygous condition were selected for deletion mapping of 17 AFLP and 28 STS markers that are specific to 7H. Of the 22 breakpoints in chromosome 7H, seven involved the short arm (7HS), 12 the long arm (7HL) and three were in the centromeric region. The seven 7HS breakpoints separated all four 7HS-specific AFLP markers and split the 21 STS markers into six groups. One breakpoint occurred between two STS markers formerly occupying the same position in the genetic map. All seven 7HS breakpoints were separated from each other by either the AFLP or STS markers. The 12 breakpoints in 7HL divided the 13 7HL-specific AFLP markers into seven groups, and the seven STS markers into three groups. On the other hand, the 12 breakpoints in 7HL were divided into six groups by the AFLP markers and into two groups by the STS markers. This deletion-based map was in accordance with previously published genetic and physical maps using the same STS markers. The breakpoints, AFLP markers and STS markers were arrayed in a consistent order. Received: 5 February 2001 / Accepted: 19 February 2001     

RFLP mapping of the barley homeotic mutant lax-a

The lax-a homeotic mutant of barley has flowers in which lodicules are replaced by stamens (giving five stamens per flower). RFLP mapping of an F₂ population from a Bonus lax-a ¹ x H. spontaneum cross showed that the mutation was on the short arm of chromosome 7(5H), closely linked to the centromere. An additional F₂ population was used to show that the lax-a mutation gave the five-stamen phenotype in all flowers of 6-rowed spikes and that hoods were elevated and reduced in size in lax-a/Hooded double-mutant plants.     

AFLP analysis of genetic relationships between barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) landraces from north Shewa in Ethiopia

Local cultivars adapted to specific environmental conditions are the chief source of seed for farmers in Ethiopia and deserve research priority. The aim of this study was, therefore, to determine the genetic relationships between different barley landraces, from north Shewa in Ethiopia so as to differentiate genotypes known by different local names and facilitate their conservation and use in breeding new varieties. Five AFLP primer combinations were analyzed for 19 barley landraces and five malting varieties. The number of scoreable fragments amplified by each AFLP primer combination varied from 49 to 118 with an average of 84.5 and polymorphic fragments for each primer combination varied from 27 to 77 with an average of 58.5. The average percent polymorphism was 69.9% with values ranging from 55.1% to 75.8%. Cluster analysis placed the accessions and malting varieties into one main group while all the farmers’ cultivars, with the exception of two, were in the other main group. It was shown that sampling of germplasm at a given locality might not represent the whole array of genetic variability of locally grown famers’ cultivars. A comprehensive study of all the farmers’ barley cultivars, grown in different parts of Ethiopia, is required to maximize the efforts of germplasm conservation and utilization in national and regional breeding programs.     

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

 Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications. Received: 30 June 1998 / Accepted: 26 October 1998     

An AFLP linkage map for Douglas-fir based upon multiple full-sib families

An amplified fragment length polymorphism (AFLP) linkage map for coastal Douglas-fir (Pseudotsuga menziesii) was constructed from eight full-sib families each consisting of 40 progeny. These families were part of the British Columbia Ministry of Forests second-generation progeny test program and represent typical family sizes used in progeny trials. For map construction, ten primer pairs using EcoRI+3 and MseI+4 were employed to identify and assay AFLP loci that segregated in backcross configurations. A new technique was used to obtain a single recombination rate for each pair of marker loci: for each locus pair, a recombination rate and log-odd value were estimated across all segregating families using a joint maximum likelihood function that considered the full dataset of segregating genotypes. The resulting matrix of recombination rates between all pairs of loci was used to construct an integrated linkage map using JoinMap. The final map consisted of 19 linkage groups spanning 938.6 cM at an average distance of 9.3 cM between markers. The simultaneous integration of data from multiple families may provide an effective way to construct a linkage map, using the genetic resources inherent in most tree improvement programs, where progeny tests of small size are conducted. The statistical property of number of families used is briefly discussed. For our data, at least three to four families greatly increased the chance of obtaining an informative locus in at least one family. Families as small as ten are adequate for closely linked loci (<10 cM), while the size used in our study (40) is adequate for loci within 30 cM.     

AFLP mapping of quantitative trait loci for yield-determining physiological characters in spring barley

 An amplified fragment length polymorphism (AFLP) map covering 965 cM was constructed using 94 recombinant inbred lines of a cross between the spring barley varieties Prisma and Apex. This map was employed to identify quantitative trait loci (QTLs) controlling plant height, yield and yield-determining physiological characters using an approximate multiple-QTL model, the MQM method. The seven physiological traits were parameters used in a process-based crop-growth model that predicts barley biomass production as affected by daily temperature and radiation. The traits were measured in experiments conducted over 2 years. Except for the relative growth rate of leaf area, all traits examined had at least one QTL in each year. QTLs and their effects were found to vary with developmental stages for one trait, the fraction of shoot biomass partitioned to leaves, that was measured at several stages. Most of the traits were associated, though to different extents, with the denso dwarfing gene (the height-reducing allele in Prisma) located on the long arm of chromosome 3. Some of the QTLs were mapped to similar positions in both years. The results in relation to effects of the dwarfing gene, the physiological basis for QTL×environment interaction, and the relative importance of the parameter traits with respect to yield, are discussed. Received: 17 September 1998 / Accepted: 28 December 1998     

Development of AFLP markers in barley

To investigate the application of amplified fragment length polymorphism (AFLP) markers in barley, 96 primer combinations were used to generate AFLP patterns with two barley lines, L94 and Vada. With seven primer combinations, only a few intense bands were obtained, probably derived from repeated sequences. With the majority of the remaining 89 primer combinations, on average about 120 amplification products were generated, and the polymorphism rate between the two lines was generally over 18%. Based on the number of amplified products and the polymorphism rate, the 48 best primer combinations were selected and tested on 16 barley lines, again including L94 and Vada. Using a subset of 24 primer combinations 2188 clearly visible bands within the range from 80 to 510 bp were generated; 55% of these showed some degree of polymorphism among the 16 lines. L94 versus Vada showed the highest polymorphism rate (29%) and Proctor versus Nudinka yielded the lowest (12%). The polymorphism rates per primer combination showed little dependence on the barley lines used. Hence the most efficient and informative primer combinations identified for a given pair of lines turned out to be highly efficient when applied to others. Generally, more than 100 common markers (possibly locus specific) among populations or crosses were easily identified by comparing 48 AFLP profiles of the parent lines. The existence of such a large number of markers common to populations will facilitate the merging of molecular marker data and other genetic data into one integrated genetic map of barley. Received: 28 October 1996 / Accepted: 27 November 1996     

Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch

Genetic linkage maps have been increasingly developed for a wide variety of plants, using segregating populations such as F₂s or backcrosses between inbred lines. These pedigrees are rarely available in outbred species like forest trees which have long generation times. Thus genetic mapping studies have to use peculiar pedigrees and markers in appropriate configurations. We constructed single-tree genetic linkage maps of European larch (Larix decidua Mill.) and Japanese larch [Larix kaempferi (Lamb.) Carr.] using segregation data from 112 progeny individuals of an hybrid family. A total of 266 markers (114 AFLP, 149 RAPD and 3 ISSR loci) showing a testcross configuration, i.e.heterozygous in one parent and null in the other parent, were grouped at LOD 4.0, θ=0.3. The maternal parent map (L. decidua)consisted of 117 markers partitioned within 17 linkage groups (1152 cM) and the paternal parent map (L. kaempferi) had 125 markers assembled into 21 linkage groups (1206 cM). The map distance covered by markers was determined by adding a 34.7-cM independence distance at the end of each group and unlinked marker. It reached 2537 cM and 2997 cM respectively for European larch and Japanese larch, and represented respectively a 79.6% and 80.8% coverage of the overall genome. A few 3:1 segregating markers were used to identify homologous linkage groups between the European larch and the Japanese larch genetic maps. The PCR-based molecular markers allowed the construction of genetic maps, thus ensuring a good coverage of the larch genome for further QTL detection and mapping studies. Received: 15 March 1999 / Accepted: 29 March 1999     

An association mapping approach to identify flowering time genes in natural populations of Lolium perenne (L.)

We describe an association mapping approach using natural populations of perennial ryegrass (Lolium perenne L.) to identify molecular markers associated with heading date, an important trait affecting seasonal production, tillering, digestibility and grassland management regimes. Twenty-three natural populations originating from throughout Europe, with heading date phenotypes ranging from very early to very late, as well as three synthetic populations (varieties) were used for molecular marker genotyping using AFLP. In total, 589 polymorphic markers were identified. Hierarchical clustering, principal coordinate and other statistical analyses identified four outlying populations forming a clearly distinct sub-group. Removal of those four populations from the subsequent analysis reduced population sub-structure twofold. However, this made relatively little difference to the result of the association analysis. Linear regression identified three markers whose frequency of occurrence correlated with the heading date phenotype. Moreover, these markers were shown to be closely linked to each other within a major QTL on Chromosome 7, explaining 70% of the total variation in heading date. Pairwise linkage disequilibrium among them was also significant. These results suggest that association mapping approaches may be feasible in L. perenne, and that the use of natural populations could provide a useful source of genetic variation in traits of importance in crop improvement.     

Chromosome mapping in barley by means of telotrisomic analysis

Summary A total of 37 genetic markers located in chromosomes 2, 3, 4 and 5 were associated with specific arms by means of telotrisomic analysis in five telotrisomics (Triplo 2 L, 2 S, 3 S, 4 S, 5 L) of barley (Hordeum vulgare L.). The genes v, gp (= gp 2), li, gs 5, tr and msg2 showed a trisomic ratio with Triplo 2 L indicating that these genes were on the long arm of chromosome 2. A disomic ratio was obtained for genes wst 4, gs 5, and v with Triplo 2 S, confirming that these genes were on the long arm of chromosome 2(2 L). A disomic ratio was observed for genes e, f(= lg), sk, and gs6 with Triplo 2 L. Two genes, f(= lg) and gs6 showed a trisomic ratio with Triplo 2S. These results indicated that genes e, f(= lg), sk, and gs 6 are on the short arm of chromosome 2 (2S). Since only one telocentric chromosome was available for chromosome 3, 4 and 5, most of the well-mapped marker genes were tested with those telocentric chromosomes. The genes cu 2, uz, wst, als, gs 2, zb,f2, and cer-zn ³⁴⁸ showed trisomic ratio with the telocentric for chromosome 3. These genes were located on the short arm of chromosome 3 (Robertson 1971). This indicated that the telocentric chromosome is for the short arm of chromosome 3(3 S). A disomic ratio was obtained for genes yst, x _c, al, yst2, a _n, ari-a ⁶ and x _s, indicating that these genes are on the long arm of chromosome 3. Two genes, f9 and K, showed trisomic ratio with the telocentric chromosome for 4, while genes gl(= gl2), br2, yh, lg 3, lg 4 and lk 5 showed disomic ratios. This indicated that the telocentric chromosome is for the short arm of chromosome 4. Two genes, fs 2 and g, were studied with Triplo 5 L. Both showed trisomic ratio, indicating that fs 2 and g are located on Triplo 5 L. The centromere position (C) on chromosome 2, 3 and 4 was thus located as (the left side of C is the short arm and the right is the long arm): chromosome 2: f — sk — gs6 — e — C — gs5 — msg2 — wst4 — v — gp — li — tr; chromosome 3: f2 — cer-zn ³⁴⁸ — uz — gs2 — als — cu2 — wst — zb — C — yst — x _c — al — yst2 — a _n — ari-a ⁶ — x _s; chromosome 4: f9 — K — C — lg4 — lg ³ — gl2 — br2 — lk5 — yh. The centromere position on chromosome 5 was not precisely located.Contribution from the Department of Agronomy, Published with the approval of the director of the Colorado State University Experiment Station as Scientific Series Paper No. 2606. This research was supported in part by by NSF Grant GB 4482X and GB 30 493 to T. Tsuchiya and Colorado State University Experiment Station Hatch Project     

Application of AFLP markers to genome mapping in poultry

The amplified fragment length polymorphism (AFLP) technique has been used to enhance marker density in the East Lansing reference chicken genome map, using a backcross family derived from a Red Jungle Fowl by White Leghorn mating with White Leghorn as the recurrent parent. To date, 204 AFLP markers have been added, expanding overall map coverage by about 25%. To the limits of our resolution, AFLP markers are distributed relatively evenly across the EL reference map. AFLP are about 60% as frequent in a cross within White Leghorns (line 7(2) x 6(3)) in comparison to the more divergent reference map population. Based on apparent identity of size, about 40% of the 7(2) x 6(3) cross AFLP fragments were also polymorphic in the reference map cross. Primer pairs in which one primer contains 3' extensions of three selective nucleotides and the other has two selective nucleotides successfully generated AFLP from chicken DNA, but such pairs appeared to amplify only a subset of those fragments to which they have an exact sequence match. Three different restriction enzymes with 4 bp recognition sites (TaqI, HinP1I and MspI) were found to work well with EcoRI as the rarer of the two AFLP restriction enzymes used, with HinP1I being the most effective of the three. AFLP markers are likely to provide an economical method with which to enhance framework linkage maps of chicken and probably other avian genomes.     

Comparison of frequency distributions of doubled haploid and single seed descent lines in barley

Summary Both doubled haploid (DH) and single seed descent (SSD) methods were used to derive homozygous lines from two crosses of barley. The frequency distributions of grain yield, heading date, and plant height of the DH and SSD lines were compared by the Mann-Whitney U test, Kolmogorov-Smirnov twosample test and Wald-Wolfowitz runs test. It was found that the DH lines distributed in the same manner as the SSD lines with respect to the three characters. The results indicated that although the SSD method had more opportunity for recombination than the DH method, it did not produce a sample of recombinants which differed significantly from the DH sample; thus both methods were equally efficient for use in deriving homozygous lines from F₁ hybrids in a relatively short time.Contribution no. 455 Charlottetown Research Station, Agriculture Canada, P.E.I. (Canada)     

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

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.     

Intrachromosomal mapping of seven biochemical loci in barley,Hordeum vulgare

Seven biochemical loci, AmpA, Amy1, Amy2, Est-H5, Hor1, Hor2, and Wsp-H1, have been intrachromosomally mapped in the barley genome using a previously published RFLP-based genetic map. In all cases, the map locations confirmed prior chromosome assignments and agreed closely with the map positions of their homoeoloci in hexaploid wheat.     

RFLP mapping of manganese efficiency in barley

In many cropping regions of the world, yield is limited by the availability of micronutrients, and micronutrient-efficient cultivars provide a yield advantage. Traditional methods of testing cultivars for micronutrient efficiency are time-consuming and laborious. Molecular markers linked to loci controlling micronutrient efficiency will allow more rapid and efficient selection and introgression of these traits than is currently possible. Using a pot-based bioassay and bulked segregant analysis of an F₂ population, we have identified several RFLPs (grouped distally on chromosome 4HS) linked to a locus for manganese efficiency in barley. This manganese efficiency locus has been designated Mel1. Pot bioassay analysis of intercrosses suggests that three useful sources of manganese efficiency are likely to be allelic at the Mel1 locus. Field evaluation of marker selected F₄ progeny supports the major role of Mel1 in the genetic control of manganese efficiency. Adoption of marker assisted selection for this trait in the Southern Australian barley breeding program has occurred. This has been facilitated by the demonstration that the Mel1 allele of Amagi Nijo can be distinguished from 95 other locally useful varieties and breeder’s lines on the basis of RFLPs identified by just two molecular markers. Received: 20 October 1999 / Accepted: 18 February 2000     

AFLP genetic maps of Eucalyptus globulus and E. tereticornis

 Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient technique for detecting large numbers of DNA markers in eucalypts. We have used AFLP markers in a two-way pseudo-testcross strategy to generate genetic maps of two clones of different Eucalyptus species (E. tereticornis and E. globulus). Of 606 polymorphic fragments scored, 487 segregated in a 1 : 1 ratio, corresponding to DNA polymorphisms heterozygous in one parent and null in the other. In the maternal E. tereticornis map, 268 markers were ordered in 14 linkage groups (919 cM); the paternal E. globulus map had 200 markers in 16 linkage groups (967 cM). Results from PGRI software were compared with MAPMAKER. The average density of markers was approximately 1 per 3.9 cM. Framework markers were ordered with an average confidence level of 90%, covering 80–100% of the estimated Eucalyptus genome size. In order to investigate the homologies between the E. tereticornis and the E. globulus genetic linkage maps, we included 19 markers segregating 3 : 1 in the analysis. Some homeologous linkage groups were recognized. The linkage data developed in these maps will be used to detect loci controlling commercially important traits. Received: 17 July 1997 / Accepted: 13 October 1997