Amplified Fragment Length Polymorphism Map Research Articles

A genetic linkage map based on AFLP and SSR markers and mapping of QTL for dry-matter content in sweetpotato

We developed a genetic linkage map of sweetpotato using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers and a mapping population consisting of 202 individuals derived from a broad cross between Xushu 18 and Xu 781, and mapped quantitative trait loci (QTL) for the storage root dry-matter content. The linkage map for Xushu 18 included 90 linkage groups with 2077 markers (1936 AFLP and 141 SSR) and covered 8,184.5 cM with an average marker distance of 3.9 cM, and the map for Xu 781 contained 90 linkage groups with 1954 markers (1824 AFLP and 130 SSR) and covered 8,151.7 cM with an average marker distance of 4.2 cM. The maps described herein have the best coverage of the sweetpotato genome and the highest marker density reported to date. These are the first maps developed that have 90 complete linkage groups, which is in agreement with the actual number of chromosomes. Duplex and triplex markers were used to detect the homologous groups, and 13 and 14 homologous groups were identified in Xushu 18 and Xu 781 maps, respectively. Interval mapping was performed first and, subsequently, a multiple QTL model was used to refine the position and magnitude of the QTL. A total of 27 QTL for dry-matter content were mapped, explaining 9.0–45.1 % of the variation; 77.8 % of the QTL had a positive effect on the variation. This work represents an important step forward in genomics and marker-assisted breeding of sweetpotato.

Detection of QTL for milk protein percentage in Italian Friesian cattle by AFLP markers and selective genotyping

We targeted quantitative trait loci (QTL) for milk protein percentage (P%) in two Italian Holstein granddaughter design families using selective genotyping in combination with high throughput amplified fragment length polymorphism (AFLP) markers. A total of 64 extreme high and low sires in respect to estimated breeding value (EBV) for P% (EBVP%) were genotyped with 25 AFLP primer combinations that revealed 305 and 291 polymorphisms in the two families. Association between markers and EBVP% was investigated by a linear model only on bands having paternal origin (105 and 96 AFLP bands in family D and S, respectively). Although no marker was significantly associated with the target trait after correction for multiple comparisons, 17 AFLP markers, significant without correction for multiple tests, were considered suggestive of the presence of a QTL. Eleven of these were successfully located on six Bos taurus (BTA) chromosomes by radiation hybrid or in-silico mapping. Ten of these mapped in the immediate neighbourhood (less than 10 cM) of already described QTL for P%. Suggestive association was verified in four regions by microsatellites analysis: one on BTA 10; one on BTA 28; and two on BTA 18. Microsatellites identified significant effects by single marker and interval mapping analyses on BTA 10 and BTA 28, while they were only suggestive of the presence of QTL on BTA 18. In summary, our results firstly indicate that AFLP markers may be used to seek QTL exploiting a selective genotyping approach in GDD, a wide used experimental design in cattle; secondly, propose two approaches for AFLP mapping, namely in-silico mapping exploiting most updated release from the bovine whole genome sequencing project, and physical mapping exploiting a panel of Bovine/Hamster Radiation Hybrids; and thirdly, provide new information on QTLs for an economic important trait in a never investigated Holstein cattle population. AFLP in combination with selective genotyping can be a useful strategy for QTL searching in minor livestock species, sometimes having large economic impact in marginal areas, where more informative markers are still poorly developed.

AFLP Mapping of Soybean Maturity Gene E4

Days to flowering and maturity are controlled by genes E1-E7 and J in soybean. Previous studies revealed that E1-E5 and E7 influence tolerances to low-temperature-induced seed coat browning in different directions at various intensities. The E4 locus is useful for the development of early maturing cultivars with chilling tolerance because the recessive allele conditions both the early-maturing habit and chilling tolerance. This study was conducted to obtain a fine map of E4 by amplified fragment length polymorphism (AFLP) analysis using a F(8:9) family segregating for E4 that was developed from a cross between photoperiod-insensitive Japanese landraces, Sakamotowase (E4) and Miharudaizu (e4). AFLP analysis using a total of 4096 primer pairs detected 20 polymorphic markers between near-isogenic lines for E4. Linkage mapping incorporated 16 AFLP markers into a previously constructed genetic map around E4 in linkage group I. Eight AFLP markers were localized to unfilled areas between E4 and the closest markers identified previously. Two AFLP markers flanking E4, e48m41-8 and e18m38-8, were mapped at positions 0.6 and 5.4 cM apart from E4, respectively. They were dominant and in cis arrangement with the recessive allele (e4) conditioning the photoperiod insensitivity and chilling tolerance. These markers can be used in developing more precise markers for fine mapping and marker-assisted selection and in isolating the underlying gene via genome walking approaches.

Amplified Fragment Length Polymorphism Mapping of Tomato Backcross Populations

Tomato backcross populations of Lycopersicon exculentum cv. Money Maker x Lycopersicon pennellii LA716 were used for construction of an Amplified Fragment Length Polymorphism (AELP) map based on pennellii markers. Ten primer combinations (7 of Eco-Mse and 3 of Pst-Mse) were used. This map spanned 893.7 cM and contained 187 AFLP markers, consisting 130 previously found markers and 57 new markers. A total of 141 markers were found from Eco-Mse primer combination and 46 from Pst-Mse primer combination. The Eco-Mse AFLP markers were not evenly distributed over the chromosomes. The Pst-Mse AFLP markers were found quite frequently and evenly distributed in the more distal part of the chromosomes. Eco-Mse markers were found quite more in numbers in chromosomes 5 and 9, whereas Pst-Mse markers in chromosomes 1 and 7. In chromosomes 4, 6, 7 and 8, the new markers and previously found markers were found quite equal in numbers. The average number of informative markers per primer combination was 18.7 ranging from 6 (E33M47) to 36(E35M48). The average size of map per chromosome was found 74.5 cM ranging from 46.2 cM of chromosome 8 and 105.5 cm of chromosome 3. Fourteen BC1 plants for BC2 and subsequently 12 BC2 plants for BC3 generation were selected to obtain inbred lines with genes of interest. BC1 plants were selected each representing about 50% Lycopersicon pennellii LA716 genome and BC2 plants were selected each representing about 25% genome coverage, and together cover 100% of the Lycopersicon pennellii genome based on scored markers J. Inst. Agric. Anim. Sci. 2003 24:21-28.

Genomic distribution of MITEs in barley determined by MITE-AFLP mapping

Miniature inverted-repeat transposable elements (MITEs) represent a large superfamily of transposons that are moderately to highly repetitive and frequently found near or within plant genes. To elucidate the organization of MITEs in the barley genome, MITEs were integrated into the genetic map of barley. In this report, we describe the use of MITEs in amplified fragment length polymorphism (AFLP) mapping, and demonstrate their superiority over conventional AFLP mapping. Barley MITEs include members of the Stowaway, Barfly, and Pangrangja families. By amplifying the flanking sequences of these MITEs, a total of 214 loci were mapped from a population of 93 doubled-haploid segregating individuals between Hordeum vulgare ssp. vulgare and H. vulgare ssp. spontaneum. The 214 MITE-AFLP and 40 anchor simple sequence repeat (SSR) loci were distributed on 7 linkage groups, covering a total map distance of 1 165 cM. The average marker density on each chromosome ranged between 3.4 and 9.6 cM per locus. Only 1 MITE-based locus was frequently found to be associated with MITE loci from the same family, resulting in clusters in chromosomal subregions. In barley, it will be possible to cover the entire genome with a limited set of MITE-based primers and to build highly dense maps of specific regions.

High-density AFLP map of nonbrittle rachis 1 (btr1) and 2 (btr2) genes in barley (Hordeum vulgare L.)

Wild relatives of barley disperse their seeds at maturity by means of their brittle rachis. In cultivated barley, brittleness of the rachis was lost during domestication. Nonbrittle rachis of occidental barley lines is controlled by a single gene (btr1) on chromosome 3H. However, nonbrittle rachis of oriental barley lines is controlled by a major gene (btr2) on chromosome 3H and two quantitative trait loci on chromosomes 5HL and 7H. This result suggests multiple mutations of the genes involved in the formation of brittle rachis in oriental lines. The btr1 and btr2 loci did not recombine in the mapping population analyzed. This result agrees with the theory of tight linkage between the two loci. A high-density amplified fragment-length polymorphism (AFLP) map of the btr1/btr2 region was constructed, providing an average density of 0.08 cM/locus. A phylogenetic tree based on the AFLPs showed clear separation of occidental and oriental barley lines. Thus, barley consists of at least two lineages as far as revealed by molecular markers linked to nonbrittle rachis genes.

Amplified fragment length polymorphism linkage analysis of common buckwheat (Fagopyrum esculentum) and its wild self-pollinated relative Fagopyrum homotropicum.

Common buckwheat (Fagopyrum esculentum) (2n = 2x = 16) and Fagopyrum homotropicum (2n = 2x = 16) were mated in an interspecific cross and amplified fragment length polymorphism (AFLP) linkage maps were constructed by analyzing segregation in the F2 population. Six hundred and sixty-nine bands were identified using 20 AFLP primer combinations, of which 462 (69%) segregated in the F2 population. The map of F. esculentum has eight linkage groups with 223 markers covering a total of 508.3 cM. The map of F. homotropicum has eight linkage groups with 211 markers covering 548.9 cM. There was one to one correspondence of the esculentum and homotropicum linkage groups. Three morphological markers, distylous self-incompatibility, shattering habit, and winged seed, were located on the AFLP map. Distylous self-incompatibility and shattering habit are tightly linked to each other (1.3 cM) and are located near the center of linkage group 1. Winged seed is located on linkage group 4.

Molecular characterization of Fusarium head blight resistance from wheat variety Wangshuibai

Fusarium head blight (FHB) is a destructive disease of wheat worldwide. FHB resistance genes from Sumai 3 and its derivatives such as Ning 7840 have been well characterized through molecular mapping. In this study, resistance genes in Wangshuibai, a Chinese landrace with high and stable FHB resistance, were analyzed through molecular mapping. A population of 104 F2-derived F7 recombinant inbred lines (RILs) was developed from the cross between resistant landrace Wangshuibai and susceptible variety Alondra‘s’. A total of 32 informative amplified fragment length polymorphism (AFLP) primer pairs (EcoRI/MseI) amplified 410 AFLP markers segregating among the RILs. Among them, 250 markers were mapped in 23 linkage groups covering a genetic distance of 2,430 cM. In addition, 90 simple sequence repeat (SSR) markers were integrated into the AFLP map. Fifteen markers associated with three quantitative trait loci (QTL) for FHB resistance (P < 0.01) were located on two chromosomes. One QTL was mapped on 1B and two others were mapped on 3B. One QTL on 3BS showed a major effect and explained up to 23.8% of the phenotypic variation for type II FHB resistance.

A preliminary genetic map of walking catfish ( Clarias macrocephalus)

We have constructed the first linkage map for walking catfish, Clarias macrocephalus, using amplified fragment length polymorphism (AFLP) markers. Forty-seven primer combinations were used to amplify DNA samples of a female C. macrocephalus and 79 haploid embryos. The AFLP map consists of 134 loci placed into 31 linkage groups, with 12 loci remaining unlinked. The number of markers per linkage group varies from 3 to 14. The map spans an estimated 2037.4 Kosambi cM, with an average spacing of 17.07 cM. This linkage map will provide the basis for a high-resolution map for walking catfish.

An AFLP-based genome-wide mapping strategy.

To efficiently determine the chromosomal location of phenotypic mutants, we designed a genome-wide mapping strategy that can be used in any crop for which a dense AFLP (Amplified Fragment Length Polymorphism) map is available or can be made. The AFLP technique is particularly suitable to initiate map-based cloning projects because it detects many markers per reaction. First a standard set of AFLP primer combinations that results in a framework of AFLP markers well dispersed over the genome is selected. These primer combinations are applied to a limited number of mutant individuals from a segregating population to register linkage and non-linkage of the AFLP markers to the gene-of-interest. Further delineation of the area of interest is accomplished by analyzing the remaining recombinants and additional mutant individuals with AFLP markers that lie within the identified region. We illustrate the usefulness of the method by mapping three rotunda ( ron) leaf-form mutant loci of Arabidopsis thaliana and show that in the initial phase of map-based cloning projects a 400-600 kb interval can be identified for the average mutant locus within a few weeks. Once such an area is identified and before initiating the more time-consuming fine-mapping procedure, it is essential to examine publicly available databases for candidate genes and known mutants in the identified region. The 390-kb interval on chromosome 4 that harbors the ron2 mutation, also carries a known flower mutant, leunig ( lug); upon crossing, the two mutants appeared to be allelic. When no such candidates are found, the mapping procedure should be continued. We present a strategy to efficiently select recombinants that can be used for fine mapping.

AFLP linkage map of the Japanese quail Coturnix japonica.

The quail is a valuable farm and laboratory animal. Yet molecular information about this species remains scarce. We present here the first genetic linkage map of the Japanese quail. This comprehensive map is based solely on amplified fragment length polymorphism (AFLP) markers. These markers were developed and genotyped in an F2 progeny from a cross between two lines of quail differing in stress reactivity. A total of 432 polymorphic AFLP markers were detected with 24 TaqI/EcoRI primer combinations. On average, 18 markers were produced per primer combination. Two hundred and fifty eight of the polymorphic markers were assigned to 39 autosomal linkage groups plus the ZW sex chromosome linkage groups. The linkage groups range from 2 to 28 markers and from 0.0 to 195.5 cM. The AFLP map covers a total length of 1516 cM, with an average genetic distance between two consecutive markers of 7.6 cM. This AFLP map can be enriched with other marker types, especially mapped chicken genes that will enable to link the maps of both species and make use of the powerful comparative mapping approach. This AFLP map of the Japanese quail already provides an efficient tool for quantitative trait loci (QTL) mapping.

Characterization of mixed disomic and polysomic inheritance in the octoploid strawberry (Fragaria x ananassa) using AFLP mapping.

A two-way pseudo-testcross strategy, combined with Single Dose Restriction Fragment (SDRF) marker analysis, was used for genetic mapping in the octoploid cultivated strawberry Fragaria x ananassa (2n = 8 x = 56). Based on a 113 full-sib progeny from a cross between the variety Capitola and the clone CF1116, we generated two parental maps using Amplified Fragment Length Polymorphism (AFLP) markers. Ninety two percent of the markers (727 out of 789) showed ratios corresponding to simplex markers (the majority being SDRF markers), and 8% (62 out of 789) fitted a multiplex ratio. Linkage maps were first established using SDRF markers in coupling phase. The female map comprised 235 markers distributed among 43 co-segregation groups, giving a map size of 1,604 cM. On the male map, 280 markers were assigned to 43 co-segregation groups, yielding a map size of 1,496 cM. Once the co-segregation groups were established, their association was tested using repulsion-phase markers. In total, taking into account associations representing the same linkage groups, 30 linkage groups were detected on the female side and 28 on the male side. On the female map, 68.3% of the pairwise marker linkages were in coupling versus 31.7% in repulsion phase, and the corresponding figures on the male map were 72.2% and 27.8%, respectively. In addition, both groups linked only in the coupling phase and groups linked in the repulsion phase were characterized. The observations suggest that the meiotic behavior of the F. x ananassa genome is neither fully disomic nor fully polysomic, but rather mixed. The genome may not be as completely diploidized as previously assumed.

Construction of a High-density AFLP and SSR Map Using Recombinant Inbred Lines of Cultivated * Weedy Soybean

A genetic linkage map of soybean was constructed by using a cross between a cultivar ‘Keburi’ and a weedy form of soybean ‘Masshokutou Kou 502’. The parents differed in their ability in somatic embryogenesis. The mapping population consisted of 117 recombinant inbred lines (F11), and generated 30 linkage groups, which covered 2089 cM, including 515 amplified-fragment-length polymorphism (AFLP) markers and 85 simple sequence repeat (SSR) markers. For AFLP analysis, a simple technique using small polyacrylamide gels combined with silver staining was applied, which enabled a single person to construct a map in a short time at a reasonable cost. Comparison of this map with previously published maps revealed that 1) the level of polymorphism was the same as in a cultivar × cultivar cross; and 2) the cultivar × weed map was 13 % shorter than the cultivar × wild map. The high-density map constructed in this study would contribute to the molecular mapping of quantitatively and qualitatively inherited characters from weedy forms of soybean.

Development of sequence-characterized amplified regions (SCARs) from amplified fragment length polymorphism (AFLP) markers tightly linked to the Vf gene in apple

Amplified fragment length polymorphism (AFLP) markers have become widely used in saturating the region of a gene of interest for the ultimate goal of map-based cloning of the gene or for marker-assisted selection. However, conversion of AFLP markers into restriction fragment length polymorphism (RFLP) or polymerase chain reaction (PCR)-based markers will greatly expand their usefulness in genetic applications. Previously, we have identified 15 AFLP markers tightly linked to the Vf gene conferring scab resistance in apple. In this study, we have successfully converted 11 of these AFLPs into sequence-characterized amplified region (SCAR) markers. Of the remaining four nonconverted AFLP markers, one, ET2MC8-1, has been found to be very short (83 base pairs) and is an A/T rich (90%) marker; a second, EA2MG11-1, has shown identical sequences between Malus floribunda 821 (the original source of the Vf gene) and scab-susceptible apple cultivars; while the other two, EA12MG16-1 and ET8MG1-1, have not been cloned. Using the 11 converted SCAR markers along with 5 previously identified SCAR markers, a high-resolution linkage map around the Vf gene has been constructed, and found to be consistent with its corresponding AFLP map. Three converted SCAR markers (ACS-3, -7, and -9) are inseparable from the Vf gene; whereas one (ACS-6) is located left of, and the remaining seven (ACS-1, -2, -4, -5, -8, -10, and -11) are located right of the Vf gene at genetic distances of 0.4 and 0.2 cM, respectively. A reliable and robust procedure for development of SCAR markers from AFLP markers is presented.Key words: apple, AFLP, SCAR, apple scab disease, Vf gene.

Development of sequence-characterized amplified regions (SCARs) from amplified fragment length polymorphism (AFLP) markers tightly linked to the Vf gene in apple.

Amplified fragment length polymorphism (AFLP) markers have become widely used in saturating the region of a gene of interest for the ultimate goal of map-based cloning of the gene or for marker-assisted selection. However, conversion of AFLP markers into restriction fragment length polymorphism (RFLP) or polymerase chain reaction (PCR)-based markers will greatly expand their usefulness in genetic applications. Previously, we have identified 15 AFLP markers tightly linked to the Vf gene conferring scab resistance in apple. In this study, we have successfully converted 11 of these AFLPs into sequence-characterized amplified region (SCAR) markers. Of the remaining four nonconverted AFLP markers, one, ET2MC8-1, has been found to be very short (83 base pairs) and is an A/T rich (90%) marker; a second, EA2MG11-1, has shown identical sequences between Malus floribunda 821 (the original source of the Vf gene) and scab-susceptible apple cultivars; while the other two, EA12MG16-1 and ET8MG1-1, have not been cloned. Using the 11 converted SCAR markers along with 5 previously identified SCAR markers, a high-resolution linkage map around the Vf gene has been constructed, and found to be consistent with its corresponding AFLP map. Three converted SCAR markers (ACS-3, -7, and -9) are inseparable from the Vf gene; whereas one (ACS-6) is located left of, and the remaining seven (ACS-1, -2, -4, -5, -8, -10, and -11) are located right of the Vf gene at genetic distances of 0.4 and 0.2 cM, respectively. A reliable and robust procedure for development of SCAR markers from AFLP markers is presented.

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.

Mapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping.

By combining the amplified fragment length polymorphism (AFLP) technique with selective genotyping, we constructed a linkage map for rice and assigned each linkage group to a corresponding chromosome. The AFLP map, consisting of 202 AFLP markers, was generated from 74 recombinant inbred lines (RIL) which were selected from both extremes of the population (250 lines) with respect to the response to complete submergence. Map length was 1756 cM, with an average interval size of 8.5 cM. To assign linkage groups to chromosomes, we used 50 previously mapped AFLP markers as anchor markers distributed over the 12 chromosomes. Other AFLP markers were then assigned to specific chromosomes based on their linkage to anchor markers. This AFLP map is equivalent to the RFLP/AFLP map constructed previously as the anchors were in the same order in both maps. Furthermore, tests with two restriction fragment length polymorphism (RFLP) markers and two sequence-tagged site (STS) markers showed that they mapped in the expected positions. Using this AFLP map, a major gene for submergence tolerance was localized on chromosome 9. Quantitative trait loci (QTL) associated with submergence tolerance were detected on chromosomes 6, 7, 11, and 12. We conclude that the combination of AFLP mapping and selective genotyping provides a much faster and easier approach to QTL identification than the use of RFLP markers.