Quantitative trait loci for lodging resistance,plant height and partial resistance to mycosphaerella blight in field pea (Pisum sativum L.)

With the development of genetic maps and the identification of the most-likely positions of quantitative trait loci (QTLs) on these maps, molecular markers for lodging resistance can be identified. Consequently, marker-assisted selection (MAS) has the potential to improve the efficiency of selection for lodging resistance in a breeding program. This study was conducted to identify genetic loci associated with lodging resistance, plant height and reaction to mycosphaerella blight in pea. A population consisting of 88 recombinant inbred lines (RILs) was developed from a cross between Carneval and MP1401. The RILs were evaluated in 11 environments across the provinces of Manitoba, Saskatchewan and Alberta, Canada in 1998, 1999 and 2000. One hundred and ninety two amplified fragment length polymorphism (AFLP) markers, 13 random amplified polymorphic DNA (RAPD) markers and one sequence tagged site (STS) marker were assigned to ten linkage groups (LGs) that covered 1,274 centi Morgans (cM) of the pea genome. Six of these LGs were aligned with the previous pea map. Two QTLs were identified for lodging resistance that collectively explained 58% of the total phenotypic variation in the mean environment. Three QTLs were identified each for plant height and resistance to mycosphaerella blight, which accounted for 65% and 36% of the total phenotypic variation, respectively, in the mean environment. These QTLs were relatively consistent across environments. The AFLP marker that was associated with the major locus for lodging resistance was converted into the sequence-characterized amplified-region (SCAR) marker. The presence or absence of the SCAR marker corresponded well with the lodging reaction of 50 commercial pea varieties.Communicated by H. F. Linskens     

High-density linkage maps and sex-linked markers for the black tiger shrimp (Penaeus monodon)

We report on the construction of sex-specific high-density linkage maps and identification of sex-linked markers for the black tiger shrimp (Penaeus monodon). Overall, we identified 44 male and 43 female linkage groups (2n = 88) from the analysis of 2,306 AFLP markers segregating in three full-sib families, covering 2,378 and 2,362 cM, respectively. Twenty-one putatively homologous linkage groups, including the sex-linkage groups, were identified between the female and male linkage maps. Six sex-linked AFLP marker alleles were inherited from female parents in the three families, suggesting that the P. monodon adopts a WZ-ZZ sex-determining system. Two sex-linked AFLP markers, one of which we converted into an allele-specific assay, confirmed their association with sex in a panel of 52 genetically unrelated animals.     

Somaclonal variation in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) analyzed using polymorphic and sequenced AFLP markers

Amplified fragment length polymorphism (AFLP) analysis of 24 in vitro regenerated rye plants was performed in order to evaluate the somaclonal variation rate in this species and to identify rye genomic regions where mutations are preferentially promoted by in vitro culture processes. Regenerated plants were obtained from cell lines derived from immature embryos and plants were regenerated by somatic embryogenesis. Twenty-three regenerants showed variation when compared against sibling plants obtained from the same cell line. A total number of 887 AFLP markers were scored, and 8.8% identified the same polymorphism in plants obtained independently from different cell lines, revealing putative mutational hot spots. Using controlled crossings and analysis of the corresponding progenies, we were able to verify the genetic stability in the next generation for only five of these polymorphisms. The nucleotide sequence of the AFLP amplicon of four of the polymorphic markers was obtained, but only the sequence of two markers was clearly identified in the databases. The sequence of marker A1-303 was identified as part of a tandemly repeated sequence, the 120-bp family, which is located at telomeric regions and is widely distributed among rye chromosomes. The marker A5-375 showed high similarity with regions of Angela retrotransposons.     

ATG-anchored AFLP (ATG-AFLP) analysis in cotton

Amplified fragment length polymorphism (AFLP) marker system has had broad applications in biology. However, the anonymous AFLP markers are mainly amplified from non-coding regions, limiting their usefulness as a functional marker system. To take advantages of the traditional AFLP techniques, we propose substitution of a restriction enzyme that recognizes a restriction site containing ATG, called ATG-anchored AFLP (ATG-AFLP) analysis. In this study, we chose NsiI (recognizing ATGCAT) to replace EcoRI in combination with MseI to completely digest genomic DNA. One specific adaptor, one pre-selective primer and six selective amplification primers for the NsiI site were designed for ligation and PCR. Six NsiI and eight MseI primers generated a total of 1,780 ATG-AFLP fragments, of which 750 (42%) were polymorphic among four genotypes from two cultivated cotton species (Upland cotton, Gossypium hirsutum and Pima cotton, G. barbadense). The number of ATG-AFLP markers was sufficient to separate the four genotypes into two groups, consistent with their evolutionary and breeding history. Our results also showed that ATG-AFLP generated less number of total and polymorphic fragments per primer combination (2-3 vs. 4-5) than conventional AFLP within Upland cotton. Using a recombination inbred line (RIL) population, 62 polymorphic ATG-AFLP markers were mapped to 19 linkage groups with known chromosome anchored simple sequence repeat (SSR) markers. Of the nine ATG-AFLP fragments randomly chosen, three were found to be highly homologous to cotton cDNA sequences. An in-silico analysis of cotton and Arabidopsis cDNA confirmed that the ATG-anchored enzyme combination NsiI/MseI did generate more fragments than the EcoRI/MseI combination.     

Mapping of AFLP markers linked to seed coat colour loci in<Emphasis Type="Italic"> Brassica juncea</Emphasis> (L.) Czern

Association mapping of the seed-coat colour with amplified fragment length polymorphism (AFLP) markers was carried out in 39 Brassica juncea lines. The lines had genetically diverse parentages and varied for seed-coat colour and other morphological characters. Eleven AFLP primer combinations were used to screen the 39 B. juncea lines, and a total of 335 polymorphic bands were detected. The bands were analysed for association with seed-coat colour using multiple regression analysis. This analysis revealed 15 markers associated with seed-coat colour, obtained with eight AFLP primer combinations. The marker E-ACA/M-CTG₃₅₀ explained 69% of the variation in seed-coat colour. This marker along with markers E-AAC/M-CTC₂₃₅ and E-AAC/M-CTA₂₅₀ explained 89% of the total variation. The 15 associated markers were validated for linkage with the seed-coat colour loci using a recombinant inbred line (RIL) mapping population. Bands were amplified with the eight AFLP primer combinations in 54 RIL progenies. Of the 15 associated markers, 11 mapped on two linkage groups. Eight markers were placed on linkage group 1 at a marker density of 6.0 cM, while the remaining three were mapped on linkage group 2 at a marker density of 3.6 cM. Marker E-ACA/M-CTG₃₅₀ co-segregated with Gene1 controlling seed-coat colour; it was specific for yellow seed-coat colour and mapped to linkage group 1. Marker E-AAC/M-CTC₂₃₅ (AFLP8), which had been studied previously, was present on linkage group 2; it was specific for brown seed-coat colour. Since AFLP markers are not adapted for large-scale applications in plant breeding, it is important to convert these to sequence-characterised amplified region (SCAR) markers. Marker E-AAC/M-CTC₂₃₅ (AFLP8) had been previously converted into a SCAR. Work is in progress to convert the second of the linked markers, E-ACA/M-CTG₃₅₀, to a SCAR. The two linked AFLP markers converted to SCARs will be useful for developing yellow-seeded B. juncea lines by means of marker-assisted selection.Communicated by H.F. Linskens     

Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties: II. Genetic and technical sources of variation in AFLP data and comparison with SSR data

Accuracy and reproducibility of genetic distances (GDs) based on molecular markers are crucial issues for identification of essentially derived varieties (EDVs). Our objectives were to investigate (1) the amount of variation for amplified fragment length polymorphism (AFLP) markers found among different accessions within maize inbreds and doubled haploid (DH) lines, (2) the proportion attributable to genetic and technical components and marker system specific sources, (3) its effect on GDs between maize lines and implications for identification of EDVs, and (4) the comparison to published SSR data from the same plant materials. Two to five accessions from nine inbred lines and five DH lines were taken from different sources of maintenance breeding or drawn as independent samples from the same seed lot. Each of the 41 accessions was genotyped with 20 AFLP primer combinations revealing 988 AFLP markers. Map positions were available for 605 AFLPs covering all maize chromosomes. On average, six (0.6%) AFLP bands were polymorphic between different accessions of the same line. GDs between two accessions of the same line averaged 0.013 for inbreds and 0.006 for DH lines. The correlation of GDs based on AFLPs and SSRs was tight (r = 0.97**) across all 946 pairs of accessions but decreased (r = 0.55**) for 43 pairs of accessions originating from the same line. On the basis of our results, we recommend specific EDV thresholds for marker systems with different degree of polymorphism. In addition, precautions should be taken to warrant a high level of homogeneity for DNA markers within maize lines before applying for plant variety protection.     

Comparative Assessment of SSR and AFLP Markers for Evaluation of Genetic Diversity and Conservation of Fig, <Emphasis Type="Italic">Ficus carica</Emphasis> L., Genetic Resources in Tunisia

This study characterises the genetic variability of fig, Ficus carica L., using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. It compares the efficiency and utility of the two techniques in detecting variation and establishing genetic relationships among Tunisian fig cultivars. Our results show that using both marker systems, the Tunisian fig germ plasm is characterised by having a large genetic diversity at the deoxyribonucleic acid level, as most of AFLP bands were detected and all SSR markers were polymorphic. In fact, 351 (342 polymorphic) and 57 (57 polymorphic) bands were detected using AFLP and SSR primers, respectively. SSR markers were the most polymorphic with an average polymorphic information content value of 0.94, while AFLP markers showed the highest effective multiplex ratio (56.9) and marker index (45.2). The effective marker index was recorded highest (4.19) for AFLP markers and lowest (0.70) for the SSR ones. Our results demonstrate that (1) independent as well as combined analyses of cluster analyses of SSR and AFLP fragments showed that cultivars are clustered independently from their geographical origin, horticultural classifications and tree sex; (2) the analysis of molecular variance allowed the partitioning of genetic variation within and among fig groups and showed greater variation within groups and (3) AFLP and SSR markers datasets showed positive correlation. This study suggests the SSR and AFLP markers are suitable for diversity analysis and cultivars fingerprinting. An understanding of the genetic diversity and population structure of F. carica in Tunisia can also provide insight into the conservation and management of this species.     

Conversion of AFLP fragments tightly linked to SCMV resistance genes Scmv1 and Scmv2 into simple PCR-based markers

In a previous study, bulked segregant analysis with amplified fragment length polymorphisms (AFLPs) identified several markers closely linked to the sugarcane mosaic virus resistance genes Scmv1 on chromosome 6 and Scmv2 on chromosome 3. Six AFLP markers (E33M61-2, E33M52, E38M51, E82M57, E84M59 and E93M53) were located on chromosome 3 and two markers (E33M61-1 and E35M62-1) on chromosome 6. Our objective in the present study was to sequence the respective AFLP bands in order to convert these dominant markers into more simple and reliable polymerase chain reaction (PCR)-based sequence-tagged site markers. Six AFLP markers resulted either in complete identical sequences between the six inbreds investigated in this study or revealed single nucleotide polymorphisms within the inbred lines and were, therefore, not converted. One dominant AFLP marker (E35M62-1) was converted into an insertion/deletion (indel) marker and a second AFLP marker (E33M61-2) into a cleaved amplified polymorphic sequence marker. Mapping of both converted PCR-based markers confirmed their localization to the same chromosome region (E33M61-2 on chromosome 3; E35M62-1 on chromosome 6) as the original AFLP markers. Thus, these markers will be useful for marker-assisted selection and facilitate map-based cloning of SCMV resistance genes.     

利用向日葵重组自交系构建遗传图谱

以向日葵自选系K55为母本、K58为父本杂交组合,通过单粒传得到的187个F_5：6代重组自交系群体为作图材料,联合应用SSR和AFLP标记构建遗传连锁图谱。经过78对SSR引物和48对AFLP引物组合选择性扩增,分别得到341和1119条带,共1460条,分别获得多态性条带184条和393条,共577条多态性条带,占所有条带的39.52%。SSR和AFLP标记各有84个和108个多态性标记偏离孟德尔分离比例(P=0.05),共192个偏分离标记。采用JoinMap4.0软件进行连锁分析,构建了1张总长度为2759.4 cM、包含17个连锁群、连锁495个多态性标记的遗传图谱,其中偏分离标记170个,标记间的平均图距为5.57 cM。每个连锁群上分布有5~72个标记,长68.88~250.17 cM。本图谱为向日葵永久性图谱,为向日葵重要性状QTL定位和基因克隆奠定基础。     

Development of a CAPS marker for the Pvr4 locus: a tool for pyramiding potyvirus resistance genes in pepper.

The Pvr4 resistance gene in pepper confers a complete resistance to the three pathotypes of potato virus Y (PVY) and to pepper mottle virus (PepMoV). In order to use this gene in a marker-assisted selection (MAS) program and to permit the pyramiding of several potyvirus resistance genes in the same cultivar, tightly linked amplified fragment length polymorphism (AFLP) markers were obtained by the bulked segregant analysis method. Eight linked AFLP markers were mapped in an interval from 2.1 +/- 0.8 to 13.8 +/- 2.9 cM around this locus. The closest codominant AFLP marker was converted into a codominant CAPS (cleaved amplified polymorphic sequence) marker using data from the alignment of the two allele sequences. We have further characterized the relevance of the CAPS marker for MAS programs in different pepper breeding lines.     

Identification of AFLP and microsatellite markers linked with an aluminium tolerance gene in barley ( Hordeum vulgare L.)

Barley is the most sensitive among the cereals to aluminium (Al) stress and breeding for more tolerant cultivars is a priority. To enhance selection efficiency for Al tolerance in barley, PCR-based AFLP and microsatellite markers linked to a locus conferring tolerance to aluminium were identified. The study used F(2) progeny derived from a single cross between Yambla (moderately tolerant of Al) and WB229 (tolerant of Al) and developed hydroponic pulse-recovery screening methods to assess tolerance of phenotypes based on root growth. The segregation ratios of tolerant and sensitive genotypes and F(3) progeny testing suggest that a single major gene controlled Al tolerance ( Alt). In order to determine the chromosomal location of the Alt gene, we used the AFLP technique coupled with bulk segregant analysis. We evaluated tolerant and sensitive bulks using 30 combinations of EcoRI/ MseI primers, and 12 of these permitted differentiation of the sensitive and tolerant bulks. More than 1,000 amplified fragments were obtained, and 98 polymorphic bands were scored. AFLP analysis of wheat-barley chromosome addition lines indicated that the Alt gene was located on barley chromosome 4H. Four chromosome 4H-specific microsatellite markers (Bmac310, Bmag353, HVM68 and HVMCABG) were tightly linked to Alt. The large allelic variation detected with microsatellite marker Bmag353 allowed us to implement this marker for routine marker-assisted selection for Al tolerance, and 396 plants could be screened on a single gel.     

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.     

Use of locus-specific AFLP markers to construct a high-density molecular map in barley

 By using 25 primer combinations, 563 AFLP markers segregating in a recombinant inbred population (103 lines, F₉) derived from L94/Vada were generated. The 38 AFLP markers in common to the existing AFLP/RFLP combined Proctor/Nudinka map, one STS marker, and four phenotypic markers with known map positions, were used to assign present AFLP linkage groups to barley chromosomes. The constructed high-density molecular map contains 561 AFLP markers, three morphological markers, one disease resistance gene and one STS marker, and covers a 1062-cM genetic distance, corresponding to an average of one marker per 1.9 cM. However, extremely uneven distributions of AFLP markers and strong clustering of markers around the centromere were identified in the present AFLP map. Around the centromeric region, 289 markers cover a genetic distance of 155 cM, corresponding to one marker per 0.5 cM; on the distal parts, 906 cM were covered by 277 markers, corresponding to one marker per 3.3 cM. Three gaps larger than 20 cM still exist on chromosomes 1, 3 and 5. A skeletal map with a uniform distribution of markers can be extracted from the high-density map, and can be applied to detect and map loci underlying quantitative traits. However, the application of this map is restricted to barley species since hardly any marker in common to a closely related Triticum species could be identified. Received: 16 June 1997 / Accepted: 9 October 1997     

Molecular mapping of the Oregon Wolfe Barleys: a phenotypically polymorphic doubled-haploid population

A phenotypically polymorphic barley (Hordeum vulgare L.) mapping population was developed using morphological marker stocks as parents. Ninety-four doubled-haploid lines were derived for genetic mapping from an F₁ using the Hordeum bulbosum system. A linkage map was constructed using 12 morphological markers, 87 restriction fragment length polymorphism (RFLP), five random amplified polymorphic DNA (RAPD), one sequence-tagged site (STS), one intron fragment length polymorphism (IFLP), 33 simple sequence repeat (SSR), and 586 amplified fragment length polymorphism (AFLP) markers. The genetic map spanned 1,387 cM with an average density of one marker every 1.9 cM. AFLP markers tended to cluster on centromeric regions and were more abundant on chromosome 1 (7H). RAPD markers showed a high level of segregation distortion, 54% compared with the 26% observed for AFLP markers, 27% for SSR markers, and 18% for RFLP markers. Three major regions of segregation distortion, based on RFLP and morphological markers, were located on chromosomes 2 (2H), 3 (3H), and 7 (5H). Segregation distortion may indicate that preferential gametic selection occurred during the development of the doubled-haploid lines. This may be due to the extreme phenotypes determined by alleles at morphological trait loci of the dominant and recessive parental stocks. Several molecular markers were found to be closely linked to morphological loci. The linkage map reported herein will be useful in integrating data on quantitative traits with morphological variants and should aid in map-based cloning of genes controlling morphological traits. Received: 23 August 2000 / Accepted: 15 December 2000     

Fine mapping of a recessive genic male sterility gene (Bnms3) in rapeseed (Brassica napus) with AFLP- and Arabidopsis-derived PCR markers

9012AB, a recessive genic male sterility (RGMS) line developed from spontaneous mutation in Brassica napus (Chen et al. in Acta Agron Sin 24:431-438, 1998), has been playing an increasing role in hybrid cultivar development in China. The male sterility of 9012AB is controlled by two recessive genes (designated Bnms3 and Bnms4) interacting with one recessive epistatic suppressor gene (esp). Previous study has identified seven AFLP markers, six of which were co-segregated with the Bnms3 gene in a small population (Ke et al. in Plant Breed 124:367-370, 2005). By cloning these AFLP markers and their flanking sequences, five of the six co-segregated markers were successfully converted into sequence characterized amplified region (SCAR) markers. For fine mapping of the Bnms3 gene, these SCAR markers were analyzed in a NIL population of 4,136 individuals. The Bnms3 gene was then genetically mapped to a region of 0.56 cM, with 0.15 cM from marker SEP8 and 0.41 from marker SEP4, respectively. BLAST analysis with these SCAR marker sequences identified a collinear genomic region in Arabidopsis chromosome 5, from which two specific PCR markers further narrowed the Bnms3 locus from an interval of 0.56 to 0.14 cM. These results provide additional information for map-based cloning of the Bnms3 gene and will be helpful for marker-assisted selection (MAS) of elite RGMS lines and maintainers.     

运用近等基因系（NIL）、AFLP、RFLP和SCAR标记对玉米S组育性恢复基因（Rf3）的研究

以1对近等基因系（NIL）及其回交群体（BC     

AFLP targeting of the 1-cM region conferring the vrs1 gene for six-rowed spike in barley, Hordeum vulgare L.

Spike morphology is a key characteristic in the study of barley domestication, yield, and use. Multiple alleles at the vrs1 locus control the development and fertility of the lateral spikelets of barley. We developed five amplified fragment length polymorphism (AFLP) markers tightly linked to the vrs1 locus using well-characterized near-isogenic lines as plant materials. The AFLP markers were integrated into three different maps, in which 'Azumamugi' was used as the maternal parent. Of the three maps, Hordeum vulgare L. 'Azumamugi' x H. vulgare 'Golden Promise' showed recombination of the AFLP markers and the vrs1 locus (closest, 0.05 cM), providing the best mapping population for positional cloning of alleles at the vrs1 locus. Conversion of AFLP bands into polymorphic sequence-tagged sites (STSs) is necessary for further high-throughput genotype scoring and for bacterial artificial chromosome (BAC) library screening. We cloned and sequenced the five AFLP bands and synthesized primer pairs. PCR amplification generated DNAs of the same size from all four parental lines for each marker. Restriction endonuclease treatment of e40m36-1110/AccIII, e34m13-260/Psp1406I, e52m32-270/FokI, and e31m26-520/MnlI revealed fragment length polymorphisms between 'Azumamugi' and all the two-rowed parents. Allelism between the AFLPs and corresponding STS markers was confirmed genetically, indicating the usefulness of the STSs as genetic markers.     

Molecular characterization and identification of markers for toxic and non-toxic varieties of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. using RAPD,AFLP and SSR markers

Jatropha curcas L., a multipurpose shrub has acquired significant economic importance for its seed oil which can be converted to biodiesel, is emerging as an alternative to petro-diesel. The deoiled seed cake remains after oil extraction is toxic and cannot be used as a feed despite having best nutritional contents. No quantitative and qualitative differences were observed between toxic and non-toxic varieties of J. curcas except for phorbol esters content. Development of molecular marker will enable to differentiate non-toxic from toxic variety in a mixed population and also help in improvement of the species through marker assisted breeding programs. The present investigation was undertaken to characterize the toxic and non-toxic varieties at molecular level and to develop PCR based molecular markers for distinguishing non-toxic from toxic or vice versa. The polymorphic markers were successfully identified specific to non-toxic and toxic variety using RAPD and AFLP techniques. Totally 371 RAPD, 1,442 AFLP markers were analyzed and 56 (15.09%) RAPD, 238 (16.49%) AFLP markers were found specific to either of the varieties. Genetic similarity between non-toxic and toxic verity was found to be 0.92 by RAPD and 0.90 by AFLP fingerprinting. In the present study out of 12 microsatellite markers analyzed, seven markers were found polymorphic. Among these seven, jcms21 showed homozygous allele in the toxic variety. The study demonstrated that both RAPD and AFLP techniques were equally competitive in identifying polymorphic markers and differentiating both the varieties of J. curcas. Polymorphism of SSR markers prevailed between the varieties of J. curcas. These RAPD and AFLP identified markers will help in selective cultivation of specific variety and along with SSRs these markers can be exploited for further improvement of the species through breeding and Marker Assisted Selection (MAS).     

Identification of DNA markers of tobacco linked to bacterial wilt resistance

Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive soil-borne diseases in the world. Breeding resistant commercial varieties of tobacco is difficult because most donor candidates' resistance is controlled by polygenes. In this paper, we demonstrate the identification of useful DNA markers for bacterial wilt-resistant tobacco breeding. One hundred and seventeen markers were identified by the amplified fragment length polymorphism (AFLP) method between W6, a burley variety with resistance originating from a Japanese domestic variety, Hatano, and Michinoku 1, a commercial burley wilt-susceptible variety, using 3,072 primer combinations. These markers were analyzed in 125 doubled haploid lines, derived from F(1) hybrids between W6 and Michinoku 1, and a linkage map consisting of ten linkage groups was drawn. The resistance phenotype of each of these lines was investigated on the basis of the average of disease severity obtained from field trials over two growing cycles. Quantitative trait loci (QTL) analysis was performed on the marker phenotypes and the resistance phenotype of each line. One QTL for the bacterial wilt resistance of W6 and DNA markers associated with this QTL were identified on a linkage group consisting of 15 markers, 32 cM in length. This QTL explained more than 30% of the variance in resistance among these lines.     

Conversion of AFLP bands into high-throughput DNA markers

The conversion of AFLP bands into polymorphic sequence-tagged-site (STS) markers is necessary for high-throughput genotype scoring. Technical hurdles that must be overcome arise from genome complexity (particularly sequence duplication), from the low-molecular-weight nature of the AFLP bands and from the location of the polymorphism within the AFLP band. We generated six STS markers from ten AFLP bands (four AFLPs were from co-dominant pairs of bands) in soybean (Glycine max). The markers were all linked to one of two loci, rhg1 on linkage group G and Rhg4 on linkage group A2, that confer resistance to the soybean cyst nematode (Heterodera glycines I.). When the polymorphic AFLP band sequence contained a duplicated sequence or could not be converted to a locus-specific STS marker, direct sequencing of BAC clones anchored to a physical map generated locus-specific flanking sequences at the polymorphic locus. When the polymorphism was adjacent to the restriction site used in the AFLP analysis, single primer extension was performed to reconstruct the polymorphism. The six converted AFLP markers represented 996 bp of sequence from alleles of each of two cultivars and identified eight insertions or deletions, two microsatellites and eight single-nucleotide polymorphisms (SNPs). The polymorphic sequences were used to design a non-electrophoretic, fluorometric assay (based on the TaqMan technology) and/or develop electrophoretic STS markers for high-throughput genotype determination during marker-assisted breeding for resistance to cyst nematode. We conclude that the converted AFLP markers contained polymorphism at a 10- to 20-fold higher frequency than expected for adapted soybean cultivars and that the efficiency of AFLP band conversion to STS can be improved using BAC libraries and physical maps. The method provides an efficient tool for SNP and STS discovery suitable for marker-assisted breeding and genomics.