Polymorphism, distribution, and segregation of AFLP markers in a doubled haploid rice population

We exploited the newly developed amplified fragment length polymorphism (AFLP) technique to study the polymorphism, distribution and inheritance of AFLP markers with a doubled haploid rice population derived from ‘IR64’/‘Azucena’. Using only 20 pairs of primer combinations, we detected 945 AFLP bands of which 208 were polymorphic. All 208 AFLP markers were mapped and distributed over all 12 chromosomes. When these were compared with RFLP markers already mapped in the population, we found the AFLP markers to be highly polymorphic in rice and to follow Mendelian segregation. As linkage map of rice can be generated rapidly with AFLP markers they will be very useful for marker-assisted backcrossing. Received: 11 April 1996 / Accepted: 14 June 1996     

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     

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     

60份优质番茄自交系遗传多样性AFLP分析

本文以AFLP技术对60份优质番茄自交系的遗传多样性进行了分析。17对AFLP引物组合共扩增得到905条带,其中多态性条带251条,平均每对引物检测到约15个多态性位点,平均多态率为27.7%;仅用E7M4和E7M10两对引物组合就可以绘制60份番茄自交系的指纹图谱。60个自交系间的Jaccard遗传相似系数变幅为0.259-0.952,平均值为0.664。采用UPGMA方法聚类,遗传相似系数变幅为0.54-0.57时,60份番茄材料可以划分为7个类群。聚类结果与来源、果实大小、果形等性状未表现出明显的相关性。     

Evaluation of AFLP in Beta

 AFLP markers were evaluated for their usefulness in the genetic analysis of sugarbeet and wild Beta species. Accessions of ten different sugarbeet breeding lines and five wild beets were screened using 256 primer combinations. Of the 11 309 bands investigated, 96.4% were polymorphic among the accessions. A strong positive correlation was found between the number of polymorphisms and AT content of the selective bases of the primer combinations. Random subsets of primer combinations were used to produce genetic distance trees. Permutation tests showed that, for the wild beets, 500 AFLP bands sufficed to obtain the best topology of the tree with a probability at any given node of more than 99%. Ten times as many bands were necessary to obtain support values of the same order of magnitude for the sugarbeet lines. The reproducibility of AFLP for seven primer combinations was investigated by repeated analysis of all steps from DNA isolation to data scoring. For 5088 comparisons, the overall reproducibility was 97.6%. Robustness to genotyping errors was investigated by including an artificial F₁ (1 : 1 DNA mixture) of two sugarbeet lines in the screen for polymorphisms. For the 3160 cases of polymorphism between the two lines, 0.2% genotyping errors were found. The general reliability and usefulness of AFLP markers are discussed in relation to the results obtained. Received: 18 May 1998 / Accepted: 28 October 1998     

Amplified fragment length polymorphisms as a tool for DNA fingerprinting sunflower germplasm: genetic diversity among oilseed inbred lines

 Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient method for producing DNA fingerprints. The AFLP diversity of sunflower has not been described, and much of the public germ plasm of sunflower has not yet been fingerprinted. Our objectives were to: (1) estimate genetic similarities, polymorphism rates, and polymorphic information contents (PICs) for AFLP markers among elite public oilseed inbred lines, and (2) assess the genetic diversity of inbred lines using genetic similarities estimated from AFLP fingerprints. We produced fingerprints for 24 public inbred lines of sunflower (Helianthus annuus L.) using six AFLP primer combinations. These primers produced a total of 359 AFLP markers or about 60 markers per primer combination. Genetic similarities ranged from 0.70 to 0.91, polymorphism rates ranged from 7 to 24%, and PICs ranged from 0.0 to 0.5. Genetic similarities were lower overall for maintainer (B)×restorer (R) crosses than for B×B or R×R crosses. Principal-coordinate and cluster analyses separated lines into two groups, one for B-lines and another for R-lines. These groupings illustrate the breeding history and basic heterotic pattern (B×R) of sunflower and the widespread practice of using B×B and R×R crosses to develop new lines. There were, nevertheless, distinct subgroups within these groups. These subgroups may represent unique heterotic groups and create a basis for formally describing heterotic patterns in sunflower. Received: 10 June 1996 / Accepted: 4 April 1997     

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. Received: 20 December 1996 / Accepted: 21 January 1997     

An integrated interspecific AFLP map of lettuce (Lactuca) based on two L. sativa × L. saligna F2 populations

AFLP markers were obtained with 12 EcoRI/ MseI primer combinations on two independent F₂ populations of Lactuca sativa ×Lactuca saligna. The polymorphism rates of the AFLP products between the two different L. saligna lines was 39%, between the two different L. sativa cultivars 13% and between the L. sativa and L. saligna parents on average 81%. In both F₂ populations segregation distortion was found, but only Chromosome 5 showed skewness that was similar for both populations. Two independent genetic maps of the two F₂ populations were constructed that could be integrated due to the high similarity in marker order and map distances of 124 markers common to both populations. The integrated map consisted of 476 AFLP markers and 12 SSRs on nine linkage groups spanning 854 cM. The AFLP markers on the integrated map were randomly distributed with an average spacing between markers of 1.8 cM and a maximal distance of 16 cM. Furthermore, the AFLP markers did not show severe clustering. This AFLP map provides good opportunities for use in QTL mapping and marker-assisted selection. Received: 13 July 2000 / Accepted: 19 January 2001     

SRAP技术研究烟粉虱遗传多样性

采用AFLP、SRAP2种标记方法分别对2个烟粉虱Bemisia tabaci Gennadius种群(一品红、甘蓝)的多态性进行分析。结果表明,(1)2种方法平均每对引物组合产生的条带数分别为29.4和21.8。(2)AFLP法每对引物组合产生10～23条多态性带,平均17.20条,多态性带的比例平均为57.93%。SRAP法每对引物组合产生5～18条多态性带,平均13.3条,多态性带的比例平均为60.59%。(3)前者的基因多样性范围为0.1503～0.2838,平均为0.2297;后者的基因多样性范围为0.0977～0.2911,平均为0.2332。证明利用SRAP技术和AFLP技术研究烟粉虱的遗传多样性是有效的。     

Genetic relationships among South-East Turkey wild barley populations and sampling strategies of Hordeum spontaneum

To assess the genetic diversity and the genetic structure of Turkish wild barley (Hordeum spontaneum Tell.) populations, 76 genotypes from ten ecologically and geographically different locations were analyzed by means of amplified fragment length polymorphism (AFLP) markers. Five primer combinations produced 187 scorable bands, of which 117 (62.6%) were polymorphic. Several population-specific and genotype-specific bands were identified, which differentiate populations or genotypes. Genetic distance, determined by Nei’s distance coefficient, varied from 0.07 to 0.21 with an average of 0.13. In the UPGMA dendrogram based on Nei genetic distances, the Hordeum spontaneum populations were separated into two major clusters. Genetic diversity was larger among (68%) than within (32%) populations. Eight AFLP bands were strongly correlated to the altitude of the collecting site, while no clear trend was detected between geographical origin and genetic diversity. Our results strongly suggest the need for a change in Hordeum spontaneum sampling strategy: more populations, rather then more individuals within population, should be sampled to appraise and safeguard genetic diversity in the wild barley gene pool.     

Genetic structure in cultivated and wild carrots (Daucus carota L.) revealed by AFLP analysis

Genetic variation within and among five Danish populations of wild carrot and five cultivated varieties was investigated using amplified fragment length polymorphism (AFLP). Ten AFLP primer combinations produced 116 polymorphic bands. Based on the marker data an UPGMA-cluster analysis and principal component analysis (PCA) separated the Daucus collections into three groups, consisting of the wild populations, the old varieties, and the recently bred varieties. The genetic distance between the wild populations reflected the physical distance between collection sites. Analysis of genetic diversity showed that the old varieties released between 1974 and 1976 were more heterogeneous than the newly developed F₁ hybrid varieties. The analysis of molecular variation (AMOVA) showed that the major part of the genetic variation in the plant material was found within populations/varieties. The presence of markers specific to the cultivated carrot makes it possible to detect introgression from cultivated to wild types. Received: 6 October 1999 / Accepted: 4 November 1999     

羊草种质基因组DNA的AFLP多态性研究

羊草是禾本科牧草之王 ,在当前我国西部生态建设和草原畜牧业发展中发挥着重要作用。用AFLP方法对2 7份我国不同地区分布的羊草 (Leymuschinensis (Trin .)Tzvel)材料进行了基因组DNA多态性分析 ,8对AFLP引物组合在 2 7个不同羊草基因型中共扩增出 5 37条带 ,产生出的DNA片段大小分布在 75bp - 5 30bp之间。其中单态性带 89条 ,占 16 .6 % ,多态性带 32 9条 ,占 6 1.3%。平均每对引物组合扩增的DNA带数为 6 6 .13,总的多态性比率为 78.84%。AFLP多态信息含量PIC值分布于 0 .0 - 0 .5之间 ,平均PIC值为 0 .2 16 ,出现的PIC最大值 (0 .5 )约占AFLP标记的 8.5 % ,说明羊草基因组DNA的多态性比较丰富。以 5 37个AFLP标记为原始数据 ,根据Nei和Li的方法对 2 7份羊草材料进行遗传变异和聚类分析的结果表明 :羊草种内有高频率的遗传变异发生 ,且与地理分布和生态环境密切相关 ;2 7份羊草不同基因型被划分为四大类群 ,不同类群相互间的遗传距离相对较大 ,在树状图中表现为较远的亲缘关系。对羊草种内遗传变异发生的原因和品种的形成进行了初步讨论。     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

Genetic differentiation of Helicoverpa armigera (Hübner) and H. assulta (Guenée) (Lepidoptera: Noctuidae) based on AFLP markers

Abstract Here we use amplified fragment length polymorphism (AFLP) to assess genetic differentiation of Helicoverpa armigera and H. assulta . The results indicated that both species-specific fingerprints and cluster analysis showed the ability of AFLP technique to discriminate the two sibling species; among a total 1963 AFLP markers amplified from nine primer combinations: 777 (39.6%) were H. armigera -specific, 602 (30.7%) were H. assulta -specific, and 584 (29.7%) were common bands. The mean number of H. armigera -specific bands was significantly more than that of H. assulta -specific bands for nine primer combinations ( P < 0.05); the intraspecific distance of H. armigera and H. assulta was 0.123 0 and 0.110 7 respectively, and the interspecific distance was 0.178 3. In addition, the percentage of polymorphic loci and estimated average heterozygosity were used to estimate genetic diversity of the two species. This study therefore demonstrates that AFLP analysis is a sensitive and reliable technique to study genetic differentiation and genetic relationships between species and provides sufficient molecular markers for future linkage map construction, location and eventual cloning of genes involved in traits differentiation.     

Efficiency of RFLP, RAPD, and AFLP markers for the construction of an intraspecific map of the tomato genome.

We have constructed a tomato genetic linkage map based on an intraspecific cross between two inbred lines of Lycopersicon esculentum and L. esculentum var. cerasiforme. The segregating population was composed of 153 recombinant inbred lines. This map is comprised of one morphological, 132 RFLP (restriction fragment length polymorphism, including 16 known-function genes), 33 RAPD (random amplified polymorphic DNA), and 211 AFLP (amplified fragment length polymorphism) loci. We compared the 3 types of markers for their polymorphism, segregation, and distribution over the genome. RFLP, RAPD, and AFLP methods revealed 8.7%, 15.8%, and 14.5% informative bands, respectively. This corresponded to polymorphism in 30% of RFLP probes, 32% of RAPD primers, and 100% of AFLP primer combinations. Less deviation from the 1:1 expected ratio was obtained with RFLP than with AFLP loci (8% and 18%, respectively). RAPD and AFLP markers were not randomly distributed over the genome. Most of them (60% and 80%, respectively) were grouped in clusters located around putative centromeric regions. This intraspecific map spans 965 cM with an average distance of 8.3 cM between markers (of the framework map). It was compared to other published interspecific maps of tomato. Despite the intraspecific origin of this map, it did not show any increase in length when compared to the high-density interspecific map of tomato.     

Genetic variation in an endemic salamander, Salamandra atra, using amplified fragment length polymorphism

The pattern of genetic differentiation of the endemic alpine salamander, Salamandra atra, has been studied using amplified fragment length polymorphism (AFLP) from 11 populations throughout the range of the two currently recognized subspecies, atra and aurorae. Five different primer combinations produced 706 bands and were analyzed by constructing a phylogenetic tree using NJ and principal component analysis. Significant genetic variation was revealed by AFLP between and within populations but, our results show a lack of genetic structure. AFLP markers seems to be unsuitable to investigate complex and recent diversification.     

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.     

Assessment of genetic diversity in 31 species of mangroves and their associates through RAPD and AFLP markers

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity in 31 species of mangroves and mangrove associates. Four AFLP primer combinations resulted in the amplification of 840 bands with an average of 210 bands per primer combination and 11 RAPD primers yielded 319 bands with an average of 29 bands per primer. The percentage of polymorphism detected was too high indicating the high degree of genetic variability in mangrove taxa both at inter- and intra-generic levels. In the dendrogram, species belonging to a particular family/ genus, taxa inhabiting similar habitats or having similar adaptations tended to be together. There were exceptions too; as many unrelated species of mangroves form clusters. The intrafamilial classification and inter-relationships of genera in the family Rhizophoraceae could be confirmed by molecular analysis. Both the markers RAPD and AFLP were found equally informative and useful for a better understanding of the genetic variability and genome relationships among mangroves and their associated species.     

Cloning and genetic mapping of wheat telomere-associated sequences

Wheat telomere-associated sequences (TASs) were cloned using a Vectorette approach and sequenced. Reverse primers specific to the TASs were combined with labelled degenerate telomere primers in PCR reactions containing total genomic DNA as template. Amplification products were separated on sequencing gels. In total, seventeen primer combinations provided 47 polymorphic fragments. Nine of these mapped beyond the most distal RFLP markers and defined the ends of seven chromosome arms. Seven of the nine terminal fragments were derived from a 118-bp tandem repeat, indicating that subtelomeric tandem repeat sequences provide an efficient means to target chromosome ends. A telomere cloning strategy and the terminal and interstitial location of TASs are discussed. Received: 13 September 1996 / Accepted: 22 January 1997