Amplified Fragment Length Polymorphism Method Research Articles

Rapid Genetic Assessment of Carrot Varieties Based on AFLP Analysis

It is necessary to regard the biodiversity of carrot as a genetic source of useful and indispensable components for the human diet. Amplified fragment length polymorphism (AFLP) markers were used to discriminate eight carrot genotypes with different root colors. With the combination of enzymes Tru9I, PstI, and three sets of primer pairs corresponding to adapters joint to the restricted sites, 92 loci were produced, including 60 polymorphic ones. Each of the three primer sets showed high efficiency, according to estimations of PIC (0.34, 0.34, and 0.41), D (0.36, 0.67, and 0.67), Rp (5.5, 11.3, and 15), and HE (0.32, 0.49, and 0.49). The genetic distances were calculated using values of Nei’s coefficient. The most genetically similar were Chantenay Coeur Rouge and Colmar a Coeur Rouge at a distance of 0.12, whereas the most distant were Saint Valery and Purple Dragon at the highest distance of 0.34. Confirming its genetic identity, Purple Dragon and Gelber Goliath with purple and yellow roots were the most detached varieties from others at distances of 0.23–0.34 and 0.23–0.28, respectively. Male sterile Berlicum breeding accessions were well distinct from other orange-colored varieties at the highest distance of 0.30 from Deep Purple F1. Slight modifications including the facilitation of gel staining enables the wide use of the AFLP method for genetic diversity assessment in carrot breeding accessions.

Numerical analysis of phenotypic properties, genomic fingerprinting, and multilocus sequence analysis of Bradyrhizobium strains isolated from root nodules of Lembotropis nigricans of the tribe Genisteae

PurposeThe aim of this study was to estimate the level of genomic and phenotypic diversity as well as the genus and species position of bacterial strains isolated from root nodules of Lembotropis nigricans (family Fabaceae).MethodsThe genomic diversity of studied L. nigricans nodule symbionts was examined by using BOX-PCR and AFLP (amplified fragment length polymorphism) fingerprinting techniques. To assign bacteria to the genus, numerical analysis of phenotypic features and comparative analysis of 16S rDNA sequences were performed. The comparative analysis of combined atpD, dnaK, gyrB, and rpoB gene sequences (multilocus sequence analysis, MLSA) was used to determine the most closely related species to the studied bacteria.ResultsBoth BOX-PCR and AFLP techniques revealed a high level of genomic heterogeneity of L. nigricans nodulators. Among 33 studied bacteria, 32 genotypes were delineated by the AFLP method and 27 genotypes were identified by the BOX-PCR fingerprinting. The numerical analysis of 86 phenotypic characteristics of L. nigricans nodule isolates and reference rhizobia showed that studied bacteria belong to the genus Bradyrhizobium. Affiliation of L. nigricans nodule isolates to the genus Bradyrhizobium was supported by comparative analysis of 16S rDNA sequences and the concatenation of atpD, dnaK, gyrB, and rpoB gene sequences. MLSA indicated also that L. nigricans microsymbionts are members of Bradyrhizobium japonicum.ConclusionL. nigricans root nodule symbionts are members of Bradyrhizobium japonicum and exhibit high phenotypic and genomic diversity important for their survival in soil.

Genetic and epigenetic changes during the invasion of a cosmopolitan species (Phragmites australis).

While many introduced invasive species can increase genetic diversity through multiple introductions and/or hybridization to colonize successfully in new environments, others with low genetic diversity have to persist by alternative mechanisms such as epigenetic variation. Given that Phragmites australis is a cosmopolitan reed growing in a wide range of habitats and its invasion history, especially in North America, has been relatively well studied, it provides an ideal system for studying the role and relationship of genetic and epigenetic variation in biological invasions. We used amplified fragment length polymorphism (AFLP) and methylation‐sensitive (MS) AFLP methods to evaluate genetic and epigenetic diversity and structure in groups of the common reed across its range in the world. Evidence from analysis of molecular variance (AMOVA) based on AFLP and MS‐AFLP data supported the previous conclusion that the invasive introduced populations of P. australis in North America were from European and Mediterranean regions. In the Gulf Coast region, the introduced group harbored a high level of genetic variation relative to originating group from its native location, and it showed epigenetic diversity equal to that of the native group, if not higher, while the introduced group held lower genetic diversity than the native. In the Great Lakes region, the native group displayed very low genetic and epigenetic variation, and the introduced one showed slightly lower genetic and epigenetic diversity than the original one. Unexpectedly, AMOVA and principal component analysis did not demonstrate any epigenetic convergence between native and introduced groups before genetic convergence. Our results suggested that intertwined changes in genetic and epigenetic variation were involved in the invasion success in North America. Although our study did not provide strong evidence proving the importance of epigenetic variation prior to genetic, it implied the similar role of stable epigenetic diversity to genetic diversity in the adaptation of P. australis to local environment.

Genetic Relationships among Different Chemotypes of Lupinus sulphureus.

Lupines (Lupinus spp.) are a common plant legume species found on western U.S. rangelands. Lupinus spp. may contain quinolizidine and/or piperidine alkaloids that can be toxic and/or teratogenic to grazing livestock. Alkaloid profiles may vary between and within a species. The objectives of this study were to (1) further explore the characteristic alkaloid profiles of Lupinus sulphureus using field collections and (2) explore the phylogenetic relationship of the different populations and chemotypes of L. sulphureus using the amplified fragment length polymorphism method of DNA fingerprinting, thus providing possible explanations to the phenomena of multiple chemotypes within a species. A total of 49 accessions of L. sulphureus were classified into seven chemotypes. The DNA profiles showed that one L. sulphureus chemotype, chemotype A, is genetically divergent from the other chemotypes of L. sulphureus, suggesting that it represents an unresolved lupine taxon, possibly a new lupine species. Additionally, the different chemotypes of L. sulphureus represented different genetic groups, as shown by Bayesian cluster analysis and principle component analysis.

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles

BackgroundIt has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP.ResultsMethylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5′ end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans.ConclusionMSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

Analysis of Genetic Diversity of Indian Tea Accessions Using Two Modified Amplified Fragment Length Polymorphism Methods

The authors compared the utility of two variants of Amplified Fragment Length Polymorphism (AFLP) technique, viz. 4-selective AFLP and three Endonuclease Amplified Fragment Length Polymorphism (TE-AFLP), for accurate assessment of genetic diversity in tea. These assays were optimized to resolve the reported discrepancies in detected levels of genetic variation in tea through conventional AFLP assay. The 4-selective AFLP assay involved the use of four selective nucleotides in MseI primer instead of three, in combination with EcoRI primers with three selective nucleotides. This led to a significant reduction in the total number of bands and improved the overall band readability as compared to conventional AFLP. However, presence of a large number of “shadow bands” of varying intensities across lanes, still disallowed an error free scoring. This was effectively resolved by TE-AFLP assay where selective adapter ligation, in addition to selective amplification, contributes to reduction in the complexity of fingerprint pattern. The major advantage of TE-AFLP over 4-select AFLP was its clear and unambiguous banding profiles that simplified the scoring of bands. Both methodologies yielded comparable results with China types clearly separating from Assam types. In contrast to previous reports, the dendrogram revealed an overall tendency of the accessions to cluster based on their morphotypes and their geographic origin. Although, 4-select AFLP detected higher polymorphism information content values, the overall reliability of TE-AFLP bands was higher due to their unambiguous nature and easy scorability. A major fraction of variation (75 %) was found to be partitioned within the morphotypes indicating predominant out-crossing in this species.

Extensive reprogramming of cytosine methylation in Oryza allotetraploids

Cytosine methylation is a prominent epigenetic modification in plants and it has been proposed to as causative for silencing some duplicates derived by polyploidization. Several synthetic polyploids have shown there is a dynamic epigenetic change following hybridization and genome doubling. Relatively few studies, however, have focused on wild natural polyploids. In this study, using a methylation-sensitive amplified fragment length polymorphism method, we investigated the levels and types of cytosine methylation in three Oryza CCDD genome allotetraploids and their related diploids with CC and EE genomes. We show an overall minor increase in methylation level in the allopolyploids, but extensive methylation reprogramming (70 %), with almost equivalently increased (22–26 %) and decreased methylation (25–27 %). Our study reveals a complicated epigenetic change in natural polyploids. The information presented in the study will be of some utility to molecular taxonomists and also rice breeders working on wide-hybridization.

Zebrafish genome instability after exposure to model genotoxicants

Sublethal exposure to environmental genotoxicants may impact genome integrity in affected organisms. It is therefore necessary to develop tools to measure the extent and longevity of genotoxicant-induced DNA damage, and choose appropriate model organisms for biomonitoring. To this end, markers of DNA damage were measured in zebrafish larvae and adults following exposure to model genotoxicants (benzo[a]pyrene and ethyl methanesulfonate). Specifically, we assessed primary DNA damage and the existence of potentially persistent genomic alterations through application of the comet assay, quantitative random amplified polymorphic DNA (qRAPD) and amplified fragment length polymorphism (AFLP) assays. Furthermore, expression of genes involved in DNA repair, oxidative stress response and xenobiotic metabolism was evaluated as well. Additionally, the AFLP method was applied to adult specimens 1year after larval exposure to the genotoxicants to evaluate the longevity of the observed DNA alterations. Large numbers of DNA alterations were detected in larval DNA using the comet assay, qRAPD and AFLP, demonstrating that zebrafish larvae are a sensitive model for revealing genotoxic effects. Furthermore, some of these genomic alterations persisted into adulthood, indicating the formation of stable genomic modifications. qRAPD and AFLP methods proved to be highly sensitive to genotoxic effects, even in cases when the comet assay indicated a lack of significant damage. These results thus support the use of zebrafish larvae as a sensitive model for monitoring the impact of genotoxic insult and give evidence of the longevity of genomic modifications induced by genotoxic agents.

Association of Aberrations in One Carbon Metabolism with Intimal Medial Thickening in Patients with Type 2 Diabetes Mellitus.

The present work was aimed to study the association of one carbon genetic variants, hyperhomocysteinemia and oxidative stress markers, i.e., serum nitrite, plasma malondialdehyde (MDA) and glutathione (GSH) on intimal medial thickening (IMT) in patients with type 2 diabetes mellitus (T2D). A total number of 76 subjects from ACS Medical College and Hospital, Chennai, India were included in the study, i.e., Group I (n=42) of T2D and Group II (n=34) of age- and sex matched healthy controls. The glycated haemoglobin was measured by ion-exchange resin method; plasma homocysteine by Enzyme Linked Immunosorbant Assay method; serum nitrite (nitric oxide, NO), plasma MDA and GSH by spectrophotometric methods; the IMT by high frequency ultrasound. The polymorphisms of one carbon genetic variants were genotyped using polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism methods. Results indicate that methyltetrahydrofolate homocysteine methyl transferase (MTR) A2756G allele was found to be protective in T2D and the other variants were not significantly associated with T2D. Glutamate carboxypeptidase II (GCP II) C1561T (r=0.34; p=0.05) and methylene tetrahydrofolate reductase (MTHFR) C677T (r=0.35; 0.04) showed positive correlation with plasma homocysteine in T2D cases. In this study, MTR A2756G allele was found to be protective in T2D; GCP II C1561T and MTHFR C677T showed positive association with plasma homocysteine in T2D cases. Among all the genetic variants, MTR A2756G was found influence IMT. RFC 1 G80A and TYMS 5'-UTR 2R3R showed synergistically interact with MTR A2756G in influencing increase in IMT.

Application of AFLP markers in fishery and seafood

Several sociological, health and conservation arguments request acorrect labelling of seafood products and fish species. Nowadays, moleculargenetics is a useful tool for food chain traceability, particularly in regardsto species identification. Among the variety of PCRbased molecular markers,AFLPs (Amplified Fragment Length Polymorphisms) have recently been used toinvestigate genomes of different complexities. This paper assesses thepotential use of the AFLP technology to determine fish and seafood species inprocessed commercial products, domestic stocks and research in the field ofmolecular ecology. In particular a species database of fish, molluscs andcrustaceans has been created with the aim to identify species of origin ofseafood products by previously defined AFLP patterns. Researchers in the fieldof molecular ecology and evolution require versatile and low-cost genetictyping methods. The AFLP method was introduced 20 years ago and shows manyfeatures that fulfil these requirements. With good quality genomic DNA at hand,it is relatively easy to generate anonymous multilocus DNA profiles in mostspecies and the start-up time before data can be generated is often less than aweek. These aspects include classical problems such as studies of populationgenetic structure and phylogenetic reconstructions, and also new challengessuch as finding markers for genes governing adaptations in wild populations andmodifications of the protocol that makes it possible to measure expressionvariation of multiple genes (cDNA-AFLP) and the distribution of DNAmethylation. We hope this review will help molecular ecologists to identifywhen AFLP is likely to be superior to other more established methods, such asmicrosatellites, SNP (single nucleotide polymorphism) analyses and multigeneDNA sequencing.

English

Amplified fragment length polymorphisms (AFLP) were used to evaluate the genetic diversity between 18 date palm (Phoenix dactylifera L.) varieties (11 females and 7 males) collected from the center of Iraq. Six primer pairs were applied to detect polymorphism between varieties. A total of 83 polymorphic AFLP fragments were detected with an average of 13.8 polymorphic fragments/primer combination. Genetic distance was estimated using Jaccard’s genetic similarity index and was ranged from 0.07 to 0.75. Unweighted pair group method with arithmetic mean UPGMA ordered date palm varieties into two main clusters independently of their origin and sex. The first cluster consisted of three sub-clusters. The first one consisted of five female varieties and one male, while the second sub-clusters consisted of five male varieties. The third one consisted of five varieties; four were females and one male. The second main cluster consisted of the remaining two female varieties. Moreover, all primer combinations contributed to the discrimination of date palm varieties, suggesting the efficiency of AFLP method in assessing genetic diversity in date palm. A large range of genetic diversity characterized Iraqi date palm germplasm.   Key words: Genetic diversity, amplified fragment length polymorphisms (AFLP) polymorphisms, molecular characterization, Phoenix dactylifera L.

Fluorescent AFLP fingerprinting of Monilinia fructicola

The fluorescent amplified fragment length polymorphism method (AFLP) has been successfully applied to one of the brown rot fungi species — Monilinia fructicola, which causes severe losses in stone fruit production. This is the first report on the use of AFLP methodology for studying genetic variability among different M. fructicola isolates. A total of 1256 scorable, well defined AFLP fragments were obtained with 20 primer pairs, of which 462 were polymorphic. An unweighted pair group (UPGMA) dendrogram was constructed based on Jaccard’s similarity coefficient. A high similarity of the two Malus isolates from the USA was observed, while the other three Prunus isolates, from Spain, Japan and New Zealand, formed a second, more variable group. Comparison of AFLP fingerprinting data between M. fructicola and M. laxa revealed completely different banding patterns and demonstrated the suitability of the AFLP marker system for cross species differentiation of brown rot fungi.

Epigenetic differentiation and relationship to adaptive genetic divergence in discrete populations of the violet Viola cazorlensis

*In plants, epigenetic variations based on DNA methylation are often heritable and could influence the course of evolution. Before this hypothesis can be assessed, fundamental questions about epigenetic variation remain to be addressed in a real-world context, including its magnitude, structuring within and among natural populations, and autonomy in relation to the genetic context. *Extent and patterns of cytosine methylation, and the relationship to adaptive genetic divergence between populations, were investigated for wild populations of the southern Spanish violet Viola cazorlensis (Violaceae) using the methylation-sensitive amplified polymorphism (MSAP) technique, a modification of the amplified fragment length polymorphism method (AFLP) based on the differential sensitivity of isoschizomeric restriction enzymes to site-specific cytosine methylation. *The genome of V. cazorlensis plants exhibited extensive levels of methylation, and methylation-based epigenetic variation was structured into distinct between- and within- population components. Epigenetic differentiation of populations was correlated with adaptive genetic divergence revealed by a Bayesian population-genomic analysis of AFLP data. Significant associations existed at the individual genome level between adaptive AFLP loci and the methylation state of methylation-susceptible MSAP loci. *Population-specific, divergent patterns of correlated selection on epigenetic and genetic individual variation could account for the coordinated epigenetic-genetic adaptive population differentiation revealed by this study.

A new simplified AFLP method for wine yeast strain typing

A simplified method of AFLP (Amplified Fragment Length Polymorphisms) is presented for typing yeast present during wine fermentations. The changes introduced allowed analysis by gel electrophoresis and considerably reduced the need for equipment. Another remarkable improvement was the use of non-labelled primers which reduces the cost of the analysis. This method was applied to reference strains from culture collection to test the reliability of the technique. A total of 180 colonies isolated from a spontaneous fermentation were typed into eleven different strains of Hanseniaspora uvarum, six of Hanseniaspora vineae, four of Candida zemplinina, and eleven of Saccharomyces cerevisiae. This method is suitable for typing yeast strains for routine grape and wine ecology analysis.

Development of SNP markers for individual identification and parentage test in a Japanese Black cattle population

This study describes the development of efficient single nucleotide polymorphism (SNP) markers for individual identification and parentage tests in a Japanese Black cattle population. An amplified fragment length polymorphism method was employed to detect informative candidate markers, and yielded 44 SNP markers from 220 primer combinations. 29 unlinked SNPs were finally selected as diagnostic markers. The allelic frequencies for each marker were estimated by using PCR-RFLP in the Japanese Black population. Based on the frequency data, the estimated identity power of these markers was 2.73 x 10(-12). Parentage exclusion probabilities, when both suspected parents' genotypes were known and when only one suspected parent was genotyped, were estimated as 0.96929 and 0.99693, respectively. This panel of SNP markers is theoretically sufficient for individual identification, and would also be a powerful tool for a parentage test in Japanese Black cattle. The markers could contribute to the management of the beef industry in Japan.

Demonstration of persistent strains of Campylobacter jejuni within broiler farms over a 1‐year period in Lithuania

The aim of the study was to investigate the flock prevalence of Campylobacter jejuni and Campylobacter coli in broiler farms in Lithuania and to identify possible persistent strains of Camp. jejuni using amplified fragment length polymorphism (AFLP) typing method. During 1 year, 42 broiler flocks from 9 broiler farms were examined to determine the prevalence of Campylobacter-positive broiler flocks in Lithuania. Among 42 broiler flocks examined, 31 flocks (73.8%) were positive for Camp. jejuni and 17 flocks (40.48%) for Camp. coli. Campylobacter jejuni isolates were genotyped by AFLP method using BspDI and BglII restriction enzymes. Typing of 190 isolates generated 50 AFLP genotypes with the highest diversity of strains found in the summer season. Each farm showed one or more predominant AFLP types, and one AFLP type (A32) was found in five broiler farms over a 1-year period. Campylobacter jejuni and Camp. coli are highly prevalent in broiler farms in Lithuania. Farm-specific genotypes were identified in all farms examined. Type A32 was present and persisted in different broiler farms, and a common source of transmission of Camp. jejuni was suspected. For the first time, Camp. jejuni in broiler flocks has been genetically characterized in Lithuania. Persistent strains of Camp. jejuni were detected over one period at the beginning of broiler meat production chain and, therefore, the identification of contamination source of such strains and the mechanism of their particular ability to persist are crucial to establish effective control measures against Camp. jejuni infection in broiler farms.

Genetic variability and population structure of endangered Panax ginseng in the Russian Primorye

BackgroundThe natural habitat of wild P. ginseng is currently found only in the Russian Primorye and the populations are extremely exhausted and require restoration. Analysis of the genetic diversity and population structure of an endangered species is a prerequisite for conservation. The present study aims to investigate the patterns and levels of genetic polymorphism and population structures of wild P. ginseng with the AFLP method to (1) estimate the level of genetic diversity in the P. ginseng populations in the Russian Primorsky Krai, (2) calculate the distribution of variability within a population and among populations and (3) examine the genetic relationship between the populations.MethodsGenetic variability and population structure of ten P. ginseng populations were investigated with Amplified Fragment Length Polymorphism (AFLP) markers. The genetic relationships among P. ginseng plants and populations were delineated.ResultsThe mean genetic variability within populations was high. The mean level of polymorphisms was 55.68% at the population level and 99.65% at the species level. The Shannon's index ranged between 0.1602 and 0.3222 with an average of 0.2626 at the population level and 0.3967 at the species level. The analysis of molecular variances (AMOVA) showed a significant population structure in P. ginseng. The partition of genetic diversity with AMOVA suggested that the majority of the genetic variation (64.5%) was within populations of P. ginseng. The inter-population variability was approximately 36% of the total variability. The genetic relationships among P. ginseng plants and populations were reconstructed by Minimum Spanning tree (MS-tree) on the basis of Euclidean distances with ARLEQUIN and NTSYS, respectively. The MS-trees suggest that the southern Uss, Part and Nad populations may have promoted P. ginseng distribution throughout the Russian Primorye.ConclusionThe P. ginseng populations in the Russian Primorye are significant in genetic diversity. The high variability demonstrates that the current genetic resources of P. ginseng populations have not been exposed to depletion.

Amplified Fragment Length Polymorphism and Multi-Locus Sequence Typing for high-resolution genotyping of Listeria monocytogenes from foods and the environment

Standardized tools for typing Listeria monocytogenes isolates are required in epidemiological surveys investigating food-borne disease outbreaks and in the food-processing environment to identify the sources of contamination and routes by which the organisms are spread. In this survey Amplified Fragment Length Polymorphism (AFLP) and Multi-Locus Sequence Typing (MLST) have been used to study 103 L. monocytogenes isolates from food and environmental sources. A total of 62 AFLP types and 66 MLST Sequence Types were identified. AFLP and MLST produced similar results in terms of discriminating power. The Discrimination Index calculated for the two techniques was 0.976 for AFLP and 0.972 for MLST. These values were appreciably higher compared to serotyping (0.739). A good congruence was observed between AFLP and MLST. The present study demonstrated that AFLP and MLST subtyping are suitable tools for studying the epidemiology of L. monocytogenes. The great advantage of MLST over AFLP and other molecular typing methods based on fragment fingerprinting lies in the unambiguity of sequence data while AFLP is less costly and highly processive. In conclusion the two methods can be perfectly integrated for high-resolution genotyping of L. monocytogenes.

Phenotypic, genomic and phylogenetic characteristics of rhizobia isolated from root nodules of Robinia pseudoacacia (black locust) growing in Poland and Japan

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98-99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771-961 kb megaplasmids.

Development of Sequence Characterized Amplified Region (SCAR) Primers for the Detection of Resistance to Sporisorium reiliana in Maize

Head smut of maize ( Zea mays L.), which was caused by Sporisorium reiliana, occurred in most of the maize growing areas of the world. The purpose of this study was to develop SCAR markers for map-based cloning of resistance genes and MAS. Two sets of BC3 progenies, one (BC3Q) derived from the cross Qi319 (resistance) × Huangzao 4 (susceptible), the other (BC3M) from Mo17 (resistance) × Huangzao 4 (susceptible), were generated. Huangzao 4 was the recurrent parent in both progenies. A combination of BSA (bulked segregant analysis) with AFLP (amplified fragment length polymorphism) method was applied to map the genes involving the resistance to S. reiliana, and corresponding resistant and susceptible bulks and their parental lines were used for screening polymorphic AFLP primer pairs. One fragment of P13M61–152 was converted into SCAR (sequence charactered amplified fragment) marker S130. The marker was mapped at chromosome bin 2.09, the interval of a major QTL region previously reported to contribute to S. reiliana resistance. Furthermore, S130 was highly associated with resistance to S. reiliana, and could be useful for marker-assisted selection and facilitate map-based cloning of resistance genes.