Integration of AFLP markers into a linkage map of sugar beet (Beta vulgaris L.)

The AFLP (amplified fragment length polymorphism) technique has been applied in establishing an extended linkage map of sugar beet. A total of 120 AFLPs were integrated into an existing linkage map based on RFLP markers. Four primer combinations yielded between 19 and 40 polymorphic bands in an F₂ population consisting of 94 plants. The AFLP loci were evenly distributed over the nine linkage groups, with the exception of linkage group V where the number of AFLPs was significantly low. The AFLPs were found to be reproducible even against the background of different combinations of Taq DNA polymerases and buffers. However, the quantity of higher molecular weight fragments (>400 bp) was reduced when using plant DNA of poor quality as a template. The results of these experiments are discussed, together with possible applications of AFLPs in sugar beet breeding.     

Evaluating genetic relationships in seed impatiens, Impatiens walleriana, using AFLP profiling

Although seed impatiens is the leading bedding plant in the US, little work has been conducted on evaluating the genetic variability present in this crop. In this study, amplified fragment length polymorphism technology was utilized to determine the level of polymorphism present across 20 commercial cultivars of seed impatiens, to assess their genetic variability, and to investigate their genetic relatedness. A total of eight EcoRI and MseI primer combinations were used for polymerase chain reaction amplification. Fluorescence‐labelled amplification products were subjected to electrophoresis and then analysed using an automated sequencer. High levels of polymorphism were detected among all 20 cultivars for all primer combinations tested. Gower's Genetic Dissimilarity estimates for the entire set of cultivars ranged from 1.000 to 0.316. The dendogram generated from these dissimilarity data revealed a number of groupings, including one major division which placed all of the cultivars into one of two groups. The implications of these results on genetic variability, genotypic relationships and genetic diversity in seed impatiens is discussed.     

Identification of polymorphic DNA markers in cultivated peanut (Arachis hypogaea L.)

The detection of DNA polymorphism in cultivated peanut (Arachis hypogaea L.) is reported here for the first time. The DNA amplification fingerprinting (DAF) and amplified fragment length polymorphism (AFLP) approaches were tested for their potential to detect genetic variation in peanut. The AFLP approach was more efficient as 43% of the primer combinations detected polymorphic DNA markers in contrast to 3% with the DAF approach. However, the number of polymorphic bands identified using primers selected in both approaches was comparable. In the DAF study, when 559 primers of varying types were screened, 17 (mostly 10-mer types) detected polymorphism producing an average of 3.7 polymorphic bands per primer with a total of 63 polymorphic markers. In the AFLP study, when 64 primer combinations (three selective nucleotides) corresponding to restriction enzymes Eco RI and Mse I were screened, 28 detected polymorphism. On an average, 6.7% of bands obtained from these 28 primer pairs were polymorphic resulting in a total of 111 AFLP markers. Our results demonstrate that both AFLP and DAF approaches can be employed to generate DNA markers in peanut and thus have potential in the marker-assisted genetic improvement and germplasm evaluation of this economically important crop. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular marker analysis of genetic variation characterizing a grasspea (Lathyrus sativus) collection from central Italy

Levels of genetic similarity characterizing 20 grasspea (Lathyrus sativus L.) populations collected in central Italy (17 populations in the Marche region and three populations in the Abruzzo region) were analysed with amplified fragment length polymorphism (AFLP) molecular markers. Two main clusters were found: one included large‐seeded populations from farms that were not market‐oriented (named Household populations) and the second, small‐seeded populations, cultivated in market‐oriented farms (named Commercial populations). Relationships among populations collected in different regions were found, although one population of the Abruzzo region was placed between the two main clusters, suggesting a possible further genetic differentiation within this grasspea germplasm collection. Principal component analysis based on AFLP marker frequency was effective in identifying polymorphic markers showing high discriminating ability between clusters H and C. In particular, seven markers showing high positive and three markers with low negative PC1 scores showed an almost cluster‐specific distribution. These results will be useful for enhancing Italian grasspea germplasm use in plant‐breeding programmes and for extending grasspea cultivation within the sustainable agricultural systems of central Italy.     

Relationship between heterosis and genetic distance based on morphological traits and AFLP markers in pepper

Genetic diversity is considered as one of the criteria for the selection of parents for hybrid breeding. The present study was undertaken to evaluate genetic divergence among seven pepper cultivars and to assess the relationship between heterosis and parental genetic distance. Twenty‐one F₁ hybrids and seven parents were evaluated for 15 morphological characters in a greenhouse and in the field. The parents were examined for DNA polymorphisms using six amplified fragment length polymorphism (AFLP) primer combinations. Cluster analysis using two genetic distance measures grouped the seven parents differently. Mid‐parent and high‐parent heterosis was observed for most characters. Most hybrids outperformed the parental lines for fruit yield, earliness and plant height. Morphological and AFLP‐based distance measurements were efficient enough to allocate pepper genotypes into heterotic groups. The correlations of morphological distances with mid‐parent heterosis were significant for days to flowering and maturity, suggesting earliness can be predicted from morphological distances of parental lines. However, the correlations of AFLP‐measured genetic distances with mid‐ and high‐parent heterosis were non‐significant for all characters, except for fruit diameter, and proved to be of no predictive value.     

珠海香蕉枯萎病菌遗传多样性的AFLP分析

为了解广东省珠海地区香蕉枯萎病菌(Fusarium oxysporum f. sp. Cubense)的种群分化和遗传变异规律,采用AFLP技术对来自珠海、湛江、台山的20株尖孢镰刀菌(Fusarium oxysporum)进行DNA指纹分析,以1株分离自粉蕉的木贼镰刀菌(F. equiseti)为外参。20株尖孢镰刀菌中17株来自珠海,包括3株非致菌、2株香蕉枯萎病1号生理小种、12株香蕉枯萎病4号生理小种,2株来自湛江的为香蕉枯萎病4号生理小种,1株来自台山的为香蕉枯萎病1号生理小种。8对AFLP引物对供试菌株扩增出253条带,其中多态性带97条,占总带数的38.34%,供试菌株的遗传距离变化在0.21~0.99间,平均为0.85。利用NTSYSpc软件中的UPGMA算法构建了21株镰刀菌的AFLP亲缘树状图,聚类结果表明：供试菌株被聚为3个大的类群,分别为1号生理小种、4号生理小种以及非致病性尖孢镰刀菌。亲缘关系分析结果表明：1号生理小种内菌株间的遗传分化大于4号生理小种,其聚类结果与菌株的地理来源有一定的相关性。总体而言,珠海地区香蕉枯萎病菌的遗传多样性较为丰富。     

AFLP analysis of genetic diversity within and between populations of perennial ryegrass (Lolium perenne L.)

Amplified fragment length polymorphism (AFLP) analysis has been used to measure genetic diversity in perennial ryegrass (Lolium perenne L.) and to relate intra- and interpopulation variation to breeding history. Cluster analysis of AFLP data from contrasting populations showed features consistent with the origins of these varieties. Significant differences in intrapopulation diversity were detected and partial separation of different cultivars was observed. Restricted base cultivars, derived from small numbers of foundation clones, were suitable for this type of study, allowing near complete discrimination of closely related cultivars. Analysis of bulked samples was based on the pooling of genomic DNA from 20 individuals from 6 selected populations. Cluster analysis of AFLP data from bulked samples produced a phenogram showing relationships consistent with the results of individual analysis. AFLP profiling provides an important tool for the detection and quantification of genetic variation in perennial ryegrass. This revised version was published online in August 2006 with corrections to the Cover Date.     

A genetic linkage map of rhodesgrass based on an F1 pseudo-testcross population

The linkage relationships between 164 polymorphic amplified fragment length polymorphism (AFLP) and 25 restriction fragment length polymorphism (RFLP) fragments assayed in a pseudo‐testcross population generated from the mating of single genotypes from two divergent cultivars were used to construct female, ‘Katambora’ (‘Kat’) and male, ‘Tochirakukei’ (‘Toch’) parental genetic maps for rhodesgrass. The ‘Kat’ genetic map consists of 84 marker loci (72 AFLP and 12 RFLP markers) distributed on 14 linkage groups and spans a total length of 488.3 cM, with an average distance of 7.8 cM between adjacent markers. The ‘Toch’ genetic map consists of 61 marker loci (52 AFLP and nine RFLP) mapped on 12 linkage groups spanning a total length of 443.3 cM, with an average spacing of 9.0 cM between adjacent markers. About 23% of the markers remained unassigned. The level of segregation distortion observed in this cross was 11.1%. In both maps, linked duplicated RFLP loci were found. These linkage maps will serve as a starting point for linkage studies in rhodesgrass with potential application for marker‐assisted selection in breeding programmes.     

Genetic diversity of some surviving on-farm Italian common bean (Phaseolus vulgaris L.) landraces

This study was undertaken to estimate the level of variation among and within 33 local populations belonging to seven Italian common bean landraces, by analysing the polymorphism of seed storage proteins, simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) molecular markers. The nutritional seed quality of the landraces studied was also investigated. Results showed that the landraces retain a considerable level of heterogeneity. Use of both biochemical and molecular approaches provided a picture of the genetic diversity of each landrace. Cluster analyses based on Nei's genetic distances and Jaccard's similarity index as defined by SSR and AFLP markers, respectively, showed that all populations clustered into two groups corresponding to the Andean and the Mesoamerican gene pools. Knowledge of the genetic structure of a landrace is fundamental in elaborating strategies, which involve the local farmers, allowing us to improve and, at the same time, safeguard the genetic integrity of landrace genetic resources.     

Genetic diversity analysis of Moroccan lentil (Lens culinaris Medik.) landraces using Simple Sequence Repeat and Amplified Fragment Length Polymorphisms reveals functional adaptation towards agro‐environmental origins

In the absence of previous molecular characterization, we assessed genetic diversity of 53 Moroccan lentil landraces including two local cultivars using simple sequence repeat (SSR) and amplified fragment length polymorphisms (AFLP). Nineteen SSRs yielded 213 alleles, and seven AFLP primer combinations gave 766 fragments of which 422 were polymorphic. Moderate to high genetic variation was observed. Several small groups of landraces were differentiated. Interestingly, one of the smallest groups contained short‐cycle landraces with high early vegetative growth. Landraces in that group were from the dry land location of Abda, where they were likely selected for adaptation to drought and heat stress over centuries. Another group contained two landraces from highland areas that may have been selected for specific adaptation to cold stress. A third group contained one landrace from the Zear region known for its seed quality and has been proposed for the protected designation of origin (PDO) quality mark. Both techniques gave evidence of differentiation of the latter landrace supporting the idea of PDO attribution. Functional grouping according to agro‐environmental origins, cycle duration and early vegetative vigour was observed.     

Comparing RAPD and AFLPTM analysis in discrimination and estimation of genetic similarities among apple (Malus domestica Borkh.) cultivars

Forty-one apple (Malus × domestica Borkh.) cultivars were screened for RAPD (Random Amplified Polymorphic DNA) and AFLP(Amplified Fragment Length Polymorphism) markers. RAPD analysis was performed with 35 arbitrary 10-mer primers, selected from 60 primers tested (kits A, C and E, Operon Technologies, Inc.). Of a total of 362bands observed, 208 (57.5%) were polymorphic. Three-hundred-and-eighty-one AFLP fragments were obtained with 8primer combinations, of which 218 (57.2%) were polymorphic. Cultivars differentiated through mutation were included in this study and showed identical patterns when analysed with both RAPD and AFLP analysis. The estimated genetic relationships were correlated (r = 73.7%) between the analysis with the two different markers. UPGMA analysis was performed and dendrograms were constructed using either the data apart from each(RAPD and AFLP) method or combined in a single joint matrix. The relationships among the forty-one studied cultivars were basically consistent with the known lineage and geographic origins of the cultivars. The four Portuguese cultivars included in this study clustered together and diverged from the other cultivars. Apparently they constitute an independent genetic pool, which could be of interest for apple plant breeders. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phenotypic variation in local populations and cultivars of meadow fescue – potential for improving cultivars by utilizing wild germplasm

The utilization of gene bank accessions for breeding purposes is generally hampered by a lack of sufficient genetic and phenotypic characterization. Results are given here of a phenotypic analysis of 30 Norwegian local populations and 10 Nordic cultivars of meadow fescue, and a combined analysis of phenotypic data with previously published molecular data based on 95 amplified fragment length polymorphic markers. Seventeen phenological and morphological, and two derived characters, were assessed on 2264 individual plants. Previous molecular diversity analyses have shown that the Norwegian local populations can be divided into three groups based on a likely history of the introduction of the species into Norway. A similar pattern was evident from relationships between the mean phenotypic values of populations and cultivars. Such a combined phenotypic and genetic characterization of germplasm can facilitate the use of gene bank material in breeding, as breeders can use these data to select populations with extreme values of a field trait, along with markers which represent potential predictors of phenotypic value.     

Bulked AFLP analysis for the assessment of genetic diversity in white clover (Trifolium repens L.)

The use of bulked leaf samples from individual plants for amplified fragment length polymorphism (AFLP) analysis was evaluated as a tool for assessment of genetic diversity in white clover (Trifolium repens L.). Bulking of leaf samples produced slightly simpler AFLP profiles compared to the combined profiles of individual plants from the same cultivar. Approximately 90% of bands which were present in individual plants were present in bulked samples of the same cultivar. The majority of those absent were rare bands, shared by less than 25% of individual plants. Replicate bulk samples gave almost identical banding patterns, demonstrating the robustness of the bulked AFLP technique. Cluster analysis of AFLP data derived from individual plants resulted in a phenogram similar to that produced from data derived from bulked samples of the same plants. AFLP analysis of bulked samples detected significant amounts of genetic variability among 52 cultivars and accessions with genetic similarity values ranging from 0.42 to 0.92. However, cluster analysis of AFLP data only partially reflected the geographic origin of cultivars and accessions and was not congruent with cluster analysis based on variation for morphophysiological characters. Bulked AFLP analysis provides a powerful tool for rapid assessment of genetic variability in white clover and may also be used for cultivar identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

AFLP in Triticum aestivum L.: patterns of genetic diversity and genome distribution

The amplified fragment length polymorphism (AFLP) procedure was applied to a diverse panel of wheat (Triticum aestivum L. em. Thell.) accessions and sixty-nine of the recombinant inbred lines (RILs) from the widely used genetic mapping population derived from the cross of Opata 85 and W7984. Most (76.8%) bands were monomorphic among T. aestivum accessions. The majority of bands monomorphic in T. aestivum also were present in the synthetic wheat parent (W7984). Ten primer pairs generated 153 polymorphic AFLP bands, 140 of which could be assigned to a chromosome location and were relatively evenly distributed on the genetic linkage map. AFLP loci in T. aestivum were distributed throughout the genome; they generally have only one detectable sequence variant; and they exhibit monogenic dominant mendelian inheritance. Frequencies of polymorphic bands in the germplasm sampled are in the range that enables informative cluster analyses as well as map-based diversity and association analysis studies. AFLP bands mapped to individual loci in the Opata 85/W7984 RIL population will frequently be polymorphic in other crosses or germplasm, irrespective of whether the band arises from the T. aestivum parent or the synthetic wheat parent. This revised version was published online in July 2006 with corrections to the Cover Date.     

Physiological and Molecular Characterization of Faba bean (Vicia faba L.) Genotypes for Adaptation to Drought Stress

Faba bean (Vicia faba L.) is one of the most important and drought sensitive grain legumes. Drought stress is thus one of major constraints in global faba bean production. In this study, twenty local and exotic faba bean genotypes were characterized on physiological and molecular basis. Seeds of faba bean genotypes (six per pot) were sown in poly venyl chloride pots. After seedling emergence, soil moisture was maintained at 100%, 50% and 25% of field capacity designated as well watered, moderate drought and severe drought, respectively. Drought stress significantly influenced the leaf area, leaf temperature, stomatal conductance, relative leaf water contents, grain yield and water‐use efficiency. Faba bean genotypes also differed for the leaf area, leaf temperature, relative leaf water contents, grain yield and water‐use efficiency. Faba bean genotypes Kamline and L.4 were better equipped to curtail water loss, maintain tissue water status, produce stable grain yield and had better water‐use efficiency under mild and severe drought stress, and may be used in breeding programmes. Amplified fragment length polymorphism markers showed high potential in detecting polymorphism and estimating genetic diversity among faba bean genotypes. Unweighted pair group method with arithmetic mean cluster analysis of the genotypes illustrated considerable association between molecular diversity, genetic background and geographic origin. In crux, high polymorphic rate and polymorphism information content values, together with the low genetic similarity observed among tested genotypes suggests a high level of heterogeneity, which may be used in breeding programmes to assemble different drought tolerance mechanisms in one genotype.     

花生黄曲霉侵染抗性的AFLP标记

本研究利用抗、感黄曲霉菌侵染的花生品种为亲本配制杂交组合“J11×中花5号”,以其F₂分离群体为研究材料，采用AFLP技术和BSA分析方法，获得了与花生黄曲霉菌侵染抗性连锁的2个分子标记，标记与抗性间的遗传距离分别为8.8 cM和6.6 cM;利用获得的分子标记对抗、感黄曲霉的花生种质资源进行了分子鉴定，实验结果表明分子标记与抗性鉴定结果具有较高的一致性，证实了两标记应用于研究群体之外的育种潜力。该抗侵染分子标记的建立为开展花生抗黄曲霉辅助选择育种提供了有效的筛选技术。     

Genetic linkage map construction for white jute (Corchorus capsularis L.) using SRAP,ISSR and RAPD markers

White jute (Corchorus capsularis) and dark jute (Corchorus olitorius) are two important cultivated crops that are used for natural fibre production. Some genetic maps have been developed for dark jute, but the genetic map information for white jute (C. capsularis) is limited. In this study, a linkage map comprising 44 sequence‐related amplified polymorphisms (SRAPs), 57 intersimple sequence repeats (ISSRs) and 18 randomly amplified polymorphic DNA (RAPD) covering 2185.7 cM with a mean density of 18.7 cM per locus was constructed in an F₂ population consisting of 185 individuals derived from a cross between two diverse genotypes of ‘Xinxuan No. 1’ and ‘Qiongyueqing’ in white jute. These markers were evenly distributed in the linkage groups without any clustering. This genetic linkage map construction will facilitate the mapping of agronomic traits and marker‐assisted selection breeding in white jute.     

Precision of genetic relationship estimates based on molecular markers

Genetic progress through selection is directly related to the amount of variability present in the population and the quality of genes contributed by the parents. Genetic relationships between lines were studied using DNA marker-based estimates of genetic similarity. A statistical methodology using the width of a confidence interval was developed to determine the number of probes to be surveyed and the precision in the estimation of genetic distance between pairs of cultivars. Precision was affected by type of genetic distance used, the number of cultivars, and amount of genetic diversity present in the studied group. The width of a (1-α)% confidence interval decreased as the number of RFLP fragments increased. Oat and wheat diversity studies were used to illustrate the methodology. This revised version was published online in July 2006 with corrections to the Cover Date.     

AFLP Analysis of Mitochondrial DNA from a Cytoplasmic Male Sterility Line and its Maintainer in Pepper （Capsicum annuum L.）

Cytoplasmic male sterility （CMS） is a maternally inherited trait characterized by the inability of a plant to produce functional pollen, which is widespread among higher plants. CMS system is a valuable tool for plant brc, ders to utilize in hybrid seed production and for molecular biologists to study the interaction between nuclear and mitochondrial genomes. Investigations into the molecular mechanism of CMS in several species have revealed that the incompatible nuclear-mitochondrial interactions leading to new chimeric mitochondrial genes such as T-urfl3 for Texas male sterility cytoplasm in maize, pcfin petunia, orf522 in sunflower, orf107 in sorghum and orf244 gene in Brassica may be responsible for CMS （Dewey et al., 1986; 1987; Young and Hanson,1987; Nivison and Hanson, 1989; Kohler et al., 1991; Moneger et al., 1994; Tang et al., 1996; Handa et al., 1995; Singh and Brown, 1991）.