Genotyping with RAPD and microsatellite markers resolves pathotype diversity in the ascochyta blight pathogen of chickpea

 The poor definition of variation in the ascochyta blight fungus (Ascochyta rabiei) has historically hindered breeding for resistance to the chickpea (Cicer arietinum L.) blight disease in West Asia and North Africa. We have employed 14 RAPD markers and an oligonucleotide probe complementary to the microsatellite sequence (GATA)₄ to construct a genotype-specific DNA fragment profile from periodically sampled Syrian field isolates of this fungus. By using conventional pathogenicity tests and genome analysis with RAPD and microsatellite markers, we demonstrated that the DNA markers distinguish variability within and among the major pathotypes of A. rabiei and resolved each pathotypes into several genotypes. The genetic diversity estimate based on DNA marker analysis within pathotypes was highest for the least-aggressive pathotype (pathotype I), followed by the aggressive (pathotype II) and the most-aggressive pathotype (pathotype III). The pair-wise genetic distance estimated for all the isolates varied from 0.00 to 0.39, indicating a range from a clonal to a diverse relationship. On the basis of genome analysis, and information on the spatial and temporal distribution of the pathogen, a general picture of A. rabiei evolution in Syria is proposed. Received: 10 January 1998 / Accepted: 23 January 1998     

Evaluation of chickpea resistance against some isolates of Ascochyta rabiei, the causal agent of ascochyta blight

Ascochyta blight, incited by Ascochyta rabiei (Pass.) Lab., is one of the most important fungal diseases that has been shown to cause significant yield losses on chickpeas in Kermanshah province, Iran. One of the most effective and reliable methods of controlling the disease has been the use of resistant cultivars worldwide. This requires the identification of the physiological races of the fungus in the region in advance. In this regard, pathogenicity of 30 isolates of A. rabiei was determined using 10 differential chickpea varieties (mean of 1-9 degree of disease in three replications) under greenhouse conditions. In this respect, isolates fell into four groups, whereas based on infection type three groups were identified using cluster analysis. Disease resistance of four chickpea cultivars, Hashem, Jam, ILC-482 and Bivanij, were evaluated against isolates from each of three groups separately and in combination in the greenhouse and field conditions (with 4 replications). The experiment was repeated twice with three replications in greenhouse. Chickpea cultivars were sprayed with fungal spore suspension (2x10(5) spores/ml). Results showed that Jam and Bivanij cultivars were susceptible under greenhouse as well as field conditions. Hashem was tolerant to group I and susceptible to groups II and III and in the field. ILC-482 was tolerant to groups II and I and susceptible to group III and the combination of groups under greenhouse condition.     

Genetics of resistance to ascochyta blight (Ascochyta lentis) of lentil and the identification of closely linked RAPD markers

 Foliar resistance to Ascochyta lentis is controlled at a single major locus by a dominant gene (AbR ₁ ) in the lentil accession ILL5588 (cv ‘Northfield’). Flanking RAPD markers that are closely linked to the resistance locus in coupling phase were identified by bulked segregant analysis. Out of 261 decanucleotide primers screened 7 produced a polymorphic marker that segregated with the resistance locus, and all markers were found to exist within a single linkage group. Five of the seven RAPD markers were within 30 cM of the resistance locus. Log likelihood analysis for detecting QTL associated with the foliar resistance revealed that a single narrow peak accounted for almost 90% of the variance of resistance between the bulks. Preliminary mapping in an F₃ population revealed that the closest flanking markers were approximately 6 and 14 centiMorgans (cM) away from the resistance locus. These markers should be useful for the discrimination of resistant germplasm through marker-assisted selection in future breeding programmes and represent the first essential step towards the map-based cloning of this resistance gene. Received: 18 December 1997 / Accepted: 9 June 1998     

Phylogenetic analysis in the genus Cicer and cultivated chickpea using RAPD and ISSR markers

Seventy five accessions belonging to 14 species of the genus Cicer were analysed with PCR-based molecular markers to determine their phylogenetic relationships. Eight of the species were annuals and included the Section Monocicer which contains cultivated chickpea (Cicer arietinum L.). The remaining six species were perennials (five from Section Polycicer and one from Section Acanthocicer). More than one accession per species was analysed in most of the wild species; within C. arietinum, 26 accessions including Kabuli and Desi types, were studied. RAPD analyses using 12 primers gave 234 polymorphic fragments. Variability within species was detected. A dendrogram based on the Jaccard similarity index showed that the distribution pattern of variability between species was related to both growth habit and geographical origin. An accession of Cicer reticulatum was closer to accessions of Cicer echinospermum than to the four remaining of C. reticulatum, suggesting the possibility of gene flow between species. Cluster analysis for cultivated chickpea differentiated Kabuli and Desi types but we did not detect a clear relationship between groups and the geographical origin of the accessions. Received: 5 April 2001 / Accepted: 13 July 2001     

Description and analysis of genetic diversity between commercial bean lines (Phaseolus vulgaris L.)

The effectiveness of RFLP, DAMD-PCR, ISSR and RAPD markers in assessing polymorphism and relationships between 24 commercial lines of Phaseolus vulgaris L.was evaluated. We have used a Phaseolus-specific minisatellite sequence as a probe, which enabled 23 of the bean lines tested to be fingerprinted. Based on the sequence information obtained, primers corresponding to the bean-specific minisatellite core sequence were used in subsequent PCR amplifications. Our observations indicated that while the DAMD-PCR was sensitive in detecting genetic variation between bean species and between accessions of P. vulgaris, when used alone it may be limited in its ability to detect genetic variation among cultivated bean lines due to the low number of loci amplified. Only one out of the five ISSR primers tested was efficient in generating multiple band profiles, which was insufficient to distinguish all the different bean lines. Reproducible RAPD profiles were obtained, and these allowed us to differentiate all the genotypes tested with seven primers. We ultimately used only results from RFLP and RAPD markers to explore the genetic diversity among commercial bean lines. Both analyses led to the same clustering of the bean lines according to their geographical origins (United States or Europe). With respect to the European lines, the results obtained from RAPD data also enable the lines to be clustered according to their creators. Received: 15 January 2000 / Accepted: 21 March 2000     

Molecular analysis of introgressive breeding in coffee (Coffea arabica L.)

Nineteen arabica coffee introgression lines (BC₁F₄) and two accessions derived from a spontaneous interspecific cross (i.e. Timor Hybrid) between Coffea arabica (2n=4x=44) and C. canephora (2n=2x=22) were analysed for the introgression of C. canephora genetic material. The Timor Hybrid-derived genotypes were evaluated by AFLP, using 42 different primer combinations, and compared to 23 accessions of C. arabica and 8 accessions of C. canephora. A total of 1062 polymorphic fragments were scored among the 52 accessions analysed. One hundred and seventy-eight markers consisting of 109 additional bands (i.e. introgressed markers) and 69 missing bands distinguished the group composed of the Timor Hybrid-derived genotypes from the accessions of C. arabica. AFLP therefore seemed to be an extremely efficient technique for DNA marker generation in coffee as well as for the detection of introgression in C. arabica. The genetic diversity observed in the Timor Hybrid-derived genotypes appeared to be approximately double that in C. arabica. Although representing only a small proportion of the genetic diversity available in C. canephora, the Timor Hybrid obviously constitutes a considerable source of genetic diversity for arabica breeding. Analysis of genetic relationships among the Timor Hybrid-derived genotypes suggested that introgression was not restricted to chromosome substitution but also involved chromosome recombinations. Furthermore, the Timor Hybrid-derived genotypes varied considerably in the number of AFLP markers attributable to introgression. In this way, the introgressed markers identified in the analysed arabica coffee introgressed genotypes were estimated to represent from 9% to 29% of the C. canephora genome. Nevertheless, the amount of alien genetic material in the introgression arabica lines remains substantial and should justify the development of adapted breeding strategies. Received: 2 February 1999 / Accepted: 12 May 1999     

Host plant resistance of different chickpea genotypes against Ascochyta rabiei (Pass.) Lab. under tunnel conditions

Host plant resistance is the most efficient and easy way to manage chickpea blight caused by Ascochyta rabiei (Pass.) Lab. For this purpose, 374 chickpea lines/varieties from various research organisations were evaluated in plastic tunnels. None of the line showed immune response against the blight; however, one line (K-01005) was found to be highly resistant. Moreover, 15 entries were resistant, 136 exhibited moderate resistant reaction, 150 were susceptible and 72 showed highly susceptible response. The genotypes found that resistance against blight can serve as a source of resistance for breeding programmes, and they could be released for commercial production directly.     

Assessment and comparison of AFLP and SSR based molecular genetic diversity in Indian isolates of Ascochyta rabiei, a causal agent of Ascochyta blight in chickpea (Cicer arietinum L.)

Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labr. (anamorph), is the most damaging disease of chickpea (Cicer arietinum L.) and is a serious biotic stress constraint for chickpea production. To understand the molecular diversity in A. rabiei populations of India, a total of 64 isolates collected from AB-infected chickpea plants from different agroclimatic regions in the North Western Plain Zone (NWPZ) of India were analyzed with 11 AFLP (amplified fragment length polymorphism) and 20 SSR (simple sequence repeat) markers. A total of 9 polymorphic AFLP primer pairs provided a total of 317 fragments, of which 130 were polymorphic and showed an average PIC value 0.28. Of the SSR markers, 12 showed polymorphism and provided a total of 29 alleles with an average PIC value 0.35. To the best of our knowledge, this is the first report on a comparison of AFLP and SSR diversity estimates in A. rabiei populations. The dendrogram developed based on AFLP and SSR data separately, as well as on the combined marker dataset, grouped the majority of AB isolates as per geographic regions. Model based population structure analysis revealed four distinct populations with varying levels of ancestral admixtures among 64 isolates studied. Interestingly, several AFLP primer combinations and SSR markers showed the locus/allele specific to AB isolates of certain regions, e.g., Hisar, Sriganganagar, Gurdaspur, and Sundarnagar. Genetic variability present in AB isolates of the NWPZ of India suggests the continuous monitoring of changes in A. rabiei population to anticipate the breakdown of AB resistance in chickpea cultivars grown in India.     

Virulence diversity of Ascochyta rabiei the causal agent of Ascochyta blight of chickpea in the western provinces of Iran

Chickpea is the third most important food legume in the world. The most important limiting factor for the chickpea production in the world, including Iran, has been the Ascochyta blight. The pathogenic variation of 40 Ascochyta rabiei isolates from the western provinces of Iran was assessed on eight chickpea differential lines. The results revealed that A. rabiei population is diverse in the western provinces of Iran and the virulence rating of isolates across differential lines showed a large but continuous pathogenic variability. Based on the statistical analysis and the continuous response in differential lines, it was not possible to categorise A. rabiei isolates in the present study into pathotypes or races. Information obtained from the current study can be valuable in developing quarantine methods aimed to prevent dissemination of highly virulent isolates and in the development of durable resistant cultivars against the Ascochyta blight of chickpea.     

Linkage mapping and QTL analysis in coconut (Cocos nucifera L.)

Different DNA marker types were used to construct linkage maps in coconut (Cocos nucifera L.; 2n = 32) for the two parents of the cross Malayan Yellow Dwarf (MYD) × Laguna Tall (LAGT). A total of 382 markers was sufficient to generate 16 linkage groups for each parent. The total genome length corresponded to 2226 cM for the LAGT map and 1266 cM for the MYD map with 4–32 markers per linkage group. Common markers allowed the association of 9 linkage groups for the two parents MYD and LAGT. QTL analysis for the trait early germination identified six loci. These QTLs correlate with early flowering and yield, representing characters which are important in coconut breeding. The co-segregation of markers with these QTLs provides the first opportunity for marker-assisted selection in coconut breeding programmes. Received: 22 September 1999 / Accepted: 29 November 1999     

Molecular diversity in pineapple assessed by RFLP markers

Pineapple, Ananas comosus (L.) Merr, is the third most important tropical fruit cultivated in all tropical and subtropical countries. Pineapple germplasm includes all seven species of the genus Ananas and the unique species of the related genus Pseudananas. A knowledge of its diversity structure is needed to develop new breeding programs. Restriction fragment length polymorphism (RFLP) was used to study molecular diversity in a set of 301 accessions, most of which were recently collected. This sample was analysed using 18 homologous genomic probes. Dissimilarities were calculated by a Dice index and submitted to Factorial Analysis. The same data were represented as a diversity tree constructed with the score method. Pseudananas sagenarius displayed a high polymorphism and shares 58.7% of its bands with Ananas. Within Ananas, variation appears continuous and was found mostly at the intraspecific level, particularly in the wild species Ananas ananassoides and Ananas parguazensis. As for the cultivated species, Ananas comosus appears relatively homogeneous despite its wide morphological variation and Ananas bracteatus, which is grown as a fence and for fruit, appears still much less variable. By contrast Ananas lucidus, cultivated by the Amerindians for fiber, displays a high polymorphism. This tree displayed a loose assemblage of numerous clusters separated by short distances. Most species were scattered in various clusters, a few of these being monospecific. Some accessions which had not been classified, as they shared morphological traits typical of different species, re-group with one or the other, and sometimes with both species in mixed clusters. No reproductive barrier exists in this germplasm and these data indicate the existence of gene flow, enhancing the role of effective sexual reproduction in a species with largely predominant vegetative mutiplication. Received: 8 March 2000 / Accepted: 14 April 2000     

Analysis of genetic diversity and relationships in East African banana germplasm

The genetic diversity and phylogenetic relationships of 29 East African highland banana (Musa spp.) cultivars and two outgroup taxa, M. acuminata Calcutta 4 and Agbagba were surveyed by RAPD analysis. A genetic similarity matrix was established based on the presence or absence of polymorphic amplified fragments. Phylogenetic relationships were determined by UPGMA cluster analysis. RAPDs showed that the highland bananas are closely related with a narrow genetic base. Nevertheless, there were sufficient RAPD polymorphisms that were collectively useful in distinguishing the cultivars. The dendrogram was divisible into a major cluster composed of all the AAA highland banana cultivars and Agbagba (AAB) and a minor cluster consisting of Kisubi (AB), Kamaramasenge (AB) and Calcutta 4 (AA). Several subgroups are recognized within the major cluster. RAPD data did not separate beer and cooking banana cultivars. Our study showed that RAPD markers can readily dissect genetic differences between the closely related highland bananas and provide a basis for the selection of parents for improvement of this germplasm. Received: 28 June 2000 / Accepted: 1 August 2000     

Application of microsatellites in wheat (Triticum aestivum L.) for studying genetic differentiation caused by selection for adaptation and use

For studying genetic differentiation caused by selection for adaptation and end-use, the allele frequencies of 42 microsatellites (MS), representative of the three wheat genomes, were analysed in a total of 60 wheat cultivars. The cultivars originate from three agroecological areas (AEAs) – Germany, Austria and Hungary – and represent equal numbers of ’quality wheats’ and ’feed wheats’ for each country. For the 42 loci, 202 alleles were detected using PAGE and silver staining. The average number of alleles per locus was 4.8, including four monomorphic loci. For 16 loci, null alleles were detected. Cluster analysis clearly differentiated the varieties according to the three AEAs and, within each AEA, into quality wheats from feed wheats. Analysis of variance revealed highly significant differences of distance data between AEAs as well as between quality groups. The correlation between genetic distance (GD) and pedigree data (coefficient of diversity, COD) was r _s=0.45. The results have proven the excellent resolving power of MS in varietal differentiation, which arises through breeding under specific environmental conditions, and for different end-use. Received: 15 March 1999 / 17 June 1999     

Variation among and within Capsicum species revealed by RAPD markers

 Germplasm characterization is an important link between the conservation and utilization of plant genetic resources. A total of 134 accessions from six Capsicumspecies maintained at the Asian Vegetable Research and Development Center were characterized using 110 randomly amplified polymorphic DNA (RAPD) markers. Ten pairs of potentially duplicated accessions were identified. Multidimensional scaling analysis of the genetic distances among accessions resulted in clustering corresponding to a previous species assignment except for six accessions. Diagnostic RAPDs were identified which discriminate among the Capsicumspecies. The diagnostic markers were employed for improved taxonomic identification of accessions since many morphological traits used in the identification of Capsicumare difficult to score. Three Capsicumaccessions, misclassified based on morphological traits, were reassigned species status based on diagnostic RAPDs. Three accessions, not previously classified, were assigned to a species based on diagnostic RAPDs. Definitive conclusions about the species assignment of three other accessions were not possible. The level of diversity between Capsicum annuumaccessions from the genebank and the breeding program were compared and no differences were observed either for RAPD variation or diversity. The utilization of genetic resources as a source of variance for useful traits in the breeding program may be the reason for the similarity of these two groups. Received: 1 September 1998 / Accepted: 28 December 1998     

Development and characterization of microsatellite markers in black poplar (Populus nigra L.)

Using an enrichment procedure, we have cloned and sequenced microsatellite loci from black poplar (Populus nigra L.) and developed primers for sequence-tagged microsatellite (STMS) analysis. Twelve primer pairs for dinucleotide repeats produced fragments of sufficient quality which were polymorphic in P. nigra. Some of them also showed amplification in other Populus species (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, and/or P. lasiocarpa). The best nine and (GT) (GA) microsatellite markers were tested on a set of 23 P. nigra genotypes from all over Europe. The microsatellites were highly polymorphic, with 10–19 different alleles per microsatellite locus among these 23 genotypes. WPMS08 sometimes amplified three fragments. Using the other eight marker loci, the level of heterozygosity among the plants was on average 0.71 (range 0.25–1.00). The microsatellite markers developed will be useful for screening the genetic diversity in natural populations and in gene bank collections. Received: 21 October 1999 / Accepted: 24 November 1999     

Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes

 Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction. Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999     

Allelic variation of the HMW glutenin subunits in Spanish accessions of spelt wheat (Triticum aestivum ssp. spelta L. em. Thell.)

Spelt wheat (Triticum aestivum ssp. spelta L. em. Thell.) is a hulled wheat of Germanic origin that survives at marginal areas in Asturias (Spain). The HMW glutenin subunit composition of 403 accessions of spelt wheat from Spain has been analysed by SDS-PAGE. Three allelic variants were detected for Glu-A1. For the Glu-B1 locus, two of seven alleles detected have not been found before; while four of nine alleles detected for the Glu-D1 are not previously described. Considering the three loci, twenty five combinations were found among all the evaluated lines. This wide polymorphism could be used to transfer new quality genes to wheat, and widen the genetic basis of them. Received: 19 September 2000 / Accepted: 20 October 2000     

Arbitrarily primed-PCR based diversity assessment reflects hierarchical groupings of Indian tetraploid wheat genotypes

Genetic diversity analysis using PCR with arbitrary decamer primers (RAPD — random amplified polymorphic DNA) was carried out in a set of 63 tetraploid wheat genotypes which comprised 24 durum landraces, 18 durum cultivars, nine dicoccum cultivars, ten less commonly cultivated species and two wild tetraploid species. The durum and dicoccum wheat genotypes are a part of the germplasm used in Indian tetraploid wheat breeding programs. A total of 206 amplification products were obtained with 21 informative primers, of which 162 were polymorphic. The highest degree of polymorphism was seen in the wild and less commonly cultivated species (68.9%). Durum released cultivars showed greater polymorphism (50.6%) than landraces (44.8%), while dicoccum cultivars showed a considerably low level of polymorphism (23.6%). Cluster analysis led to the separation of wild and cultivated genotypes, and among cultivated emmer wheat distinct groups were formed by the durum cultivars, durum landraces and dicoccum cultivars. The subgroupings of landraces had no relation to their geographical distribution. The durum cultivars formed subgroups based on common parentage in their pedigree. Among species, wild timopheevi wheat (T. araraticum) and its cultivated form (T. timopheevi) formed a distinct group distant from all other genotypes. The present study is a first attempt at determining the genetic variation in Indian tetraploid wheats at the molecular level. Received: 10 January 1999 / Accepted: 30 January 1999