Interspecific reproductive barriers and genomic similarity among the rough-seeded Lupinus species

The nature of reproductive barriers and meiotic behaviour of chromosomes were studied in interspecific hybrids among the six rough-seeded Lupinus species. Out of 30 different interspecific crosses attempted in all possible combinations, eight produced viable F, seeds. These successful crosses involved L. cosentinii, L. digitatus, L. atlanticus and L. pilosus. Crosses of L. princei with other species resulted in shrivelled F, seed in all combinations. In contrast to previous reports, crosses of L. palaestinus with all other rough-seeded lupins were incompatible as no F₁ hybrids were obtained. Barriers to interspecific reproduction were identified as nucleo-cytoplasmic and embryo-endosperm incompatibility in unsuccessful crosses, and chromosomal imbalances in F₁ hybrids. Gene transfer is possible among L. cosentinii, L. digitatus and L. atlanticus, which produced F₂ seed. Patterns of chromosome configurations in F₁ meiotic cells suggested that these species have at least two genomes partially in common, but include inversions and translocations. The genome of L. atlanticus is closer to L. digitatus than to L. cosentinii, and that of L. pilosus is closer to L. atlanticus than to L. cosentinii. L. princei appears to have an isolated genome within the rough-seeded Lupinus species.     

Flower-Colour Polymorphism in Lupinus pilosus in Israel

Four rare flower-colour mutants were detected in natural populations of Lupinus pilosus in Israel. The breeding systems and genetic bases of three oif them were examined. Mutants were tagged in wild populations and seed obtained upon open pollination was collected and grown in a, net house which was insect-proof. Mutant progenies were used in crosses with each other and with wild-type plants. Our results indicate the possibility that all three mutants are simple Mendelian recessives to the wild-type deep-violet colour and that in L. pilosus this colour is not monogenic but determined by at least two loci. All these mutants show a high degree of selling when open-pollinated in nature.     

Genetic diversity in cornsalad (Valerianella locusta) and related species as determined by AFLP markers

Fifteen amplified fragment length polymorphism (AFLP) EcoRI/MseI‐based primer combinations with five selective bases (Eco RI‐ANN, MseI‐CN) were used to estimate genetic diversity among 45 line varieties of cultivated cornsalad and 19 genebank accessions classified into nine different species related to cornsalad. Polymorphic fragments were scored for calculation of Jaccard's coefficient of genetic similarity (GS). The average GS estimate in elite germplasm (GS = 0.90) was substantially higher than in exotic germplasm (GS = 0.47). UPGMA‐cluster analysis revealed genetic relationships among recently bred varieties, old varieties and genebank accessions. Analysis of molecular variance indicated almost threefold variability within sets compared with between sets due to a high level of polymorphism among wild species. Sources for increasing genetic diversity in elite germplasm of cornsalad were suggested and a duplicate among the genebank accessions was detected. AFLPs could be considered a powerful tool for genetic diversity estimation in cornsalad germplasm and are recommended for systematic fingerprinting of remaining cornsalad species.     

Assessment of genetic diversity of cultivated chickpea using microsatellite-derived RFLP markers: Implications for origin

Restriction fragment length polymorphism (RFLP) analysis was performed on 30 accessions of cultivated chickpea (Cicer arietinum L.) collected from 11 different countries representing the Near East, Central Asian and Hindustani regions. A synthetic digoxygenated oligonucleotide (GATA)₄ complementary to a microsatellite DNA sequence was used as a probe. The results revealed that simple repetitive sequences are abundant and polymorphic in the chickpea genome. The fragments detected were used lo estimate the genetic diversity within accessions and a similarity index between the genotypes of the accessions. The genetic distance data were used to construct a dendrogram depicting genetic relationships among the different accessions. The results indicate that the greatest genetic diversity occurs in Pakistan, Iraq, Afghanistan, south-east Russia, Turkey and Lebanon. Lower genetic diversity was found in Iran, India, Syria, Jordan and Palestine, Based on DNA markers, it is concluded that there are three centres of diversity for chickpea: Pakistan-Afghanistan. Iraq Turkey and Lebanon. India, which was previously considered as a secondary center of diversity for chickpea, showed lower diversity than the above regions.     

Detecting genetic diversity in diploid bananas using PCR and primers from a highly repetitive DNA sequence

Summary The polymerase chain reaction (PCR) was used to detect polymorphisms among 29 diploid clones of Musa acuminata Colla. from Papua New Guinea. Primer sequences were derived from a 520 bp highly repetitive DNA sequence isolated from M. acuminata ssp. malaccensis. Primers, used individually, detected a total of 48 polymorphisms that were scored as unit characters and used to generate a Jaccard's similarity index. Principal coordinate analysis (PCO) was used to cluster clones and the unweighted paired-group method of analysis (UPGMA) was used to compute genetic distance among the materials examined. The abundance of diversity within the PNG diploids examined reflects the extreme genetic variability within the M. acuminata gene pool. PCR, utilizing primers from a highly repetitive sequence, is a rapid means of detecting genetic diversity in M. acuminata.     

Genetic relationships among 23 Ficus accessions using inter-simple sequence repeat markers

Ficus genus is widely distributed in all the climatic stages and great diversity. The exploration of genetic diversity is a pre-requisite for genome organization in the wild species and the related domesticated ones. Inter-Simple Sequence Repeat (ISSR) markers were used to assess the identification of 23 important Ficus accessions and determination of the genetic relationships among these accessions. Out of 21 ISSR primers tested, five primers produced 116 detectable fragments, of which 106 (91.3%) were polymorphic across the accessions. Each of the five primers produced a fingerprint profile unique to each of the accessions studied, and thus could be solely used for their identification. Thirteen unique bands specific to nine species were detected. These may be converted into species-specific probes for identification purposes. Genetic relationships among these accessions were evaluated by generating a similarity matrix based on the Dice coefficient and the Unweighted Pair Group Method with Arithmetic Average (UPGMA) dendogram. The results showed a clear cut separation of the 23 Ficus accessions and were in broad agreement with the morphology. Both molecular and morphological markers will be useful for preservation of the Ficus germplasm.     

Application of the TRAP technique to lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) genotyping

Summary To demonstrate the applicability of the target region amplification polymorphism (TRAP) marker technique to lettuce genotyping, we fingerprinted 53 lettuce (Lactuca sativa L.) cultivars and six wild accessions (three from each of the two wild species, L. saligna L. and L. serriola L.). Seven hundred and sixty-nine fragments from 50 to 900 bp in length were amplified in 10 PCR reactions using 10 fixed primers in combination with four fluorescent labeled arbitrary primers. Three hundred and eighty-eight of these fragments were polymorphic among the 59 Lactuca entries and 107 fragments were polymorphic among the 53 lettuce cultivars and the six wild accessions; 251 fragments were present only in the wild species. These markers not only discriminated all cultivars, but also revealed the evolutionary relationship among the three species: L. sativa, the cultivated species, is more closely related to L. serriola than to L. saligna. Cluster analysis grouped the cultivars by horticultural types with a few exceptions. These results are consistent with previous findings using RFLP, AFLP, and SAMPL markers. The TRAP markers revealed significant differences in genetic variability among horticultural types, measured by the average genetic similarity among the cultivars of the same type. Within the sample set, the leaf type and butterhead types possessed relatively high genetic variability, the iceberg types had moderate variability and the romaine types had the lowest variability. The genetic behavior of TRAP markers was assessed with a mapping population of 45 recombinant inbred lines (RILs) derived from an interspecific cross between L. serriola and L. sativa. Almost all the markers segregated in the expected 1:1 Mendelian ratio and are being incorporated into the existing lettuce linkage maps. Our results indicate that the TRAP markers can provide a powerful technique for fingerprinting lettuce cultivars. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Genetic diversity and taxonomic relationships within the genus Lens as revealed by allozyme polymorphism

Summary A survey of allozyme polymorphism at 11 loci was carried out on 439 accessions from the genus Lens. This comprised 153 Lens culinaris subsp. orientalis, 35 L. odemensis, 117 L. ervoides, 32 L. nigricans, 2 of a differentiated cytotype of L. nigricans and 100 landrace accessions of the cultivated lentil (L. culinaris subsp. culinaris), from 10 different countries. The aim of the survey was to determine intra-specific genetic diversity and species relationships, based on phylogenetic and phenetic analyses, particularly regarding the position of L. odemensis and the differentiated cytotype of L. nigricans. Diversity was described by three statistics. The level of diversity in the cultivated taxon was lower than in any of the wild species according to two of these statistics, the percentage of polymorphic loci and mean number of alleles per locus. For the third measure (Nei's mean genetic diversity) it was only greater than L. ervoides. Genetic diversity statistics of the wild species indicated differences in the nature of between-population genetic diversity within the different taxa. Phylogenetic analysis suggests that L. odemensis and L. ervoides evolved from a common ancestor, and L. culinaris subsp. orientalis subsequently evolved from L. odemensis. Phenetic analysis, however, places L. odemensis closer to L. culinaris subsp. orientalis than to L. ervoides. Nei's mean genetic distance of L. odemensis from both L. culinaris subsp. culinaris (0.204) and L. culinaris subsp. orientalis (0.110) was greater than the distance between them (0.062). This evidence is not conclusive in determining whether L. odemensis should retain its specific status. Further crossability studies should be carried out on a range of genotypes to assess the potential for gene flow. The evidence presented shows the differentiated cytotype of L. nigricans to be quite distinct from other L. nigricans accessions, both phenetically and phylogenetically. This indicates that the differentiated cytotype of L. nigricans may constitute a new taxon. Discriminant function analysis reveals that isozymes may be useful in validating species classification.     

Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F₁ and F₂ progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessment of genetic diversity in sesame (Sesamum indicum L.) genotypes, using EST-derived SSR markers

A total of 16,619 ESTs sequences (SSRs) of sesame (Sesamum indicum L.) were mined from Genbank. From sequences, 156 primer pairs were designed and characterized to determine the diversity among 49 sesame accessions. Twenty SSRs were found to be polymorphic and the number of alleles ranged from two to five per locus. The allele size varied from 101 to 399 bp. The average PIC value of the 20 SSR loci was 0.72 ranging from 0.49 (SEM-12-68) to 0.90 (SEM-12-27). Dendrogram analysis grouped the 49 genotypes into five separate clusters exhibiting a genetic similarity coefficient from 0.59 to 1.0. Hence, these EST-derived SSRs markers could be useful in assessing the diversity of sesame accessions and could also help in identifying diverse parents for sesame improvement programs.     

Genetic diversity among tea cultivars from China, Japan and Kenya revealed by ISSR markers and its implication for parental selection in tea breeding programmes

Tea plant [Camellia sinensis (L.) O. Kuntze] is an important beverage crop in the world. In recent years many clonal tea cultivars have been released, and they play major roles in improving the production and quality of tea. It is important to understand the genetic diversity and relatedness of these cultivars to avoid inbreeding and narrow genetic basis in future tea breeding. In the present study, genetic diversity and relationship of 48 tea cultivars from China, Japan and Kenya were evaluated by inter‐simple sequence repeat (ISSR) markers. A total of 382 ISSR bands were scored, of which 381 (99.7%) were polymorphic. The ISSR primers showed high ability to distinguish between tea cultivars according to their high Resolving Power (R_P) with an average of 7.4. The mean of Nei’s gene diversity (H) and Shannon’s information index (I) were 0.22 and 0.35, respectively. More abundant diversity was revealed among cultivars in China than those in Japan and Kenya. Within Chinese populations, the level of diversity in east China was higher than that in other regions. The coefficient of genetic differentiation (G_ST) was 0.202, which indicates a high degree of genetic variation within populations. This result was further confirmed by analysis of molecular variance, which revealed the variance component within the populations (92.07%) was obviously larger than that among populations (7.93%). The level of gene flow (N_m) was estimated to be 2.0. This could be explained by frequent natural cross‐pollination and seed dispersal among tea populations. The pairwise similarity coefficient between the cultivars varied from 0.162 to 0.538. A dendrogram of 48 tea cultivars was constructed where all the tested cultivars were divided into two groups. Our data show that the genetic relationship among tea cultivars can be determined by the ISSR markers. This will provide valuable information to assist parental selection in current and future tea breeding programmes.     

Diversity analysis and genotyping in Pisum with sequence tagged microsatellite site (STMS) primers

Pisum sativum specific sequence tagged microsatellite site primers were used to amplify genomic profiles from 15accessions of P. sativum L. that represented the genetic base of the Australian field pea-breeding program and five accessions of the wild related species P. fulvum. The STMS primers were used to assess genetic relationships among the Pisum accessions in two ways. Firstly, to produce RAPD-like multiple banding marker profiles using an adapted RAMS method, for intra- and interspecific diversity analysis. From the 14 flanking primer pairs assessed, 133 markers were obtained. Conservation and reproducibility of markers among individuals within accessions was demonstrated. The largest distance observed among P. sativumaccessions was 22% and among P.fulvum accessions was 40%, similar to that revealed with other PCR-based methods. The maximum distance between P.sativum and P. fulvum accessions was 46%. Phylogenetic clustering of P. sativum accessions, using the neighbour joining method and based on simple matching distances, was distinct and distant to P. fulvum. Secondly, PCR with a higher annealing temperature and fluorescent labeling identified simple and allelic loci markers useful for creating agenotype/fingerprint database for P. sativum cultivars. This is the first report to demonstrate the use of Pisum specific STMS sequences for both diversity analysis and genotype identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

SCAR and CAPS markers flanking the Brassica oleracea L. Pp523 downy mildew resistance locus demarcate a genomic region syntenic to the top arm end of Arabidopsis thaliana L. chromosome 1

We recently mapped the Pp523 locus that includes a single, dominant gene conferring resistance to downy mildew expressed in adult plants to a 75.1 cm long linkage group on a genetic linkage map of Brassica oleracea L. More recently, we identified a new AFLP marker 2.8 cm downstream from the resistance gene. The five DNA markers within an 8.5 cm region encompassing the Pp523 gene were cloned and sequenced. Three of these markers were transformed into SCARs (sequence characterised amplified regions), however, two among them were monomorphic and were analysed as CAPS (cleaved amplified polymorphic sequence) markers among the mapping population. Searched against genomic databases, the five B. oleracea DNA-marker sequences matched Arabidopsis thaliana L. gene sequences that delimit a conserved syntenic region in the top arm end of chromosome 1 of this last species. Considering the close genetic relatedness between both species, the information on this specific genomic region in A. thaliana is particularly useful for the construction of a fine-scale map of the corresponding genomic region in B. oleracea. The identified SCAR and CAPS markers can be used for marker assisted selection (MAS) in breeding programs aimed at the introgression of the Pp523 resistance locus, allowing the reliable indirect identification of plants harbouring the resistance gene with a margin of error of approximately six in ten-thousand selected plants.     

Genetic diversity and phylogenetic relationships among the annual Cicer species as revealed by isozyme polymorphism

Summary There are few estimates of genetic variability within and among populations of the nine annual Cicer species and for the wild species this information is based on few accessions. The present study was undertaken to examine genetic variation within and between annual Cicer species. One hundred and thirty-nine accessions of nine annual Cicer species were used for electrophoretic analysis at ICARDA. High levels of polymorphism in all eight wild annual Cicer species was found. This is in contrast to earlier research which had shown high polymorphism only in C. reticulatum. Cicer reticulatum had the highest proportion of polymorphic loci. However, for the cultigen, among 14 loci assayed, only two were polymorphic, ADH and PGD2. The nine species formed four phylogenetic groups based on the neighbor-joining method. The first group comprised C. arietinum, C. Reticulatum and C. echinospermum, the second C. bijugum, C. judaicum and C. pinnatifidum, the third C. chorassanicum and C. yamashitae; and the fourth group consisted of one species, C. cuneatum. The phylogenetic tree developed from the neighbor-joining technique illustrated that C. reticulatum is the probable progenitor of C. arietinum and that C. echinospermum split off from a common ancestor at an earlier stage in the evolutionary history of Cicer. Genetic diversity data showed that the greatest diversity was within C. reticulatum and the lowest with the cultigen, C. arientinum. With the exception of C. reticulatum, genetic diversity increased with genetic distance from the cultigen. Little geographic variation in genetic diversity was found.     

MicroRNA‐based molecular markers: a novel PCR‐based genotyping technique in Brassica species

DNA‐based molecular markers play a significant role in gene mapping, genetic diversity analysis, germplasm evaluation and molecular marker‐assisted selection. A combination of desirable marker characteristics such as abundant polymorphism, good stability, ease of production and high efficiency is difficult to achieve when utilizing traditional molecular marker systems. MicroRNAs are a type of endogenous non‐coding RNAs prevalent in the genomes of many organisms. The high conservation of miRNA and pre‐miRNA sequences provides an opportunity to develop a novel molecular marker type. We downloaded 82 miRNA sequences from the Brassica miRBase database and designed 46 single miRNA‐based primers which could be randomly combined to generate primer pairs. A proportion of these primer pairs were validated for transferability and polymorphism using DNA from 16 varieties of six Brassica species. The percentage of polymorphic primer pairs were 28.1%, and the average polymorphism information content (PIC) value for the 34 primer pairs was 0.43. Good transferability was verified across species. These results indicate that miRNA‐based markers provide a novel genotyping technique with low costs, high efficiency, stability and good transferability.     

Relationship between morphological variation and geographical origin or selection history in Lupinus pilosus

Patterns of morphological diversity were examined in Lupinus pilosus in relation to geographical origins or selection history of accessions. There was significant variation among accessions for most architectural and reproductive characters in a field experiment in Perth, Western Australia. Late flowering was associated with resetted early growth, profuse branching and collection in higher rainfall sites. High yield and profuse podding on the main stem was associated with large pods and leaves, many seeds per pod, greater height to the first main-stem pod, and collection in higher altitude sites. Ten groups of accessions, identified by hierarchical cluster analysis, accounted for 79% of genotype and 69% of genotype x character sums of squares. One group, possibly taxonomically distinct from the others, consisted exclusively of wild types from northern Israel with poor pod set and low seed yield, poor nodulation, pale foliage, and small leaves. Wild types from Turkey and Crete had rosetted early growth and were late flowering, and the Turkish group had extremely rapid mid-season growth. Ornamental types from Europe or Australia with pink, purple or white flowers clustered together in an early flowering group with vigorous early growth. A smooth-seeded type of L. pilosus was discovered in a group of short, late flowering and rough-seeded types from Syria.     

Changes in genetic diversity during seven cycles of recurrent selection for grainyield in oat, Avena sativa L.

Variability for desirable alleles within elite breeding populations is a requirement for long-term genetic improvement. Changes in genetic diversity were evaluated in a recurrent selection programme for enhanced grain yield in oat, Avena sativa L., using coefficient of parentage (r_p) and restriction fragment length polymorphism (RFLP) based genetic similarity (S) estimates. Both r_p and s increased during seven cycles of recurrent selection, indicating a reduction in the level of genetic diversity within the population. The relationship between genetic diversity and agronomic performance was also examined. Genetic similarity measures were poor predictors of the near-homozygous progeny performance and general and specific combining ability effects for three agronomic traits in the recurrent selection programme. In addition, there was no apparent trend for preferential selection of progeny from either more similar or more diverse parents within a given cycle of selection. The overall reduction in genetic diversity in this population has not affected selection response for grain yield. Variability remaining at important loci or de novo variation are possible explanations for the continued selection progress.     

Assessment of genetic relatedness of the genera Ananas and Pseudananas confirmed by RAPD markers

Genetic relationships among 18 accessions, including 16 of Ananas and two of Pseudananas, were investigated using RAPD molecular markers. The procedure for DNA extraction was adapted from the method of Dellaporta et al. (1983) where an incubation in proteinase K and a purification step were included. From the total of 148 markers scored,132 (89.2%) were polymorphic. The similarity matrix was used for cluster analysis. The phenogram developed from the RAPD bands showed that for most of the cases, the accessions within a species grouped together. Nevertheless, a moderate infraspecific genetic variation was observed. For example, DNA data grouped all A. comosus accessions with a mean similarity coefficient of 0.85. Comparable results were obtained with all other species investigated. The highest genetic divergence was found withinA. lucidus where the mean similarity coefficient among accessions was0.75. A similar level of genetic polymorphism was observed among species,therefore, a definition about which species were involved in the constitution of A. comosus genotypes was not possible. These results agree with the breeders standpoint suggesting that all Ananas species belong to the primary gene pool of pineapple. This revised version was published online in July 2006 with corrections to the Cover Date.