Genetic diversity in Ethiopian hexaploid and tetraploid wheat germplasm assessed by microsatellite markers

The genetic diversity of a subset of the Ethiopian genebank collection maintained at the IPK Gatersleben was investigated applying 22 wheat microsatellites (WMS). The material consisted of 135 accessions belonging to the species T. aestivum L. (69 accessions), T. aethiopicum Jacubz. (54 accessions) and T. durum Desf. (12 accessions), obtained from different collection missions. In total 286 alleles were detected, ranging from 4 to 26 per WMS. For the three species T. aestivum, T. aethiopicum and T. durum on average 9.9, 7.9 and 7.9 alleles per locus, respectively, were observed. The average PIC values per locus were highly comparable for the three species analysed. Considering the genomes it was shown that the largest numbers of alleles per locus occurred in the B genome (18.4 alleles per locus) compared to A (10.1 alleles per locus) and D (8.2 alleles per locus) genomes. Genetic dissimilarity values between accessions were used to produce a dendrogram. All accessions could be distinguished, clustering in two large groups. Whereas T. aestivum formed a separate cluster, no clear discrimination between the two tetraploid species T. durum and T. aethiopicum was observed.     

Variation of cultivated flax (Linum usitatissimum L. subsp.usitatissimum) and its wild progenitor pale flax (subsp.angustifolium (Huds.) Thell.)

The domestication of cultivated flax (Linum usitatissimum L. subsp.usitatissimum) is briefly discussed. Using data documented as a matter of routine in genebank work, 63 accessions of cultivated flax from the flax germplasm collection of the Gatersleben Genebank are compared with 73 accessions of its wild progenitor pale flax (subsp.angustifolium (Huds.) Thell.), which have been observed in systematic field trials. Range of variation, genetically based variation, heritability and correlation of several characters are considered, especially with respect to the influence of domestication. Cultivated flax shows higher variation in the characters of generative plant parts, while pale flax varies more in the vegetative parts of the plant. The character correlations are similar in both subspecies. Of the 29 characters studied in pale flax the parameters describing tillering, height of plant, weight of seeds, width of petals and width of leaves are especially suitable for distinguishing between different accessions. In spite of the high heritabilities of most of the characters, the influence of the environment is significant in most cases.     

Measurement of nuclear DNA content of the genus <Emphasis Type="Italic">Trifolium</Emphasis> L. as a measure of genebank accession identity

The collection, identification and maintenance of genebank accessions of the genus Trifolium is a major task because of the large number of genera and their occasional morphological similarity. We investigated whether the measurement of nuclear DNA content can serve as an additional criterion for identification of mislabeled accessions. Relative nuclear DNA content was determined by flow cytometry measurements for a total of 151 genebank accessions of 23 Trifolium species with notable agronomical value. Among 23 species analyzed, 15 were found to possess a uniform relative nuclear DNA content, with intraspecific variability of the majority of analyzed species lower than 5%. Within six Trifolium species, 1–2 atypical accessions with outstanding differences in relative DNA content, chromosome number and morphological features were found. In T. hybridum and partially in T. ambiguum these outstanding differences could be ascribed to variations in accession ploidy level. For the remaining atypical accessions, the determined nuclear DNA content, chromosome number and morphological features were not related to those characteristic of the species. Additionally taxonomic identity of atypical accessions was determined using ITS region sequencing and morphological observations. We propose flow cytometric measurements of nuclear DNA content as simple and reliable technique which can be used at seedling stage to verify identity and genetic stability of Trifolium genebank accessions.     

Patterns of SSR variation in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) seeds under ex situ genebank storage and accelerated ageing

Maintaining seed viability and germplasm integrity is a challenging task in conservation of plant genetic resources, as seeds under storage will lose viability and genetic changes will occur. Attempt was made to analyze the patterns of genetic changes in wheat germplasm under ex situ genebank storage and accelerated ageing treatments. A set of 16 naturally aged wheat accessions under ex situ genebank storage since 1994 were sampled. Four recently regenerated wheat accessions were selected, four random seed samples were chosen from each accession, and three of them were exposed to three different accelerated ageing treatments. These 32 seed samples in two germplasm sets displayed a range of germination rates from 4 to 98 %. Thirty-seven microsatellite markers representing 21 wheat chromosomes were applied to screen 12 seeds of each sample and 449 SSR alleles were scored. Large SSR variation was found in each germplasm set. There was 73.1 % of the total SSR variation present among the naturally aged samples and 78.2 % present among the accelerated ageing samples. Several analyses for genetic association consistently revealed no clear genetic separations among samples of high or low germination rates in both germplasm sets. Samples under different accelerated ageing treatments did not show much genetic differentiations from the original sample of each accession. Mantel tests revealed non-significant associations between SSR variability and sample germination rates for both germplasm sets. These findings are useful for understanding seed deterioration under different ageing conditions and suggest that genome-wide SSR variability may not provide sensitive markers for the monitoring of wheat seed viability.     

Allelopathy variation in dryland winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) accessions grown on the Loess Plateau of China for about fifty years

In this study, we investigated the genetic variation of allelopathic potential, its grey correlation with important agronomic characters, and rank analysis on allelopathic stability of fifteen wheat accessions commonly grown in arid regions of the Loess Plateau in China. The genetic variation of allelopathic property in dryland winter wheat showed significant differences between accessions. Allelopathic effects exhibited high heritability (55–95%) throughout the life cycle of wheat. Heritability was highest in the tillering stage and weakest in the seed filling stage. Allelopathic potential varied and was discontinuous throughout the wheat life cycle. Grey correlation analysis indicated a close relationship between allelopathic potential dependant on growth phase and agronomic characters. Allelopathic expression during some growth periods induced a partial correlation effect on some important agronomic characters that affected wheat yield. Allelopathic heritability and its degree of influence on yield were more evident in the vegetative growth stage compared to the reproductive stage. A multiple linear regression was built between allelopathic potential during different growth periods and agronomic characters pursued in wheat breeding. Allelopathic potential had a linear effect on production traits cultivated in wheat breeding. When allelopathic intensity varied from 0 to 1 in the reproductive stage, plant height ranged from 44 to 108 cm, spike length from 6.4 to 9.2 cm, number of spikelets with seeds from 13.4 to 21.0, mean seed number per spike from 41.5 to 50.3 and thousand seed weight from 36.2 to 38.3 g. Based on rank analysis, we concluded that there was a synergistic relationship between allelopathic potential in wheat and genetic, chemical and ecological factors. Triticum aestivum L. ‘No 6 Lankao’ and T. aestivum L. ‘No 22 Xiaoyan’ were identified as stable and relatively strong allelopathic accessions, whereas T. aestivum L. ‘Lankao 95–25’ was a stable but relatively weak allelopathic cultivar. Other varieties showed unstable allelopathic potential.     

AFLP similarities among historic Danish cultivars of fodder beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L. subsp. <Emphasis Type="Italic">vulgaris</Emphasis>var. <Emphasis Type="Italic">rapacea</Emphasis> Koch)

Dead seeds of a fodder beet cultivar ‘Elvetham’ stored under ambient conditions since 1880 were compared to a homonymous sample preserved in an on-farm situation in Denmark. DNA was isolated from single seeds and successfully applied to Amplified Fragment Length Polymorphism (AFLP) analysis of the accessions. Six primer pairs were used to determine the similarity between the two accessions based on 112 polymorphic bands. Furthermore, similarity among seven cultivars of fodder beets representing the main types used in Scandinavia at the end of the 19th century was determined. This analysis was based on 152 polymorphic bands. Differentiation among the seven cultivars was determined to a mean G _ST value of 0.438, while G _ST between the two ‘Elvetham’ accessions was 0.266. A principal coordinate analysis based on jaccards similarity index illustrates that the two ‘Elvetham’ accessions are different from each other. The differentiation is higher than the value found between two separate ‘Eckerndorfer’ accessions. The results indicate that the cultivated accession has changed. Additionally, the value of applying old dead seed material for documentation in gene banks is demonstrated. During the analysis it was found that DNA isolated from seeds and leaves behaved differently in the AFLP process, however, the two fractions assigned to their common accession.     

Phenotypic diversity of durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> Desf.) from Jordan

Durum wheat landrace genotypes are disappearing from the main wheat areas in Jordan, because of spreading of new uniform cultivars and the serious reduction in wheat cultivation. This study was conducted to evaluate genetic diversity in durum wheat landraces from Jordan and to identify desirable agronomic traits. Landraces were collected from two target areas: Ajloun and Karak. The collected material was grown under rainfed conditions using an augmented design with five blocks and four repeated check cultivars. Data were collected for 14 morphological and agronomic traits. Phenotypic diversity index (H′) was estimated, and the relationships among accessions were measured using cluster analysis and dendrogram similarity matrix. The results revealed the presence of a wide range of variability among landraces., which possess high levels of variability for biological yield, fertile tillers, number of seeds per spike, seed weight per spike and weight of 1000 seeds. These landraces must be considered as a reservoir of genes that plant breeders need in their wheat improvement programs and should be conserved both ex situ and in situ.     

Identification of duplicates and fingerprinting of primary and secondary wild annual <Emphasis Type="Italic">Cicer</Emphasis> gene pools using AFLP markers

Wild annual Cicer gene pools contain valuable germplasm for chickpea improvement programs. Previous research showed that duplication might exist in accessions collected from these gene pools, which would hinder chickpea breeding and related research. AFLP (amplified fragment length polymorphism) markers were used to fingerprint the world collections of the primary and secondary gene pools including C. reticulatum Lad., C. bijugum K.H. Rech., C. judaicum Boiss. and C. pinnatifidum Jaub. et Sp. Duplicates were detected in a total of 24 accessions in both the gene pools, highlighting the necessity to fingerprint the germplasm. Genotypic difference was detected as gene pool specific, species specific and accession specific AFLP markers. These were developed into fingerprinting keys for accession identification between and within species and gene pools. Use of AFLP markers to detect duplicates and to identify accessions is a reliable method which will assist in the characterisation and use of wild annual Cicer germplasm in chickpea improvement programs. We recommend the procedure presented in this paper as a standard approach for the precise genetic identification and characterisation of future world collections of wild Cicer, to keep germplasm integrity and to benefit chickpea breeding and related research programs.     

Intra-specific DNA polymorphism in pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> (L.) Merr.) assessed by AFLP markers

Pineapple (Ananas comosus (L.) Merr.) cultivars, often derived from somatic mutations, are propagated vegetatively. It has been suggested by isozyme data that there is little genetic variation among Smooth Cayenne cultivars. A thorough investigation of the genetic variation within the cultivated speciesAnanas comosus, particularly among commercial cultivars, will provide critical information needed for crop improvement and cultivar protection. One-hundred and forty-eight accessions ofA. comosus and 14 accessions of related species were evaluated with AFLP markers. The average genetic similarity ofA. comosus was 0.735 ranging from 0.549 to 0.972, suggesting a high degree of genetic variation within this species. With AFLP markers, discrete DNA fingerprints were detected for each commercial cultivar, breeding line, and intra-specific hybrid. Self-incompatibility, high levels of somatic mutation, and intraspecific hybridization may account for this high degree of variation. However, major cultivar groups of pineapple, such as Cayenne, Spanish, and Queen, could not be distinctively separated. These cultivar groups are based on morphological similarity, and the similar appearance can be caused by a few mutations that occurred on different genetic background. Our results suggest that there is abundant genetic variation within existing pineapple germplasm for selection, and discrete DNA fingerprinting patterns for commercial cultivars can be detected for cultivar protection. The genetic diversity and relationships of fourAnanas species are also discussed.     

Genetic diversity of the D-genome in <Emphasis Type="Italic">T. aestivum</Emphasis> and <Emphasis Type="Italic">Aegilops</Emphasis> species using SSR markers

Simple sequence repeats (SSRs), highly dispersed nucleotide sequences in genomes, were used for germplasm analysis and estimation of the genetic relationship of the D-genome among 52 accessions of T. aestivum (AABBDD), Ae. tauschii (D^tD^t), Ae. cylindrica (CCD^cD^c) and Ae. crassa (MMD^cr1D^cr1), collected from 13 different sites in Iran. A set of 21 microsatellite primers, from various locations on the seven D-genome chromosomes, revealed a high level of polymorphism. A total of 273 alleles were detected across all four species and the number of alleles per each microsatellite marker varied from 3 to 27. The highest genetic diversity occurred in Ae. tauschii followed by Ae. crassa, and the genetic distance was the smallest between Ae. tauschii and Ae. cylindrica. Data obtained in this study supports the view that genetic variability in the D-genome of hexaploid wheat is less than in Ae. tauschii. The highest number of unique alleles was observed within Ae. crassa accessions, indicating this species as a great potential source of novel genes for bread wheat improvement. Knowledge of genetic diversity in Aegilops species provides different levels of information which is important in the management of germplasm resources.     

Sources of Resistance to Stem Rust (<Emphasis Type="Italic">Puccinia graminis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis>) in Ethiopian Tetraploid Wheat Accessions

Seedlings of 41 emmer (Triticum dicoccon Schrank) and 56 durum (T. durum Desf.) wheat accessions were evaluated for their response to stem rust (Puccinia graminis f. sp. tritici) infection under greenhouse condition at Kulumsa Agricultural Research Center, Ethiopia. The objectives were to identify tetraploid wheat accessions that could serve as sources of resistance to stem rust, and postulate the stem rust (Sr) resistance genes through multipatotype testing. The test included screening of accessions for stem rust resistance and multipatotype testing. To ensure vigorous screening, a mixture of six isolates (Si-1a, Am-2, Ku-3, Dz-4a, Ro-4 and Na-22) that were collected from severely infected emmer, durum, and bread wheat (Triticum aestivum L.) varieties of major wheat growing areas of Ethiopia was used as inocula. Out of the tested accessions, 18 emmer and 6 durum accessions exhibited low infection types (0–2) response and hence selected as a source of resistance to stem rust infection. Multipatotype testing was done to postulate Sr genes in the selected accessions. In the test, 10 different stem rust races (A2, A9, A11, A14, A16, A17, B3, B7, B15, and B21), 33 stem rust differential lines, and a universal susceptible check variety, Morocco were used, The high (3–4) and low infection type reaction patterns of the tested accessions and differential lines were used to postulate the genes that exhibit gene-for-gene relationship. The presence of Sr 7b, 8b, 9a, 9b, 10, 14, 24, 27, 28, 29, 30, 31, 32 and Tt-3+10 genes were postulated in 16 selected emmer and 5 durum wheat accessions. Efforts to transfer these valuable Sr genes from cultivated tetraploid wheats could be rewarding to get stem rust resistant varieties and boost wheat production.     

Taxonomic status and geographic provenance of germplasm accessions in the Solanum nigrum L. complex: AFLP data

Amplified Fragment Length Polymorphisms (AFLPs) were employed within the taxonomically difficult Solanum nigrum L. complex in order to characterize the genetic diversity present in a collection of the Gatersleben Genebank, to classify taxonomically unknown material, and to correlate the clustering of the examined accessions with their geographic origin. The results from AFLP analyses using two primer combinations on 44 entries from five species led to the detection of four major clusters, simultaneously uncovering significant differences in the levels of genetic diversity within or between species. S. americanumexhibited the highest infraspecific variation despite close geographic origins, simultaneously being placed in a clearly separated cluster in comparison to the other examined species of the complex. In addition, these other species showed even less interspecific variation than was found at the infraspecific level in S. americanum. In terms of taxonomy, the application of AFLPs helped in the classification of 13 black nightshade accessions formerly only listed as Solanum sp. This also was confirmed by morphological determination. Furthermore, one accession formerly classified as S. physalifolium var. nitidibaccatum i) clustered with AFLPs and ii) was identified morphologically as S. villosum. Contradictory classifications remain for two further entries from the same species, found within the S. nigrum cluster after AFLP analyses, while belonging to S. physalifolium var. nitidibaccatum according to herbarium specimen. Finally, as indicated by the information on provenance in geographically separated subclusters in S. americanum and partially in S. villosum, clues on the currently unknown origin of accessions from the genebank seem feasible by AFLP data.     

Processes affecting genetic structure and conservation: a case study of wild and cultivated <Emphasis Type="Italic">Brassica rapa</Emphasis>

When planning optimal conservation strategies for wild and cultivated types of a plant species, a number of influencing biological and environmental factors should be considered from the outset. In the present study Brassica rapa was used to illustrate this: to develop Scandinavian conservation strategies for wild and cultivated B. rapa, DNA-marker analysis was performed on 15 cultivated and 17 wild accessions of B. rapa plus 8 accessions of the cross compatible B. napus. The B. rapa cultivars were bred in Sweden and Finland in 1944–1997 and the wild B. rapa material was collected from Denmark, Sweden and United Kingdom. The B. napus accessions were bred within the last 20 years in the Scandinavian countries. Results were based on scoring of 131 polymorphic ISSR markers in the total plant material. A Bayesian Markov chain Monte Carlo (MCMC) approach implemented in NewHybrids demonstrated a clear distinction of B. rapa and B. napus individuals except for three individuals that seemed to be backcrosses. The backcrossed hybrids descended from two Swedish populations, one wild and one escaped. The overall pattern of genetic variation and structure in B. rapa showed that cultivated and wild B. rapa accessions formed two almost separated clusters. Geographical origin and breeding history of cultivars were reflected in these genetic relationships. In addition, wild populations from Denmark and Sweden seemed to be closely related, except for a Swedish population, which seemingly was an escaped cultivar. The study point to that many processes, e.g. spontaneous introgression, naturalisation, breeding and agricultural practise affected the genetic structure of wild and cultivated B. rapa populations.     

Perennial kales: collection rationalization and genetic relatedness to other Brassica oleracea crop types

Perennial kale is a rare leafy vegetable and forage crop that is mainly vegetatively propagated and therefore expensive to conserve ex situ. A genebank collection of 47 perennial kales and 34 reference samples from the main Brassica oleracea crop types were characterized with seven microsatellite markers in order to verify potential redundancies and to obtain more insight in the position of perennial kales within B. oleracea. Based on the obtained results and on data from previous studies, the collection was reduced with 49% to 24 perennial kales. Considering this level of reduction, it was estimated that the investments made for the final verification by microsatellite analysis are returned after only 4-year time. A principal coordinate plot clearly separated the perennial kales from the other crop types of B. oleracea, except in one case. This deviating accession of vegetatively preserved perennial kale clustered closely together with the single seed-preserved accession of perennial kale included in the study. These two accessions occupied an intermediate position between the group of vegetatively propagated perennial kales and the group of seed-propagated Brassica accessions, suggesting a hybridization background with another B. oleracea crop type. The microsatellite study demonstrated a close genetic relationship among the investigated perennial kales and their unique position within B. oleracea.     

Powdery mildew resistance in Aegilops tauschii coss. and synthetic hexaploid wheats

Summary A collection of 400 Ae. tauschii (syn. Ae. squarrosa) Coss. accessions were screened for powdery mildew resistance based on the response patterns of 13 wheat cultivars/lines possessing major resistance genes to nine differential mildew isolates. 106 accessions showed complete resistance to all isolates, and 174 accessions revealed isolate-specific resistance, among which were 40 accessions exhibiting an identical response pattern as wheat cultivar Ulka/^*8Cc which is known to possess resistance gene Pm2. Expression of both complete and isolate-specific resistance from Ae. tauschii was observed in some synthetic hexaploid wheats derived from four mildew susceptible T. durum Desf. parents, each crossed with five to 38 resistant diploid Ae. tauschii accessions. Synthetic amphiploids involving different combinations of T. durum and Ae. tauschii generally showed a decrease in resistance compared with that expressed by the Ae. tauschii parental lines.     

Evaluating landraces of bread wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. for tolerance to aluminium under low pH conditions

Bread wheat (Triticum aestivum L.) landraces held within ex situ collections offer a valuable and largely unexplored genetic resource for wheat improvement programs. To maximise full utilisation of such collections the evaluation of landrace accessions for traits of interest is required. In this study, 250 accessions from 21 countries were screened sequentially for tolerance to aluminium (Al) using haematoxylin staining of root tips and by root regrowth measurement. The staining test indicated tolerance in 35 accessions, with an intermediate response to Al exhibited in a further 21 accessions. Of the 35 accessions classified as tolerant, 33 also exhibited increased root length following exposure to Al. The tolerant genotypes originated from Bulgaria, Croatia, India, Italy, Nepal, Spain, Tunisia, and Turkey. AFLP analysis of the 35 tolerant accessions indicated that these represent diverse genetic backgrounds. These accessions form a valuable set of germplasm for the study of Al tolerance and may be of benefit to breeding programs for expanding the diversity of the gene pool from which tolerant cultivars are developed.