A genetic linkage map for tef [Eragrostis tef (Zucc.) Trotter]

Tef [Eragrostis tef (Zucc.) Trotter] is the major cereal crop in Ethiopia. Tef is an allotetraploid with a base chromosome number of 10 (2n = 4x = 40) and a genome size of 730 Mbp. Ninety-four F₉ recombinant inbred lines (RIL) derived from the interspecific cross, Eragrostis tef cv. Kaye Murri × Eragrostis pilosa (accession 30-5), were mapped using restriction fragment length polymorphisms (RFLP), simple sequence repeats derived from expressed sequence tags (EST–SSR), single nucleotide polymorphism/insertion and deletion (SNP/INDEL), intron fragment length polymorphism (IFLP) and inter-simple sequence repeat amplification (ISSR). A total of 156 loci from 121 markers was grouped into 21 linkage groups at LOD 4, and the map covered 2,081.5 cM with a mean density of 12.3 cM per locus. Three putative homoeologous groups were identified based on multi-locus markers. Sixteen percent of the loci deviated from normal segregation with a predominance of E. tef alleles, and a majority of the distorted loci were clustered on three linkage groups. This map will be useful for further genetic studies in tef including mapping of loci controlling quantitative traits (QTL), and comparative analysis with other cereal crops.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Expressed sequence tag analysis in tef (Eragrostis tef (Zucc) Trotter).

Tef (Eragrostis tef (Zucc.) Trotter) is the most important cereal crop in Ethiopia; however, there is very little DNA sequence information available for this species. Expressed sequence tags (ESTs) were generated from 4 cDNA libraries: seedling leaf, seedling root, and inflorescence of E. tef and seedling leaf of Eragrostis pilosa, a wild relative of E. tef. Clustering of 3603 sequences produced 530 clusters and 1890 singletons, resulting in 2420 tef unigenes. Approximately 3/4 of tef unigenes matched protein or nucleotide sequences in public databases. Annotation of unigenes associated 68% of the putative tef genes with gene ontology categories. Identification of the translated unigenes for conserved protein domains revealed 389 protein family domains (Pfam), the most frequent of which was protein kinase. A total of 170 ESTs containing simple sequence repeats (EST-SSRs) were identified and 80 EST-SSR markers were developed. In addition, 19 single-nucleotide polymorphism (SNP) and (or) insertion-deletion (indel) and 34 intron fragment length polymorphism (IFLP) markers were developed. The EST database and molecular markers generated in this study will be valuable resources for further tef genetic research.     

Multivariate analysis of diversity of tef (Eragrostis tef (Zucc.) Trotter) germplasm from western and southern Ethiopia

Sixty tef germplasm populations consisting of 3,000 panicle-derived lines from six western and southern regions of Ethiopia were evaluated for 17 pheno-morphic and agronomic traits on pellic Vertisols at Debre Zeit Agricultural Research Center during the 1999 main season. The objectives were to study the extent and pattern of variation of the germplasm with respect to regions and altitude zones, to classify the populations into relatively homogenous groups and to identify the major traits contributing to the overall diversity of the populations. At 75 % similarity level, the 60 populations aggregated into nine complexes of two to 10, with 12 populations remaining un-grouped. Five principal components (PC) extracted about 81 % of the gross variance among the populations. About 40 % of the variance accounted for by the first PC alone resulted largely from variations in diameters of the two basal culm internodes, grain yield and number of spikelets/panicle, shoot phytomass and grain yield/plant, and number of culm internodes. The entire regional as well as the clinal (altitude zone) variation was explained by five and two PCs, respectively. The discriminant analyses depicted about 77 % correct grouping of the 47 populations into nine clusters and about 62 % and 68 % correct origin-based classification of the germplasm in terms of altitude zones and regions, respectively. In general, the study demonstrated the existence of regional and clinal (altitude zone) variation patterns in the tef germplasm populations. The broad trait variation in the germplasm implies ample opportunities for genetic improvement of tef through selection and hybridization.     

Flow cytometric analysis of nuclear genome of the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter]

Flow cytometric analysis of nuclear DNA content was performed by using nuclei isolated from young leaf tissue of tef (Eragrostis tef). The method was very useful for rapid screening of ploidy levels in cultivars and lines of tef representing the phenotypic variability of this species in Ethiopia. The results of the analysis showed that all cultivars were tetraploid. Flow cytometry was also used to determine nuclear DNA content in absolute units (genome size) in four tef cultivars. Nuclei isolated from tomato (Lycopersicon esculentum, 2C=1.96 pg) were used as an internal reference standard. The 2C DNA content of individual tef cultivars ranged from 1.48 to 1.52 pg (1C genome size: 714 Mbp-733 Mbp), the differences among them being statistically nonsignificant. The fact that the nuclear genome of tef is only about 50% larger than that of rice should make it amenable for analysis and mapping at the molecular level.     

A genetic linkage map of tef [Eragrostis tef (Zucc.) Trotter] based on amplified fragment length polymorphism

A genetic linkage map of tef was constructed with amplified fragment length polymorphism (AFLP) markers using F₅ recombinant inbred lines (RILs) derived by single seed descent from the intraspecific cross of ’Kaye Murri’×’Fesho’. A total of 192 EcoRI/MseI primer combinations were screened for parental polymorphism. Around three polymorphic fragments per primer combination were detected, indicating a low polymorphism level in tef. Fifty primer combinations were selected to assay the mapping population, and 226 loci segregated among 85 F₅ RILs. Most AFLP loci behaved as dominant markers (presence or absence of a band), but about 15% of the loci were codominant. Significant deviations from the expected Mendelian segregation ratio were observed for 26 loci. The genetic linkage map comprised 211 markers assembled into 25 linkage groups and covered 2,149 cM of genome. AFLP is an efficient marker system for mapping plant species with low polymorphism such as tef. This is the first genetic linkage map constructed for tef. It will facilitate the mapping of genes controlling agronomically important traits and cultivar improvement in tef. Received: 27 April 1998 / Accepted: 4 January 1999     

RFLP linkage map of the Ethiopian cereal tef [Eragrostis tef (Zucc) Trotter]

Tef [Eragrostis tef (Zucc)Trotter] is one of the most important cereal crops in Ethiopia. It is an allotetraploid species with a genome size of 720 Mbp. In this paper we report results of genetic linkage-map construction for E. tef using tef and heterologous cDNA probes for the first time. One hundred and sixteen F₈ recombinant inbred lines (RILs) from the cross E. tef cv Kaye Murri×Eragrostis pilosa (accession30–5) were used for mapping. Parental lines were digested with nine restriction enzymes and screened using 159 tef cDNA and 162 heterologous probes including the grass genome anchor probes. The polymorphism level between parental lines was 66.9%. One hundred and thirty nine polymorphic probes were hybridized against 116 RILs. Both the tef and the heterologous probes hybridized well against tef genomic DNA. The linkage map defined 1,489 cM of the tef genome comprising 149 marker loci distributed among 20 linkage groups. The average interval between markers was 9.99 cM. A fraction (14.8%) of the markers deviated significantly from the expected segregation. Such a genetic linkage map is useful for tagging economically useful genes in tef because a wide range of agronomically important traits is segregating within this population. This would enable the use of a marker assisted breeding strategy which, in turn, will enhance breeding efficiency. Alignment of the tef RFLP map with the rice RFLP map indicates that a number of syntenic chromosomal fragments exist between tef and rice in which the gene order was for the most part collinear. The comparative mapping information should enable tef scientists to take advantage of whatever genetic progress is made on the cereal model species rice. Received: 9 June 2000 / Accepted: 31 August 2000     

Screening of ethyl methane sulphonate mutagenized tef [Eragrostis tef (Zucc.) Trotter] population identifies Al-tolerant lines

About 15,000 M₂ seeds of ethyl-methane-sulphonate (EMS)-mutagenized population were screened along with Al-tolerant and sensitive checks and the M₀ variety. Strongly acidic soil with an external application of a toxic Al-solution and exposure to moisture stress was used to maximize selection pressure. Twenty-one M₂ plants with root lengths of greater than the mean of the tolerant check were selected and planted for seed production. Candidate M₃ plants were investigated for Al-tolerance and for morpho-agronomic traits under greenhouse and field conditions, respectively. Highly significant differences were observed for Al-tolerance between the candidate mutant lines and the M₀ (P?<?.001), and between mutant lines and the sensitive check (P?<?.001). Similarly, significant differences were observed between the mutant lines for 16 of the 20 quantitative traits measured. This study is the first to report successful induction of enhanced Al-tolerance in tef by using EMS mutagenized population.     

Variation of nuclear ribosomal RNA genes in Eragrostis tef (Zucc.) Trotter.

Variation in the ribosomal RNA genes (rDNA) was examined to assess the genetic variability among 314 plants representing 28 accessions of Eragrostis tef, an important food crop. A restriction site map was constructed for the species by localization of the BamHI, BglII, DraI, EcoRI, EcoRV, NdeI, SacI, SpeI, XbaI, and XhoI sites. A comparison of this map with those of other grasses showed conservation of sites, especially in the coding region. However, a unique EcoRI site combined with a BamHI site in the 18S region may be of diagnostic value for the species. A BamHI fragment that spans the intergenic spacer was used as an indicator of length variation of rDNA repeat units. rDNA repeat units in E. tef ranged in size from 8.4 to 11.07 kbp. Considerable size variation of rDNA repeats was present among accessions, between individual plants within some accessions, and within single plants. A total of 19 spacer length (sl) phenotypes was observed in 16 accessions in which 11-42 plants were analyzed. A single restriction site polymorphism was detected in PI442115 that was also distinguished by having a single sl variant. Variation in the rRNA genes is a useful indicator of genetic diversity in E. tef germplasm.     

Somatic embryogenesis and plant regeneration in callus culture of tef, Eragrostis tef (Zucc.) Trotter

The study was carried out to establish in vitro culture conditions for plant regeneration of tef, Eragrostis tef (Zucc.) Trotter. Mature seeds of two Ethiopian varieties, DZ-01-354 and DZ-01-196, were used to initiate callus cultures on Murashige and Skoog (MS) medium with different auxins. Four- and 8-week-old calli induced on a medium with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were subcultured onto various media to induce somatic embryogenesis. Compact, nodulated, embryogenic callus was observed after transfer onto MS-callus proliferating (CP) medium. Embryogenic tissue appeared on soft and amorphous callus and developed into somatic embryos during a subsequent subculture to MS embryo-promoting (EP) media. Various growth regulator combinations were tested in CP and EP media to obtain a high efficiency of somatic embryo formation. The highest frequency of calli forming somatic embryos (56.1–68.3%) was observed when CP media with 2.0 or 4.0 mg/l 2,3,5-triiodobenzoic acid were employed and then cultures were transferred to EP media with 0.5 mg/l 2,4-D and 0.5 mg/l kinetin followed by 0.5 mg/l indole-3-acetic acid and 0.5 mg/l N⁶-benzyladenine. Plant development from somatic embryos was obtained on MS medium supplemented with 1.0 mg/l gibberellic acid. On average, 71.2% of calli displaying somatic embryos converted into plants. Regenerated plants were successfully transferred to soil. Neither chlorophyll-deficient plants nor morphological variants were found among regenerants. All regenerated plants were fertile. Received: 9 May 1997 / Revision received: 25 September 1997 / Accepted: 3 January 1998     

Variation and inter-relationships of quantitative traits in tef (Eragrostis tef (Zucc.) Trotter) germplasm from western and southern Ethiopia

Three thousand tef (Eragrostis tef (Zucc.) Trotter) lines representing 60 germplasm populations from western and southern Ethiopia were sown on pellic Vertisols at Debre Zeit Agricultural Research Center during the 1999/2000 main season. The objectives were to assess the variation with respect to regions and altitude zones of origin and to study the inter-relationships of 17 pheno-morphic and agronomic traits. The populations showed significant (p < or = 0.05) regional variation in 10 (59%) of the quantitative traits, but clinal variation among altitude zones was significant (p < or = 0.05) only for six (35%) of the traits. On the other hand, the populations revealed consistent variation (p < or = 0.05) within both regions and altitude zones in all the traits evaluated. Likewise, the variation among lines within populations of both regions and altitude zones was significant (p < or = 0.05) in most of the traits. The number of characters showing substantial correlation depicted regional and clinal variation mainly depending on the number of populations. Based on the mean of the populations, grain yield panicle and shoot phytomass plant showed negative correlation with harvest index, and positive correlation with most of the remaining traits. Individual plant grain yield was positively correlated with all the other traits except harvest index, days to maturity, grain filling period and number of primary panicle branches. Overall, the tef germplasm populations showed substantial phenotypic variation which can be utilized in the genetic improvement of the crop.     

Relationships of the Ethiopian Cereal T'ef (Eragrostis tef (Zucc.) Trotter): Evidence from Morphology and Chromosome Number

The relationships of the cereal Eragrostis tef (Gramineae: Eragrostoideae)have been investigated. Nine species are closely related toT'ef on morphological grounds. Their chromosome numbers arereported. E. pilosa and some of the E. cilianensis complex areclosest to E. tef. These taxa may be useful sources of genesfor T'ef improvement. Ethiopian cereal, T'ef, Eragrostis, Gramineae: Eragrostoideae, chromosome number, poly-ploidy, improvement     

Assessment of genetic diversity in Salvadora persica L. based on inter simple sequence repeat (ISSR) genetic marker

Studies on the genetic variation in marginal populations and differentiation between them are essential for assessment of best gene conservation strategies and sampling schemes. In this study, ISSR markers were used to establish the level of genetic relationships and polymorphism 50 genotypes of Salvadora persica collected from 6 different regions of Hormozgan province. The ISSR analysis with 9 anchored primers also generated 105 scorable loci, of which 85 were polymorphic (80.95%). Parameters of genetic diversity and its partitioning were calculated. The genetic analysis demonstrated that S. persica maintain relatively high genetic diversity (PIC was 0.63, Na was 1.27 and Ho and He were 0.15 and 0.17 respectively). The coefficient of genetic differentiation among populations based on FST equaled 0.20. Genetic identities between population's pairs were high (mean I?=?0.88). These values are high as compared with other widespread congener species. Cluster analysis based on the Unweighted Pair Group Method with Arithmetic Averages (UPGMA) revealed 3 main clusters for the ISSR data. The levels of genetic diversity maintained within populations of S. persica indicate that an appropriate sampling design for ex situ safeguarding should capture the majority of genetic diversity found within these taxa to help ensure the long term viability of this species. Furthermore, it could be inferred that ISSR markers are suitable tools for the evaluation of genetic diversity and relationships within the Salvadora persica.     

Inter simple sequence repeat analysis of genetic diversity and relationships in cultivated barley of Nordic and Baltic origin

This study evaluates putative changes of genetic diversity and relationships of barley in the Nordic and Baltic countries that might have taken place during the last century as a result of commercial breeding. Four ISSR primers were used to analyse 227 accessions, yielding a total of 47 polymorphic loci. Shannon-Weaver diversity values for each locus ranged from 0.012 to 0.693. Overall, there were no significant changes of genetic diversity observed over time. A significant decrease of diversity was, however, observed in material from the southern parts of the Nordic and Baltic countries. In material from the northern parts no decrease of diversity was observed. The genetic diversity of six-rowed barley bred in the middle of the 20th century was low, but there was no significant difference between modern accessions and landraces or old cultivars. The magnitude in changes of genetic diversity differed also in material from different countries of origin. A cluster analysis clearly separated the material into two groups. The first cluster included 86.5% of all six-rowed accessions, whereas the second cluster contained 97.4% of all two-rowed accessions.     

Gynogenic plant regeneration from unpollinated flower explants of Eragrostis tef (Zuccagni) Trotter

Tef [Eragrostis tef (Zucc.) Trotter] is the most important cereal in Ethiopia. In its wild relative E. mexicana, regeneration of six green plants resulted from culture of 121 non-pollinated immature pistils. In the allotetraploid crop species tef, however, only callus and root formation was obtained by this method. By contrast, immature spikelets and panicle segments of E. tef proved amenable to gynogenic plant regeneration. Upon step-wise optimization of the protocol, efficient plant formation was achieved in all three cultivars tested. In cv. DZ-01-196, culture of 1305 immature spikelets resulted in formation of 159 green plants. Flow cytometric analysis revealed (di)haploid, triploid, tetraploid and octoploid regenerants, from which the vast majority was tetraploid. Tef-breeding programs will likely benefit substantially from efficient generation of true-breeding plants.     

白黄侧耳Pleurotus cornucopiae微卫星间区(ISSR)分析

本试验对我国1982年至2004年22年间栽培的白黄侧耳Pleurotus cornucopiae 30个菌株进行了锚定ISSR分析,试验表明,引物P4和P5都能对白黄侧耳P.cornucopiae进行多态性扩增,P4将供试菌株扩增出45个条带,大小在200～20000bp,P5将供试菌株扩增出39个条带,大小在500～15000bp,扩增出的条带100%具多态性。聚类分析在遗传相似性61%的水平下将30个供试菌株划分为15个类群,即15个具一定遗传差异的菌株;具有相同ISSR图谱、遗传相似性程度100%的可能为同一菌株,属于同物异名。试验表明我国的食用蕈菌野生环境面临人工栽培种质的污染,采自河北、山东、云南自然环境下的白黄侧耳P.cornucopiae与此前大量栽培的一些商业品种具完全相同的ISSR指纹图谱,聚类分析相似性系数100%。     

Analysis of the genetic diversity of garlic (Allium sativum L.) by simple sequence repeat and inter simple sequence repeat analysis and agro-morphological traits

The genetic diversity of 39 garlic accessions was investigated using eight simple sequence repeat (SSR) primer combinations and 17 inter-simple sequence repeat (ISSR) primer combinations. A total of 109 polymorphic loci were detected among these accessions, with an average of 4.63 polymorphic loci per SSR primer combination and 4.29 polymorphic loci per ISSR primer combination. The mean effective number of alleles, the mean Nei's genetic diversity, and the mean Shannon's information index for SSR were 1.4799, 0.2870, and 0.4378, respectively; and those for ISSR were 1.4847, 0.2898 and 0.4415, respectively. Cluster analysis, using the unweighted pair-group method with arithmetic averages (UPGMA) based on the allele frequency data, classified the accessions into three groups. The results of principal component analysis (PCA) were consistent with those of the cluster analysis. PCA showed that each of these three groups exhibited significant variation in agro-morphological traits. These findings suggest that the eight SSR and 17 ISSR primers identified could define valuable markers for genetic diversity for use by plant breeders.     

QTL mapping of agronomic traits in tef [<Emphasis Type="Italic">Eragrostis tef</Emphasis>(Zucc) Trotter]

Background  

Tef [Eragrostis tef (Zucc.) Trotter] is the major cereal crop in Ethiopia. Tef is an allotetraploid with a base chromosome number of 10 (2n = 4× = 40) and a genome size of 730 Mbp. The goal of this study was to identify agronomically important quantitative trait loci (QTL) using recombinant inbred lines (RIL) derived from an inter-specific cross between E. tef and E. pilosa (30-5).     

Variation in response of accessions of minor millets,Pennisetum americanum (L.) Leeke (pearl millet) and Eleusine coracana (L.) Gaertn (finger millet), and Eragrostis tef (Zucc.) Trotter (tef) to salinity in early seedling growth

The response to increasing NaCl concentration of seedlings of 25 accessions of Ethiopian land races of each of Pennisetum americanum (L.) Leeke (pearl millet) and Eleusine coracana (L.) Gaertn (finger millet), and 15 accessions of Eragrostis tef (Zucc.) Trotter (tef), was examined after two week's growth in NaCl solution culture. Although increasing NaCl concentration significantly reduced seedling root lengths, there was considerable variation within, and between accessions within each species.Analysis based upon a non-linear least square inversion method, using root length data, revealed significant differences in accessions of P. americanum and E. tef on the basis of the estimated salinity threshold, C _t , the NaCl concentrations at which root length begins to decrease. C _t did not differ significantly between E. coracana accessions. Estimates of C₅₀ and C₀, mininum concentrations causing a 50% decrease in root length, and zero root growth respectively, revealed differences between and within accessions for all three species. Overall, finger millet was more tolerant than tef, which was more tolerant than pearl millet. There is clear evidence that differences in tolerance are genetically based from broad sense heritability estimates.     

Genetic diversity and genetic relationships of Chukrasia spp. (Meliaceae) as revealed by inter simple sequence repeat (ISSR) markers

Key message

ISSR characterization of Chukrasia populations from the natural range revealed two distinct groups of populations consonant with morphological differentiation. Results suggest the current taxonomic classification of the genus should be reviewed.

Abstract

There are different views as to whether the genus Chukrasia (Meliaceae) consists of one species, C. tabularis, or two species C. tabularis and C. velutina. Despite a clear pattern of variation in many morphological characteristics such as leaves and bark, some authors regard the latter merely an ecotype of the former in seasonal forest. In the present study, we used ISSR markers to determine the genetic diversity and population structure among 23 Chukrasia subpopulations from across the natural range in Asia. Molecular analysis clearly differentiated two distinct groups of subpopulations, corresponding to the putative species, as well as well-defined subpopulations corresponding to geographic regions within the two groups. The molecular results are in concordance with morphological differentiation and corresponded to the two recognized taxa. The present study suggests that current taxonomic classification of the genus Chukrasia should be reviewed.