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 .     

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     

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.     

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     

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.     

A genetic map of an interspecific cross in Allium based on amplified fragment length polymorphism (AFLPTM) markers

Segregation of 692 polymorphic AFLP^TM (amplified fragment length polymorphism) fragments was determined in an F₂ of the interspecific cross A. roylei x A. cepa. Two different enzyme combinations were used, PstI/MseIand EcoRI/MseI; in the latter one extra selective nucleotide was added to the MseI primer. The map based on A. cepa markers consisted of eight linkage groups with 262 markers covering 694 cM of the expected 800 cM. The map based on A. roylei markers comprised 15 linkage groups with 243 markers and had a length of 626 cM. The two maps were not integrated, and 25% of the markers remained unlinked. One of the alliinase genes and a SCAR marker linked to the disease resistance gene to downy mildew are present on this map. Of the AFLP markers, 50—80% were polymorphic between A. cepa and A. roylei; the level of polymorphic markers between different A. cepa accessions was4-8%. Received: 28 August 1998 / Accepted: 31 March 1999     

A genetic map of melon (Cucumis melo L.) based on amplified fragment length polymorphism (AFLP) markers

 Genetic maps facilitate the study of genome structure and evolution, and the identification of monogenic traits or Mendelian components of quantitative traits. We evaluated 228 RAPD, microsatellite and AFLP markers for linkage analysis in melon (Cucumis melo L.) varieties MR-1 (resistant to Fusarium wilt, powdery and downy mildews) and Ananas Yokneum (AY; susceptible to these diseases) and constructed a detailed genetic map. The mapping population consisted of 66 backcross progenies derived from AY×(MR-1×AY). Despite a relatively low level of polymorphism in the species, AFLP markers were found to be more efficient in mapping the melon genome than RAPD or microsatellite markers. The map contains 197 AFLPs, six RAPDs and one microsatellite marker assigned to 14 major and six minor linkage groups, and covers 1942 cM with the average distance between adjacent markers of approximately 10 cM. The maximum distance allowed between markers is 27.5 cM. About 11% of the intervals (20 out of 173) are over 20 cM (but less than 27.5 cM). The map has immediate utility for identifying markers linked to disease resistance genes that are suitable for marker-assisted breeding. The use of microsatellite markers for integration with other maps is also discussed. Received: 12 March 1997 / Accepted: 20 May 1997     

A Genetic linkage map of Pinyon pine (Pinus edulis) based on amplified fragment length polymorphisms

 Amplified fragment length polymorphisms (AFLP) were used to rapidly generate a dense linkage map for pinyon pine (Pinus edulis). The map population consisted of 40 megagametophytes derived from one tree at Sunset Crater, Arizona. A total of 78 primer combinations, each with three to five selective nucleotides, amplified 542 polymorphic markers. Of these, 33 markers showed significant deviation from the expected Mendelian genotypic segregation ratio of 1 : 1, and 164 showed complete linkage with another marker. This resulted in 338 unique markers mapping to 25 linkage groups, each of which ranged from 2 to 22 markers, averaging 80 centiMorgans (cM) in size and covering 2,012 cM (2,200 cM with the inclusion of 25 cM for each of 7 unlinked markers). Pairwise linkage values gave a genome size estimate of 2,390 cM, suggesting comprehensive coverage of the genome. A search for subsets of primer combinations giving the best map coverage found 10 primer combinations which together marked 72% of the linkage map to within 10 cM; an additional 10 primer combinations increased this percentage to 85%. Our map represents an initial step towards the identification of quantitative trait loci associated with pest resistance and water stress in pinyons and will further allow us to examine introgression rates between P. edulis and P. californiarum. Received: 14 October 1997 / Accepted: 29 April 1998     

Inter simple sequence repeat (ISSR) analysis of genetic diversity in tef [Eragrostis tef (Zucc.) Trotter

The DNA polymorphism among 92 selected tef genotypes belonging to eight origin groups was assessed using eight inter simple sequence repeat (ISSR) primers. The objectives were to examine the possibility of using ISSR markers for unravelling genetic diversity in tef, and to assess the extent and pattern of genetic diversity in the test germplasm with respect to origin groups. The eight primers were able to separate or distinguish all of the 92 tef genotypes based on a total of 110 polymorphic bands among the test lines. The Jaccard similarity coefficient among the test genotypes ranged from 0.26 to 0.86, and at about 60 % similarity level the clustering of this matrix using the unweighted pair-group method based on arithmetic average (UPGMA) resulted in the formation of six major clusters of 2 to 37 lines with further eight lines remaining ungrouped. The standardized Nei genetic distance among the eight groups of origin ranged between 0.03 and 0.32. The UPGMA clustering using the standardized genetic distance matrix resulted in the identification of three clusters of the eight groups of origin with bootstrap values ranging from 56 to 97. The overall mean Shannon Weaver diversity index of the test lines was 0.73, indicating better resolution of genetic diversity in tef with ISSR markers than with phenotypic (morphological) traits used in previous studies. This can be attributed mainly to the larger number of loci generated for evaluation with ISSR analysis as compared to the few number of phenotypic traits amenable for assessment and which are further greatly affected by environment and genotype x environment interaction. Analysis of variance of mean Shannon Weaver diversity indices revealed substantial (P < or = 0.05) variation in the level of diversity among the eight groups of origin. In conclusion, our results indicate that ISSR can be useful as DNA-based molecular markers for studying genetic diversity and phylogenetic relationships, DNA fingerprinting for the identification of varieties or cultivars, and also for genome mapping in tef.     

大粒裸燕麦(Avena nuda L.)遗传连锁图谱的构建

以“元莜麦”和“555”杂交得到的281个F2单株为作图群体,利用20对AFLP引物、3对SSR引物和1个穗型性状构建了一张大粒裸燕麦遗传连锁图。该图谱全长1544.8cM,包含19个连锁群,其上分布有92个AFLP标记、3个SSR标记和1个穗型形态标记,不同连锁群标记数为2-14个,长度在23.7-276.3cM之间,平均长度为81.3cM,标记间平均距离为20.1cM。穗型标记分离比符合3：1,11个AFLP标记表现为偏分离,偏分离比为11.5%。该图谱符合遗传连锁框架图的要求,为今后大粒裸燕麦的QTL定位、分子标记辅助育种和比较基因组学等研究奠定基础。     

An integrated genetic map of Populus deltoides based on amplified fragment length polymorphisms

Amplified fragment length polymorphism (AFLP) is an efficient molecular technique for generating a large number of DNA-based genetic markers in Populus. We have constructed an integrated genetic map for a Populus backcross population derived from two selected P. deltoides clones using AFLP markers. A traditional strategy for genetic mapping in outcrossing species, such as forest trees, is based on two-way pseudo-testcross configurations of the markers (testcross markers) heterozygous in one parent and null in the other. By using the markers segregating in both parents (intercross markers) as bridges, the two parent-specific genetic maps can be aligned. In this study, we detected a number of non-parental heteroduplex markers resulting from the PCR amplification of two DNA segments that have a high degree of homology to one another but differ in their nucleotide sequences. These heteroduplex markers detected have served as bridges to generate an integrated map which includes 19 major linkage groups equal to the Populus haploid chromosome number and 24 minor groups. The 19 major linkage groups cover a total of 2,927 cM, with an average spacing between two markers of 23. 3 cM. The map developed in this study provides a first step in producing a highly saturated linkage map of the Populus deltoides genome. Received: 10 September 1999 / Accepted: 3 November 1999     

家蚕高密度AFLP连锁图谱的构建

为了进行家蚕Bombyx mori数量性状的QTL定位研究,以白色茧系品种C₁₀₀ (♀)和近交系大造(P50)(♂)杂交得到F₁,用F₁(♂)与双隐性标记的C₁₀₀ (♀)回交,得到回交一代(BC₁),用改进的AFLP分子标记方法,经96组选择性扩增引物扩增,获得分离比为1∶1(P≤0.05)的1 744个AFLP位点。用Map Manager QTXb19（Version 0.29）连锁图谱构建软件,构建了具有814个标记,36个连锁群的家蚕高密度AFLP分子标记连锁图谱。该连锁图谱覆盖的家蚕基因组长度为13 005 cM,连锁群长度变化范围为109.0～1 573.7 cM,连锁群的平均长度为361.25 cM,其标记间平均图距15.98 cM,最小图距2.3 cM,最大图距47.7 cM,标记间大于30 cM的gap共有39个。该连锁图平均每个连锁群23个标记,最多一个连锁群有92个标记,最少8个标记。该连锁图谱确定了与经典实验遗传图谱第15连锁群和W染色体连锁群相对应的两个连锁群。     

Effect of moxidectin selection on the genetic variation within Cylicocyclus nassatus based on amplified fragment length polymorphism (AFLP)

Cyathostomins are among the most important intestinal nematodes of horses, yet, the literature on the molecular genetics of these worms is scarce. In this study, the technique of amplified fragment length polymorphism (AFLP) was applied to study the genetic diversity as well as to determine the effect of moxidectin selection on the population genetic diversity for Cylicocyclus nassatus, one of the most common cyathostomin species. Genomic DNAs from 30 individual male worms were used from each of two populations: an avermectin-milbemycin (AM)-naive population (Population-S) and a population derived from Population-S following 21 treatments with moxidectin (Population-Mox). Three selective primer pairs were used for each worm, yielding a total of 229 AFLP markers. Calculation of average pair wise Jaccard indices revealed a high degree of genetic variation within both populations using all three primer combinations. In addition, selection by moxidectin during a 3-year period caused a significant decrease in the level of genetic diversity as evidenced by analysis of AFLP markers for two primer combinations but not for the third. A dendrogram of relationships among individuals based on AFLP markers did not show a clear classification of individuals in separate groups. It was concluded that a high degree of genetic intrapopulation variation exists in C. nassatus and that moxidectin selection has a significant effect on the genetic composition of C. nassatus.     

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.     

Population structure and genetic diversity of Huperzia serrata (Huperziaceae) based on amplified fragment length polymorphism (AFLP) markers

The levels and pattern of the genetic variation within and among natural populations of Huperzia serrata were investigated using amplified fragment length polymorphism markers. Seven primer combinations used in the study amplified 615 discernible bands with 532 (86.5%) being polymorphic, indicating a considerable high level of genetic diversity at the species level. AMOVA analysis revealed a low level of genetic differentiation among the ten populations. The UPGMA cluster of all samples showed that individuals from the same population occasionally failed to cluster in one distinct group. A Mantel test showed no significant correlation between genetic distance and geographical distance (r = 0.278, P = 0.891), suggesting that the gene flow was not restricted geographically. A number of factors that might affect the genetic profiles of H. serrata included clonal growth, selective effect of niche and outcrossing, as well as the effective wind-dispersal of spores.     

A genetic linkage map of lentil (Lens sp.) based on RAPD and AFLP markers using recombinant inbred lines

 A genetic linkage map of Lens sp. was constructed with 177 markers (89 RAPD, 79 AFLP, six RFLP and three morphological markers) using 86 recombinant inbred lines (F_6:8) obtained from a partially interspecific cross. The map covered 1073 cM of the lentil genome with an average distance of 6.0 cM between adjacent markers. Previously mapped RFLP markers were used as anchor probes. The morphological markers, pod indehiscence, seed-coat pattern and flower-color loci were mapped. Out of the total linked loci, 8.4% showed segregation distortion. More than one-fourth of the distorted loci were clustered in one linkage group. AFLP markers showed more segregation distortion than the RAPD markers. The AFLP and RAPD markers were intermingled and clustering of AFLPs was seldom observed. This is the most extensive genetic linkage map of lentil to-date. The marker density of this map could be used for the identification of markers linked to quantitative trait loci in this population. Received: 6 November 1997 / Accepted: 10 February 1998     

DNA fingerprinting of Leonurus cardiaca L. germplasm in Iran using amplified fragment length polymorphism and inter-retrotransposon amplified polymorphism

In the present study, the extent of inter and intra-population genetic variation was evaluated in Leonurus cardiaca accessions naturally growing in Iran by AFLP and IRAP markers. The fingerprints corresponding to AFLP and IRAP markers revealed high levels of heterozygosity, indicating that L. cardiaca is predominantly an out-crossing species. The average percentage polymorphism was detected as 58% and 90.8% on utilizing AFLP and IRAP data, respectively. Gene diversity values within populations varied 0.14 to 0.20 for AFLP and 0.12 to 0.21 for IRAP. The overall levels of genetic variation present in the L. cardiaca germplasm in Iran were finally determined by combining the AFLP and IRAP datasets to ensure wide genome coverage. The phenogram depicted that the accessions of Dargaz population were genetically distinct from other populations. Based on AFLP and IRAP analysis, it is concluded that L. cardiaca maintains high levels of genetic variation at inter and intra-population level.     

Genetic diversity and structure of natural Pinus tabulaeformis populations in North China using amplified fragment length polymorphism (AFLP)

Chinese pine (Pinus tabulaeformis carr.), endemic to China, is a conifer species with extensive and fragmented distribution in North China. In this study, the genetic diversity and structure of 20 natural populations of this species were investigated using amplified fragment length polymorphism (AFLP) markers. A total of 445 fragments were revealed with 8 pairs of primers, 379 (85.17%) of which were polymorphic. A moderate level of genetic diversity was detected at the species level (Shannon's information index I = 0.356, Nei's gene diversity H_E = 0.271) and at the population level (I = 0.219, H_E = 0.206). Most of genetic variation was within populations while a considerable level of genetic differentiation was detected (G_ST = 0.352, Ф_ST = 0.304). The high differentiation could be attributed to the complex and fragmented habitats, and a limited gene flow among populations (N_m = 0.572). The Mantel test indicated that there was significant correlation (r = 0.455, P < 0.001) between Nei's genetic distance and geographical distance among all the populations. The results suggested that proper countermeasures should be taken to prevent the habitat further deterioration and maintain the genetic diversity of this species.     

Assessment of genetic diversity among and within Achillea species using amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity of 57 Achillea accessions belonging to five species, A. millefolium, A. filipendulina, A. tenuifolia, A. santolina and A. biebersteinii. Nine AFLP primer combinations were used, which produced 301 polymorphic bands. In most species, a high level of genetic variation was detected among the genotypes. The Jaccard's similarity indices (J), based on AFLP profiles, were subjected to UPGMA cluster analysis. Application of Mantel's test for cophenetic correlation to the cluster analysis indicated the high fitness of the accessions to a group (r = 0.918). The dendrogram generated revealed five major groups corresponding to five species. The principle coordinate analysis (PCoA) data confirmed the results of the clustering. Among the species, A. teunifolia and A. santolina showed the greatest and the least genetic diversity, respectively. A. filipendulina accessions were acquired primarily from the same ecological regions of western Iran. Accessions belonging to A. biebersteinii originated from the Isfahan province and were separated from other species at the root of the dendrogram. The results of the clustering method, based on AFLP markers, corresponded closely with the geographical origins of the genotypes. The results of the present study could contribute to a better understanding and management of conservation and exploitation of the Achillea germplasm.