Comparative chloroplast genome analyses of cultivated and wild Capsicum species shed light on evolution and phylogeny.

The chloroplast (cp.) genome, also known as plastome, plays crucial roles in plant survival, adaptation, and evolution. The stable genetic structure of cp. genomes provides an ideal system for investigating species evolution. We sequenced three complete cp. genome sequences of Capsicum species and analyzed them using sequences of various Capsicum species retrieved from the NCBI database. The cp. genome of Capsicum species maintains a well-preserved quadripartite structure consisting of two inverted repeats (IRs) flanked by a large single copy (LSC) region and a small single copy (SSC) region. The sizes of cp. genome sequences ranged from 156,583 bp (C. lycianthoides) to 157,390 bp (C.pubescens). A total of 127-132 unique genes, including 83-87 protein-coding, 36-37 tRNA, and eight rRNA genes, were predicted. Comparison of cp. genomes of 10 Capsicum species revealed high sequence similarity in genome-wide organization and gene arrangements. Fragments of trnT-UGU/trnL-UAA, ccsA, ndhD, rps12, and ycf1 were identified as variable regions, and nucleotide variability of LSC and SSC was higher than that of IR. Phylogenetic speciation analysis showed that the major domesticated C. annuum species were the most extensively divergent species and closely related to C. tovarii and C. frutescens. Analysis of divergent times suggested that a substantial range of speciation events started occurring ~ 25.79 million years ago (Mya). Overall, comparative analysis of cp. genomes of Capsicum species not only offers new insights into their genetic variation and phylogenetic relationships, but also lays a foundation for evolutionary history, genetic diversity, conservation, and biological breeding of Capsicum species.

Comparative Analysis of Polyphenolic Compounds in Different Amaranthus Species: Influence of Genotypes and Harvesting Year.

Amaranth is a nutritionally valuable crop, as it contains phenolic acids and flavonoids, yielding diverse plant secondary metabolites (PSMs) like phytosterol, tocopherols, and carotenoids. This study explored the variations in the contents of seventeen polyphenolic compounds within the leaves of one hundred twenty Amaranthus accessions representing nine Amaranthus species. The investigation entailed the analysis of phenolic content across nine Amaranthus species, specifically A. hypochondriacus, A. cruentus, A. caudatus, A. tricolor, A. dubius, A. blitum, A. crispus, A. hybridus, and A. viridis, utilizing ultra performance liquid chromatography with photodiode array detection (UPLC-PDA). The results revealed significant differences in polyphenolic compounds among accessions in which rutin content was predominant in all Amaranthus species in both 2018 and 2019. Among the nine Amaranthus species, the rutin content ranged from 95.72 ± 199.17 µg g-1 (A. dubius) to 1485.09 ± 679.51 µg g-1 (A. viridis) in 2018 and from 821.59 ± 709.95 µg g-1 (A. tricolor) to 3166.52 ± 1317.38 µg g-1 (A. hypochondriacus) in 2019. Correlation analysis revealed, significant positive correlations between rutin and kaempferol-3-O-ß-rutinoside (r = 0.93), benzoic acid and ferulic acid (r = 0.76), and benzoic acid and kaempferol-3-O-ß-rutinoside (r = 0.76), whereas gallic acid showed consistently negative correlations with each of the 16 phenolic compounds. Wide variations were identified among accessions and between plants grown in the two years. The nine species and one hundred twenty Amaranthus accessions were clustered into six groups based on their seventeen phenolic compounds in each year. These findings contribute to expanding our understanding of the phytochemical traits of accessions within nine Amaranthus species, which serve as valuable resources for Amaranthus component breeding and functional material development.

The First Domesticated 'Cheongju Sorori Rice' Excavated in Korea.

Archaeological excavations led by Yung-jo Lee and Jong-yoon Woo were carried out twice at the Sorori paleolithic site, Cheongju, in the Republic of Korea, at the upper stream of the Geumgang river, the Miho riverside. A total of 127 rice seeds were excavated, including 18 ancient rice and 109 Quasi-rice, in 1998 and 2001. At the first excavation, eleven short japonica-type ancient rice and one slender smooth ancient rice with two kinds of Quasi-rice were excavated. The average length of the 11 short rice grains obtained from the first and second excavation was 7.19 mm and the average width was 3.08 mm, respectively. The Quasi-rice are apparently different from the rice and do not have bi-peak protuberances on their glume surface. At the second excavation, six short ancient rice chaffs and some Quasi-rice 2 were found. These short-grained ancient rice were comparable to the ancient rice that were excavated at the Illsan Neolithic site. Geologists and radiologists confirmed that the peat layer in which the rice found was older than 15,000 years. In this study, the morphological characteristics, crushing, and DNA band patterns related to the genetic polymorphism of rice grains in Cheongju Sorori were compared and analyzed for genetic similarities and differences with wild rice, weed rice, and modern rice. The morphological, ecological, and physiological variations in rice grains excavated from the Sorori site were presumed to denote the origin of rice domestication in Korea. It is also suggested that the results of the DNA sequencing of excavated rice are very important clues in estimating the origin of the early domestication of rice.

Implication of high variance in germplasm characteristics.

The beauty of conserving germplasm is the securement of genetic resources with numerous important traits, which could be utilized whenever they need to be incorporated into current cultivars. However, it would not be as useful as expected if the proper information was not given to breeders and researchers. In this study, we demonstrated that there is a large variation, both among and within germplasm, using a low-cost image-based phenotyping method; this could be valuable for improving gene banks' screening systems and for crop breeding. Using the image analyses of 507 accessions of buckwheat, we identified a wide range of variations per trait between germplasm accessions and within an accession. Since this implies a similarity with other important agronomic traits, we suggest that the variance of the presented traits should be checked and provided for better germplasm enhancement.

A chromosome-level genome assembly of Korean mint (Agastache rugosa).

Agastache rugosa, also known as Korean mint, is a perennial plant from the Lamiaceae family that is traditionally used for various ailments and contains antioxidant and antibacterial phenolic compounds. Molecular breeding of A. rugosa can enhance secondary metabolite production and improve agricultural traits, but progress in this field has been delayed due to the lack of chromosome-scale genome information. Herein, we constructed a chromosome-level reference genome using Nanopore sequencing and Hi-C technology, resulting in a final genome assembly with a scaffold N50 of 52.15 Mbp and a total size of 410.67 Mbp. Nine pseudochromosomes accounted for 89.1% of the predicted genome. The BUSCO analysis indicated a high level of completeness in the assembly. Repeat annotation revealed 561,061 repeat elements, accounting for 61.65% of the genome, with Copia and Gypsy long terminal repeats being the most abundant. A total of 26,430 protein-coding genes were predicted, with an average length of 1,184 bp. The availability of this chromosome-scale genome will advance our understanding of A. rugosa's genetic makeup and its potential applications in various industries.

Potential Use of Colored LED Lights to Increase the Production of Bioactive Metabolites Hedyotis corymbosa (L.) Lam.

Hedyotis corymbosa (L.) Lam is a wild herb that is used in traditional Indian, Chinese, and African medicine. Light-emitting diode (LED) technology is paving the way to enhance crop production and inducing targeted photomorphogenic, biochemical, or physiological responses in plants. This study examines the efficiency of H. corymbosa (L.) Lam production under blue 450 nm and red 660 nm LED lights for overall plant growth, photosynthetic characteristics, and the contents of metabolite compounds. Our research showed that blue LED lights provided a positive effect on enhancing plant growth and overall biomass. In addition, blue LED lights are more effective in controlling the production of sucrose, starch, total phenolic compounds, and total flavonoid compared to red LED lights. However, blue and red LED lights played essential but different roles in photosynthetic characteristics. Our results showed the potential of colored LED light applications in improving farming methods and increasing metabolite production in herbs.

Marker-Assisted Backcrossing (MABc) to Improve Eating Quality with Thin Seed Coat and Aleurone Layer of Non-Glutinous Japonica Variety in Rice.

Brown rice is composed of rice bran, pericarp, seed coat, and aleurone layers, and the rice bran layer contains a large number of substances useful for the human body, such as dietary fiber, α-tocopherol, α-tocotrienol, and vitamins. However, more than 90% of these substances are removed when polished, and white rice has the disadvantage of losing food-related ingredients, such as umami-related amino acids, when compared to the unpolished group. In this study, we tried to develop new breeding lines with a thinner seed coat and aleurone layer to provide high eating quality with softer chewing characteristics and processability in rice grain. We detected an SNP for foreground selection for the backcross population by comparing genome sequences between Samgwang and Seolgaeng and developed high eating quality brown rice breeding lines by applying marker-assisted backcrossing (MABC) breeding programs to backcross populations between Samgwang and Seolgaeng using KASP markers. SNP markers for foreground selection were identified to improve eating and processability through SNP mapping of Samgwang and Seolgaeng with SSIIa as a target gene in this study. Line selection according to genotype of KASP markers was successful in BC1F1 and BC2F1 generations, with the recurrent parent genome recovery ratio ranging from 91.22% to 98.65%. In BC2F1 seeds of the selected lines, thickness of the aleurone layer was found to range from 13.82 to 21.67 µm, which is much thinner than the 30.91 µm of the wild type, suggesting that selection by MABc could be used as an additional breeding material for the development of highly processed rice varieties. These lines will be useful to develop new brown rice varieties with softer chewing characteristics and processability in rice grain.

Comparative Transcriptomics for Genes Related to Berberine and Berbamine Biosynthesis in Berberidaceae.

Berberine and berbamine are bioactive compounds of benzylisoquinoline alkaloids (BIAs) present in Berberis species. The contents of berbamine are 20 times higher than berberine in leaf tissues in three closely related species: Berberis koreana, B. thunbergii and B. amurensis. This is the first report on the quantification of berberine compared to the berbamine in the Berberis species. Comparative transcriptome analyses were carried out with mRNAs from the leaf tissues of the three-species. The comparison of the transcriptomes of B. thunbergii and B. amurensis to those of B. koreana, B. thunbergii showed a consistently higher number of differentially expressed genes than B. amurensis in KEGG and DEG analyses. All genes encoding enzymes involved in berberine synthesis were identified and their expressions were variable among the three species. There was a single copy of CYP80A/berbamunine synthase in B. koreana. Methyltransferases and cytochrome P450 mono-oxidases (CYPs) are key enzymes for BIA biosynthesis. The current report contains the copy numbers and other genomic characteristics of the methyltransferases and CYPs in Berberis species. Thus, the contents of the current research are valuable for molecular characterization for the medicinal utilization of the Berberis species.

Plant Variety Protection: Current Practices and Insights.

Breeders persistently supply farmers with the best varieties in order to exceed consumer demand through plant-breeding processes that are resource-intensive. In order to motivate continuous innovation in variety development, a system needs to provide incentives for plant breeders to develop superior varieties, for example, exclusive ownership to produce and market those varieties. The most common system is the acquisition of intellectual property protection through plant variety protection, also known as the breeder's right. Most countries have adopted the system established by the International Union for the Protection of New Varieties of Plants (UPOV). To be granted plant variety protection, the variety should prove to be unique by meeting three requirements: distinctness, uniformity, and stability. This review summarizes (1) the plant variety protection via UPOV convention, (2) technical methods for distinctness, uniformity, and stability testing via phenotype, molecular markers, and sequencing as well as their challenges and potentiality, and (3) additional discussions in essentially derived variety, value for cultivation and use testing, and open source seed initiative.

Corn Starch: Quality and Quantity Improvement for Industrial Uses.

Corn starch serves as food, feed, and a raw material for industrial use. Starch makes up most of the biomass of the corn hybrid and is the most important and main yield component in corn breeding programs. Starch is composed of two polymers, branched amylopectin and linear amylose, which normally constitute about 75% and 25% of the corn starch, respectively. Breeding for corn starch quality has become economically beneficial because of the development of niche markets for specialty grains. In addition, due to the increased demands of biofuel production, corn ethanol production is receiving more attention. Consequently, improving starch quantity has become one of the most important breeding objectives. This review will summarize the use of corn starch, and the genetics and breeding of grain quality and quantity for industrial applications.

Breeding of High Cooking and Eating Quality in Rice by Marker-Assisted Backcrossing (MABc) Using KASP Markers.

The primary goals of rice breeding programs are grain quality and yield potential improvement. With the high demand for rice varieties of premium cooking and eating quality, we developed low-amylose content breeding lines crossed with Samgwang and Milkyqueen through the marker-assisted backcross (MABc) breeding program. Trait markers of the SSIIIa gene referring to low-amylose content were identified through an SNP mapping activity, and the markers were applied to select favorable lines for a foreground selection. To rapidly recover the genetic background of Samgwang (recurrent parent genome, RPG), 386 genome-wide markers were used to select BC1F1 and BC2F1 individuals. Seven BC2F1 lines with targeted traits were selected, and the genetic background recovery range varied within 97.4-99.1% of RPG. The amylose content of the selected BC2F2 grains ranged from 12.4-16.8%. We demonstrated the MABc using a trait and genome-wide markers, allowing us to efficiently select lines of a target trait and reduce the breeding cycle effectively. In addition, the BC2F2 lines confirmed by molecular markers in this study can be utilized as parental lines for subsequent breeding programs of high-quality rice for cooking and eating.

Gene Expression and Isoform Identification of PacBio Full-Length cDNA Sequences for Berberine Biosynthesis in Berberis koreana.

Berberis koreana is a medicinal plant containing berberine, which is a bioactive compound of the benzylisoquinoline alkaloid (BIA) class. BIA is widely used in the food and drug industry for its health benefits. To investigate the berberine biosynthesis pathway, gene expression analysis was performed in leaves, flowers, and fruits at different stages of growth. This was followed by full-length cDNA sequencing analysis using the PacBio sequencer platform to determine the number of isoforms of those expressed genes. We identified 23,246 full-length unigenes, among which 8479 had more than one isoform. The number of isoforms ranged between two to thirty-one among all genes. Complete isoform analysis was carried out on the unigenes encoding BIA synthesis. Thirteen of the sixteen genes encoding enzymes for berberine synthesis were present in more than one copy. This demonstrates that gene duplication and translation into isoforms may contribute to the functional specificity of the duplicated genes and isoforms in plant alkaloid synthesis. Our study also demonstrated the streamlining of berberine biosynthesis via the absence of genes for enzymes of other BIAs, but the presence of all the genes for berberine biosynthesize in B. koreana. In addition to genes encoding enzymes for the berberine biosynthesis pathway, the genes encoding enzymes for other BIAs were not present in our dataset except for those encoding corytuberine synthase (CTS) and berbamunine synthase (BS). Therefore, this explains how B. koreana produces berberine by blocking the pathways leading to other BIAs, effectively only allowing the pathway to lead to berberine synthesis.

What Traits Should Be Measured for Biomass in Kenaf?

Kenaf (Hibiscus cannabinus L.) is widely used as an important industrial crop. It has the potential to act as a sustainable energy provider in the future, and contains beneficial compounds for medical and therapeutic use. However, there are no clear breeding strategies to increase its biomass or leaf volume. Thus, to attain an increase in these parameters, we examined potential key traits such as stem diameter, plant height, and number of nodes to determine the relationship among them. We hypothesized that it would be easier to reduce the amount of time and labor required for breeding if correlations among these parameters are identified. In this study, we found a strong positive correlation between height and number of nodes (Spearman's Rho = 0.67, p < 0.001) and number of nodes and stem diameter (Spearman's Rho = 0.65, p < 0.001), but a relatively low correlation (Spearman's Rho = 0.34, p < 0.01) between height and stem diameter in the later stages of kenaf growth. We suggest that an efficient breeding strategy could be devised according to the breeding purpose, considering the correlations between various individual traits of kenaf.

QTL mapping of agronomic traits in tef [Eragrostis tef (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 = 4x = 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). RESULTS: Twenty-two yield-related and morphological traits were assessed across eight different locations in Ethiopia during the growing seasons of 1999 and 2000. Using composite interval mapping and a linkage map incorporating 192 loci, 99 QTLs were identified on 15 of the 21 linkage groups for 19 traits. Twelve QTLs on nine linkage groups were identified for grain yield. Clusters of more than five QTLs for various traits were identified on seven linkage groups. The largest cluster (10 QTLs) was identified on linkage group 8; eight of these QTLs were for yield or yield components, suggesting linkage or pleotrophic effects of loci. There were 15 two-way interactions of loci to detect potential epistasis identified and 75% of the interactions were derived from yield and shoot biomass. Thirty-one percent of the QTLs were observed in multiple environments; two yield QTLs were consistent across all agro-ecology zones. For 29.3% of the QTLs, the alleles from E. pilosa (30-5) had a beneficial effect. CONCLUSION: The extensive QTL data generated for tef in this study will provide a basis for initiating molecular breeding to improve agronomic traits in this staple food crop for the people of Ethiopia.

Nonrandom distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley.

BACKGROUND: Earlier comparative maps between the genomes of rice (Oryza sativa L.), barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were linkage maps based on cDNA-RFLP markers. The low number of polymorphic RFLP markers has limited the development of dense genetic maps in wheat and the number of available anchor points in comparative maps. Higher density comparative maps using PCR-based anchor markers are necessary to better estimate the conservation of colinearity among cereal genomes. The purposes of this study were to characterize the proportion of transcribed DNA sequences containing simple sequence repeats (SSR or microsatellites) by length and motif for wheat, barley and rice and to determine in-silico rice genome locations for primer sets developed for wheat and barley Expressed Sequence Tags. RESULTS: The proportions of SSR types (di-, tri-, tetra-, and penta-nucleotide repeats) and motifs varied with the length of the SSRs within and among the three species, with trinucleotide SSRs being the most frequent. Distributions of genomic microsatellites (gSSRs), EST-derived microsatellites (EST-SSRs), and transcribed regions in the contiguous sequence of rice chromosome 1 were highly correlated. More than 13,000 primer pairs were developed for use by the cereal research community as potential markers in wheat, barley and rice. CONCLUSION: Trinucleotide SSRs were the most common type in each of the species; however, the relative proportions of SSR types and motifs differed among rice, wheat, and barley. Genomic microsatellites were found to be primarily located in gene-rich regions of the rice genome. Microsatellite markers derived from the use of non-redundant EST-SSRs are an economic and efficient alternative to RFLP for comparative mapping in cereals.

Haplotyping and mapping a large cluster of downy mildew resistance gene candidates in sunflower using multilocus intron fragment length polymorphisms.

Downy mildew (Plasmopara halstedii (Farl.) Berlese et de Toni) is a serious foliar pathogen of cultivated sunflower (Helianthus annuus L.). Genetic resistance is conditioned by several linked downy mildew resistance gene specificities in the HaRGC1 cluster of TIR-NBS-LRR resistance gene candidates (RGCs) on linkage group 8. The complexity and diversity of the HaRGC1 cluster was assessed by multilocus intron fragment length polymorphism (IFLP) genotyping using a single pair of primers flanking a hypervariable intron located between the TIR and NBS domains. Two to 23 bands were amplified per germplasm accession. The size of the included intron ranged from 89 to 858 nucleotides. Forty-eight unique markers were distinguished among 24 elite inbred lines, six partially isogenic inbred lines, nine open-pollinated populations, four Native American land races, and 20 wild H. annuus populations. Nine haplotypes (based on 24 RGCs) were identified among elite inbred lines and were correlated with known downy mildew resistance specificities. Sixteen out of 39 RGCs identified in wild H. annuus populations were not observed in elite germplasm. Five partially isogenic downy mildew resistant lines developed from wild H. annuus and H. praecox donors carried eight RGCs not found in other elite inbred lines. Twenty-four HaRGC1 loci were mapped to a 2-4 cM segment of linkage group 8. The multilocus IFLP marker and duplicated, hypervariable microsatellite markers tightly linked to the HaRGC1 cluster are powerful tools for distinguishing downy mildew resistance gene specificities and identifying and introgressing new downy mildew resistance gene specificities from wild sunflowers.

Transferability of cereal EST-SSR markers to ryegrass.

A large number of expressed sequence tags (ESTs) in public databases have provided an opportunity for the systematic development of simple sequence repeat (SSR) markers. EST-SSRs derived from conserved coding sequences show considerable cross-species transferability in related species. In the present study, we assessed the utility of cereal EST-SSRs in ryegrass (Lolium spp.). A total of 165 cereal EST-SSRs were tested; a high rate of transferability (57%) and polymorphism (67% of functional EST-SSRs) was demonstrated between cereals and ryegrass. A total of 46 segregating loci derived from 37 EST-SSRs were mapped on an existing ryegrass genetic map. The mapped loci were uniformly distributed across all seven linkage groups without significant clustering at the distal regions of linkage groups. Sequences of ryegrass amplicons generated by randomly selected 16 EST-SSRs were aligned with reference sequences of cereal EST-SSRs. The SSR motifs and repeat lengths of the cereal EST-SSR markers were different from the majority of ryegrass amplicons. Furthermore, a majority of EST-SSRs amplified different flanking sequences of SSRs in ryegrass than the original cereal sequences. Our results suggest that the high degree of cereal EST-SSR transferability to ryegrass can be a useful enhancement to the molecular database of PCR-based markers but sequence analysis is essential before transferring genetic information using comparative mapping.

Impact of plant breeding on genetic diversity of the Canadian hard red spring wheat germplasm as revealed by EST-derived SSR markers.

Genetic diversity changes in wheat germplasm have been studied using different molecular markers, but little is known about the impact of plant breeding on the transcribed segments of the wheat genome. The objective of this study was to assess diversity changes in 75 Canadian hard red wheat cultivars released from 1845 to 2004 using 37 EST-derived microsatellite (eSSR) markers. These markers were derived from at least 19 transcribed sequences with putative functions assigned and sampled 17 wheat chromosomes. A total of 138 eSSR alleles was detected, and their allelic frequencies ranged from 0.01 to 0.99 with an average of 0.41. Allelic counts were significantly reduced at three loci for cultivars released after 1990. Sixteen alleles at 14 loci in pre-1910 cultivars were lost in cultivars released after 1990. The lost alleles had frequencies ranging from 0.03 to 0.17 and averaging 0.07. Partitioning the eSSR variation showed the four ancestral families accounted for 14.7% of the variation, followed by the six breeding periods with 12.8% and the eight breeding programs with 5.8%. A genetic shift was observed in the cultivars released over the six breeding periods, reflecting the various breeding efforts. These results illustrate the impact of the Canadian wheat breeding on the transcriptional segments of the wheat genome. These findings, along with those from genomic SSR markers, suggest the Canadian wheat breeding programs have reduced genetic diversity in the hard red spring wheat.

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.

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 F(9) recombinant inbred lines (RIL) derived from the interspecific cross, Eragrostis tef cv. Kaye Murri x 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.