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BioMed Central Page 1 of 13 (page number not for citation purposes) BMC Plant Biology Open Access Research article QTL mapping of agronomic traits in tef [Eragrostis tef (Zucc) Trotter] Ju-Kyung Yu 1,3 , Elizabeth Graznak 1 , Flavio Breseghello 1,4 , Hailu Tefera 2 and Mark E Sorrells* 1 Address: 1 Department of Plant Breeding and Genetics, Cornell University, Ithaca NY 14853, USA, 2 Debre Zeit Agricultural Research Center, P.O. Box 32, Debre Zeit, Ethiopia, 3 Syngenta Seeds Inc. 317 330th Street, Stanton, MN 55018, USA and 4 Embrapa Arroze Feijão, Caixa Postal 179, Santo Antônio de Goiás, GO 75375-000, Brazil Email: Ju-Kyung Yu - ju-kyung.yu@syngenta.com; Elizabeth Graznak - egraznak@coin.org; Flavio Breseghello - flavio@cnpaf.embrapa.br; Hailu Tefera - hailutefera@yahoo.com; Mark E Sorrells* - mes12@cornell.edu * Corresponding author Abstract 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). 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. Background Tef, Eragrostis tef (Zucc.) Trotter, is a major food grain in Ethiopia but is a minor cereal crop worldwide. The pri- mary use of tef is for grinding into flour to make injera, a spongy fermented flat bread that is a staple food for most Ethiopians. The vegetative portions of the plant are also an important source of fodder for livestock. In Ethiopia for the crop year 2003–2004, it occupied two million hec- tares, which represented 28% of the area grown with eight cereal crops in the country [1]. The ability of tef to perform Published: 12 June 2007 BMC Plant Biology 2007, 7:30 doi:10.1186/1471-2229-7-30 Received: 23 October 2006 Accepted: 12 June 2007 This article is available from: http://www.biomedcentral.com/1471-2229/7/30 © 2007 Yu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Plant Biology 2007, 7:30 http://www.biomedcentral.com/1471-2229/7/30 Page 2 of 13 (page number not for citation purposes) well on both waterlogged Vertisols in the highlands as well as water-stressed areas in the semi-arid regions throughout the country is one of the reasons for which tef is preferred over other grain crops such as maize or barley [2]. In addition, tef generally suffers less from biotic stresses compared to most other cereal crops grown in Ethiopia and it contains high levels of proteins and min- eral [3]. Tef is an allotetraploid species with a base chromosome number of 10 (2n = 4× = 40). It belongs to the family Poaceae, sub-family Eragrostidae and genus Eragrostis. The genus contains approximately 350 species [4]. The exact diploid progenitors of tef are still unknown; however, most researchers agree that E. pilosa is the species most closely related to E. tef and is considered the direct wild tetraploid progenitor of tef [5]. It is also the only species known to be cross-compatible with modern tef varieties. Flow cytometry research has shown that tef has a genome size of 730 Mbp [6], which is roughly the same size as dip- loid sorghum and about 60% larger than the diploid rice genome. It has also the smallest chromosomes reported among the Poaceae ranging from 0.8 to 2.9 μm [6], which has significantly hindered the cytogenetic research of this species. Understanding the genetic control of agronomic traits is essential for the sustained improvement of tef. Lodging is the number one cause of yield loss in tef; even with good crop management practices. Recent studies in tef have shown strong correlations between lodging, panicle type, culm thickness, and grain yield [2,7]. Important agro- nomic traits in tef, as in most crop species, are quantitative inherited [7,8], which complicates genetic analysis. Quan- titative trait locus (QTL) analysis allows the identification of discrete chromosome segments controlling complex traits [9]. The significance of identifying QTLs that corre- spond with certain traits is that the information can be used for marker-assisted selection (MAS) program. This is the most comprehensive report of QTL analyses for agro- nomic traits in tef to date. Cultivated tef and the wild species, E. pilosa, differ greatly for most agronomic traits and the close relationship betweenthese two species facilitate hybridization provid- ing a unique opportunity to develop a new pool of genetic variation. The study by Tefera et al. [7] has demonstrated that E. pilosa has contributed useful breeding traits, such as earliness and short stature. Therefore, utilization of E. pilosa as a donor in an inter-specific cross is a useful strat- egy for broadening the genetic diversity of the existing gene pool in cultivated tef. The purpose of this research was to identify and character- ize QTLs controlling 22 agronomic traits; eight yield- related traits and 14 morphological traits, in the inter-spe- cific cross between E tef, cv. Kaye Murri and E. pilosa (30- 5). Results Trait analysis Effects of years and locations were highly significant (p < 0.001) for all traits evaluated in multiple locations (data not shown). The variance among lines was highly signifi- cant (p < 0.001) for all traits except RPR1, RPR2, and Crush1 (data not shown). The mean value of the two par- ents, Kaye Murri and E. pilosa (30-5) were significantly dif- ferent for all 22 traits (Table 1). As expected for an inter- specific cross, distribution of phenotypic values in the progeny showed bi-directional transgressive segregants for all traits, except Crush1 and Crush2, which showed transgressive segregants towards the E. pilosa (30-5) parent only. Phenotypic correlations were estimated between the over- all means of the 22 phenotypic traits. All traits, except RPR1 and RPR2, were highly correlated (p < 0.001) with at least one other trait. Significant positive correlations were identified between yield and most agronomic traits except PedL and Dia in this population (Table 2). Lodging was not correlated with traits supposedly lodging related, such as PH, RPR1, 2 and Crush1, 2 (Table 2). The fre- quency distributions of most of traits fit the normal distri- bution, however, seven traits (PWt, PSWt, GY, SB, HD, RPR1 and RPR2) were significantly skewed, and transfor- mation was applied prior to QTL analysis except RPR1 and 2. The traits, RPR1, RPR2 and Crush1 were excluded for QTL analyses which did not show variances among lines thus, 19 traits were evaluated for QTL analyses. A total of 99 QTLs for 19 traits was identified by three analyses in common; SMR, CIM and MT-CIM. The map positions of the QTLs together with the additive effects and R 2 values from CIM are presented in Fig. 1 and Table 3. The QTLs were distributed over all linkage groups except 4, 5, 12, 14, 15, and 17 (Fig. 1). Two or more QTLs were identified for all traits except HD, CD2 and Dia. The number of chromosomes with significant QTL for the spe- cific traits ranged from one (HD, CD2 and Dia) to 12 (GY). The number of significant QTL for the specific chro- mosomes ranged from zero (LG4, 5, 12, 14, 15, and 17) to 14 (LG2) (Fig. 1). The wild relative, E. pilosa (30-5) alle- les had an increasing effect on 29.3% of the QTLs in the present study. A test for potential interactions between significant QTL marker loci for all traits identified a relatively small number of epistatic interactions between loci. A total of 20 interactions consisting of 18 marker loci for four traits BMC Plant Biology 2007, 7:30 http://www.biomedcentral.com/1471-2229/7/30 Page 3 of 13 (page number not for citation purposes) Table 1: Traits, phenotypes of RIL population, parents (E. tef cv. Kaye Murri, KM and E. pilosa (30-5), Ep), and evaluation environments. RIL Parent Trait Abbv. Unit Mean Min Max SD KM Ep Norm. Experiments Yield and Yield Related Traits Heading date*** HD days 30.50 21.25 47.50 7.13 44.00 32.50 log e09,10,11 Marturity date*** MD days 81.75 65.25 107.00 11.18 96.00 84.25 e09,10,11 Panicle weight*** PWt g 0.32 0.06 1.16 0.16 0.71 0.23 log e01,02,03,04,05,06,07,08,09,10,11 Panicle seed weight*** PSWt g 0.18 0.01 0.66 0.11 0.47 0.12 log e01,02,03,04,05,06,07,08,09,10,11 100 seed weight*** 100sw mg 17.25 6.50 32.50 4.23 26.75 17.75 e01,02,03,04,05,07,08 Grain yield*** GY g 156 7.25 707.50 130.23 319 165 sqrt e01,02,03,04,05,06,07,08,09,10,11 Shoot biomass*** SB g 986 196 4050 755 1650 750 sqrt e01,02,03,04,05,06,07,08,09,10,11 Lodging index*** Lodg score 71.00 35.00 99.50 14.61 65.13 81.50 e01,02,03,04,05,07,08,09,10,11 Morphological and Plant Height Related Triats Culm length*** CulmL cm 44.50 22.02 71.55 9.32 56.40 42.25 e01,02,03,04,05,06,07,08,09,10,11 Culm diameter1 a *** CD1 cm 1.30 0.72 2.10 0.23 1.72 1.04 e01,02,03,04,05,06,07 Culm diameter2 b *** CD2 cm 1.29 0.66 2.08 0.24 1.76 1.11 e01,02,03,04,05,06,07 Peduncle length*** PedL cm 19.35 9.75 29.65 3.63 19.93 17.80 e01,02,03,04,05,06,07,08,09,10,11 Panicle length*** PanL cm 23.75 12.50 39.75 4.35 30.90 20.95 e01,02,03,04,05,06,07,08,09,10,11 Plant height*** PH cm 71.45 37.80 99.95 12.30 88.23 61.70 e01,02,03,04,05,06,07 Number of internodes*** Ninter score 3.00 2.25 4.45 0.40 3.35 2.88 e01,02,03,04,05,06,07 1st internode length*** Inter1 cm 6.65 2.70 13.75 1.62 8.40 6.20 e01,02,03,04,05,06,07,08,09,10,11 2nd internode length*** Inter2 cm 10.45 5.40 16.95 2.20 12.88 9.83 e01,02,03,04,05,06,07,08,09,10,11 Crown diameter*** Dia cm 1.55 0.83 2.23 0.28 2.08 1.14 e01,03,07 Rind penetrometer1 c RPR1 lbs 0.54 0.28 0.83 0.11 1.15 0.45 e04 Rind penetrometer2 d RPR2 lbs 0.36 0.24 0.65 0.08 0.74 0.30 e04 Crush strength1 e Crush1 lbs 4.88 1.98 6.88 1.05 9.49 3.59 e04 Crush strength2 f *** Crush2 lbs 4.06 1.17 7.67 1.11 9.64 3.24 e04 Abbr. = abbreviation of trait; Norm. = transformation used to achieve normality. Eight locations representing three agro-ecologies in Ethiopia; Akaki (AK), Alemtena (AL), Debre Zeit Black Soil (DZBS), Debre Zeit Light Soil (DZLS), Denbi (DE), Melkasa (MEL), Chefe (CH) and Holetta (HO), wet semi-arid in higher than 1900 masl altitude (C2-1; AK, CH, HO), wet semi-arid in 1700–1900 masl altitude (C2-2; DZBS, DZLS, DE), dry semi-arid in lower than 1700 masl altitude (C3-3; AL, MEL). Each experiment representing the combination of different environments and years for each trait evaluation; AK and 2000 (e01), AL and 2000 (e02), DZBS and 2000 (e03), DZLS and 2000 (e04), DE and 2000 (e05), MEL and 2000 (e06), CH and 2000 (e07), HO and 2000 (e08), AK and 1999 (e09), AL and 1999 (e10) and DZBS and 1999 (e11). a culm diameter of 1 st internode b culm diameter of 2 nd internode c measurement of penetration strength in 1 st internode rind d measurement of penetration strength in 2 nd internode rind e measurement of crushing strength in 1 st internode f measurement of crushing strength in 2 nd internode *** The analysis of variance for traits among lines and experiments at significance of 0.001 probability level were identified across nine linkage groups and three unlinked loci (Table 4). QTL for grain yield and yield related traits Heading date (HD) and maturity date (MD) Two MD QTLs were identified at three locations represent- ative of all three agro-ecologies. The MD QTL on LG2 at 24.8 cM explained 0.34 of R 2 , and was associated with yield related traits such as PWt and SB (Fig. 1). Early matu- rity is a common characteristic of wild relatives of tef and E. pilosa (30-5) matured on average 12 days earlier than Kaye Murri. On the other hand, at the QTL for HD, the allele from E. pilosa contributed longer cycle. Panicle weight (PWt) Five QTLs were identified for PWt on LG2, 8, 10, 19 and 20 and R 2 ranged from 14% to 23%. The QTL interval on LG2 (RZ876 to RZ962c), was associated with two yield related traits and six morphological traits. All five QTLs were overlapped or closely located with the QTLs for PSWt. Three of the QTLs were positively affected by Kaye Murri resulting in weight increase. Panicle seed weight (PSWt) Nine QTLs were identified for PSWt covering all three agro-ecologies with six locations. Out of seven QTLs that were associated with GY, five Kaye Murri QTLs showed a BMC Plant Biology 2007, 7:30 http://www.biomedcentral.com/1471-2229/7/30 Page 4 of 13 (page number not for citation purposes) positive effect. Four PSWt QTLs were associated with PWt and two overlapped with GY QTLs. However, there was no QTL associated with 100sw. 100 seed weight (100sw) Four QTLs were identified for 100sw, all of which were increased by the alleles of the cultivated parent. No 100sw QTL were associated with PWt, PSWt or GY QTL. Grain yield (GY) The largest number of QTLs was identified for GY, among the traits studied. Twelve QTLs were identified in nine linkage groups. The highest LOD score was 6.39 for ISSR549b explaining 0.2 of R 2 . Two QTLs in LG3, 50 cM apart, were significant in six locations representing three agro-ecologies. The E. pilosa (30-5) alleles in LG18 (ISSR840b) and LG20 (RZ588) increased grain yield. The rest of the QTLs were positively affected by the Kaye Murri alleles. Shoot biomass (SB) The most significant QTLs for SB were found on LG3, 8 and 10 with a LOD > 6 and R 2 > 0.19. One QTL on LG20 (RZ588) explained 0.22 of R 2 and the positive allele was from E. pilosa (30-5). This QTL co-located with PWt, PSWt and GY QTLs, all with same positive alleles from E. pilosa (30-5). Lodging index (Lodg) Three QTLs were located on LG1 and 8, and two QTLs were associated with unlinked loci. All five QTL alleles contributed by Kaye Murri increased lodging. The two QTLs (PALb and TCD323) on LG8 were located in the dis- tal region of the linkage group. PALb showed the highest R 2 (0.38) and highest LOD score (5.5) and co-segregated with MD. TCD323 co-located with SB and GY, and was located near eight other QTLs, including lodging related traits, such as Crush2. QTL for morphological and plant height related traits Culm length (CulmL) Eight significant CulmL QTLs were identified on seven linkage groups and one unlinked locus (Table 3). The R 2 ranged from 0.12 to 0.34. Except for RZ251 on LG13, increasing effects of all significant QTLs came from Kaye Murri. The strongest CulmL QTL is TCD95 on LG3 with a LOD score of 5.92 and an R 2 value of 0.21. This locus was associated with PSWt, Inter2, GY and SB. Culm diameter 1 st and 2 nd internode (CD1 and CD2) Two and one QTLs were associated with CD1 and CD2, respectively and were identified only in the C2-2 agro- ecology zone. These traits share common QTL regions on LG2 and the allele for thicker culms was contributed by Kaye Murri. Peduncle length (PedL) Eleven significant QTLs were identified on six linkage groups and five of the QTLs were associated with unlinked loci. The R 2 for PedL ranged from 0.11 to 0.35. At seven QTLs, E. pilosa (30-5) alleles increased PedL. Among these, two QTLs in LG10 and 21 were negatively associated with other traits (100sw and SB in LG10 and GY in LG21). Panicle length (PanL) Seven QTLs were identified for PanL, with a maximum R 2 of 0.22 and LOD = 4 for RZ588 in LG20. Kaye Murri alle- les increased PanL in all QTLs, except for RZ251 (LG13) and RZ588 (LG20). Six PanL QTLs were associated with several yield-related traits. Plant height (PH) Four significant QTLs were identified with R 2 ranging from 0.13 to 0.26. Kaye Murri alleles at QTLs in LG2, 7, and 8 increased PH while the E. pilosa (30-5) allele increased PH at RZ588 (LG20). All PH QTLs were associ- ated with QTLs for multiple yield-related traits. Number of internodes (Ninter) Three QTLs were associated with Ninter. The most signifi- cant QTL (LOD = 4.97, R 2 = 0.20) was on LG2 which was associated with PH. Table 2: Trait correlations for grain yield and lodging index. GY Lodg HD 0.50*** 0.14 MD 0.48*** -0.06 PWt 0.67*** 0.21* PSWt 0.75*** 0.28** 100sw 0.50*** 0.41*** GY 0.51*** SB 0.87*** 0.37*** Lodg 0.51*** CulmL 0.60*** 0.25* CD1 0.42*** -0.06 CD2 0.42*** -0.06 PedL -0.19 -0.28** PanL 0.52*** 0.07 PH 0.58*** 0.16 Ninter 0.53*** 0.23* Inter1 0.41*** 0.15 Inter2 0.46*** 0.23* Dia 0.16 -0.22* RPR1 0.05 -0.13 RPR2 0.04 -0.13 Crush1 0.14 -0.09 Crush2 0.45*** -0.02 *, ** and *** significant at the 0.05, 0.01 and 0.001 probability level, respectively. BMC Plant Biology 2007, 7:30 http://www.biomedcentral.com/1471-2229/7/30 Page 5 of 13 (page number not for citation purposes) Table 3: QTLs detected by composite interval mapping in the RIL population from the cross 'E. tef × E. pilosa (30-5)' Trait a Chrom. Closest locus/loci b Peak c LOD R2 Add d Exp. e HD 13 RZ251 4.47 0.16 0.01 e11 MD 2 RZ876 ~ RZ962c RZ876 3.08 0.34 -1.70 e09, e10 8 PALb 3.42 0.20 -3.14 e11 PWt 2 RZ876 ~ RZ962c RZ876 3.18 0.15 -0.06 e05 8 ISSR548a 3.97 0.15 -0.06 e06 10 TCD52 3.51 0.19 -0.08 e04 19 RZ698b 3.25 0.14 0.07 e01 20 RZ588 3.30 0.23 0.09 e07 PSWt 2 PALa 4.41 0.17 -0.10 e04 3 TCD95 3.80 0.21 -0.10 e09 7 ISSR811b ~ ISSR840a ISSR811b 5.20 0.24 -0.12 e06, e10 8 ISSR548a 3.35 0.13 -0.07 e06 10 TCD52 3.99 0.27 -0.12 e04 13 RZ251 3.02 0.11 0.10 e08 18 ISSR840b 5.45 0.24 0.13 e04, e8 19 RZ698b 3.17 0.14 0.09 e01 20 RZ588 3.51 0.20 0.11 e07 100sw 6 RM170b 5.21 0.26 -2.22 e02, e03 10 ISSR842c ~ TCD327b ISSR842c 6.23 0.21 -1.90 e03, e07 un CNLT127-T04 5.25 0.21 -1.75 e05 un KSUM222 5.78 0.30 -2.49 e01 GY 2 CNL53 ~ ISSR547 CNL53 4.06 0.11 -1.13 e04, e06, e08 2 BCD880 5.84 0.24 -1.47 e02 3 TCD248 ~ TCD95 TCD95 6.34 0.28 -1.07 e03, e05, e07, e09, e10, e11 3 PRSC1_022 3.98 0.12 -1.23 e01 6 TCD308 ~ ISSR842b ISSR549b 6.36 0.20 -1.61 e08, e09 7 ISSR840a 5.94 0.25 -1.67 e01, e06, e10 8 TCD227a ~ ISSR548a ISSR548a 4.88 0.15 -1.02 e04, e11 8 TCD323 4.92 0.15 -1.12 e02 16 RZ395 ~ RM134 RZ395 3.35 0.12 -0.59 e05, e09 18 ISSR840b 3.48 0.12 0.97 e07 20 RZ588 3.98 0.18 1.10 e06, e11 21 lfm256 5.03 0.14 -1.29 e05, e03 SB 2 BCD880 3.94 0.14 -2.33 e05 2 RZ962c 3.04 0.15 -2.40 e04 3 TCD248 ~ ISSR549a ISSR549a 6.57 0.19 -1.63 e01, e02, e09, e10, e11 6 RM176 ~ ISSR549b RM176 4.72 0.16 -2.39 e04, e05, e06, e08, e09 6 ISSR841b 3.13 0.11 -1.90 e03 7 CNLT145 4.60 0.26 -2.88 e03 8 ISSR548a ~ TCD323 ISSR548a 6.60 0.21 -2.28 e06, e11 10 TCD52 ~ CNLT78 6.32 0.26 -0.90 e07, e09 10 TCD327b 3.07 0.12 -1.97 e03 11 DupW4 ~ ISSR842e DupW4 3.28 0.15 -1.42 e10, e11 20 RZ588 4.36 0.22 1.71 e10 Lodg 1 TCD99b 3.87 0.25 -6.12 e03 8 PALb 5.50 0.38 -7.12 e02 8 TCD323 5.53 0.23 -6.00 e05 un BCD944a 5.68 0.32 -7.21 e05 un TCD182a 4.90 0.23 -5.60 e02 CulmL 2 RZ876 3.67 0.23 -2.18 e02 3 TCD95 5.92 0.21 -2.28 e09, e10 6 RM170b 3.34 0.12 -1.92 e08 7 RM124b ~ CNLT145 CNLT145 3.44 0.34 -2.64 e02 8 ISSR548a 3.46 0.12 -1.49 e11 BMC Plant Biology 2007, 7:30 http://www.biomedcentral.com/1471-2229/7/30 Page 6 of 13 (page number not for citation purposes) 11 DupW4 ~ ISSR842e ISSR842e 5.66 0.28 -2.16 e07, e09, e11 13 RZ251 3.32 0.24 2.20 e07, e08 un KSUM222 3.57 0.22 -2.50 e01 CD1 2 RZ876 ~ RZ962c RZ876 4.67 0.33 -0.12 e05 13 RZ251 3.39 0.21 0.09 e04 CD2 2 RZ876 ~ RZ962c RZ876 4.86 0.33 -0.12 e05 PedL 1 CDO1160 ~ TCD45 CDO1160 4.23 0.19 -0.86 e07 3 RM170a 5.90 0.25 1.40 e08 7 CNLT145 3.17 0.11 -0.94 e08 9 ISSR842h 3.43 0.12 -0.68 e01 10 ISSR842c 5.35 0.21 1.08 e10 21 lfm256 3.72 0.11 0.78 e02, e04, e11 un BCD944a 4.11 0.17 1.06 e04 un CNLT12 4.05 0.17 0.98 e02, e03 un DupW216 3.32 0.35 -1.12 e09 un ISSR842d 3.16 0.17 1.00 e11 un CNLT142-T03 3.50 0.13 0.72 e01 PanL 2 RZ876 3.36 0.11 -1.27 e03 6 RM176 3.52 0.10 -1.22 e03, e06 7 RM124b 3.92 0.14 -1.07 e10 8 ISSR548a ~ CD038 ISSR548a 4.73 0.14 -1.46 e03 13 RZ251 4.38 0.19 1.78 e05 20 RZ588 4.00 0.22 1.36 e07 un ISSR842d 3.25 0.12 -0.98 e10 PH 2 RZ876 ~ RZ962c RZ962c 3.63 0.26 -3.79 e01, e05 7 CNLT145 3.14 0.13 -3.26 e04 8 ISSR548a 4.35 0.14 -2.91 e03 20 RZ588 3.82 0.15 2.52 e07 Ninter 2 RZ876 ~ RZ962c RZ962c 4.97 0.20 -0.11 e01, e02, e05 10 TCD52 4.61 0.16 -0.13 e04 un ISSR842d 3.51 0.17 -0.11 e01 Inter1 13 RZ69 ~ RZ251 RZ69 4.20 0.22 0.62 e02 un CNLT17 4.61 0.24 0.58 e02 un CNLT142-T03 3.95 0.18 -0.30 e03, e07 un RZ961 3.66 0.33 0.84 e04, e08 Inter2 1 RZ909a 4.30 0.14 -0.35 e07 3 TCD95 ~ ISSR549a TCD95 4.72 0.21 -0.75 e06, e07 7 CNLT145 3.02 0.14 -0.57 e08 10 CNLT78 3.31 0.15 -0.51 e01 13 RZ467a ~ RZ69 RZ69 3.80 0.26 0.78 e01, e02 un CNLT12 4.46 0.34 0.71 e02, e03 un RZ961 3.53 0.16 0.38 e07 Dia 8 ISSR548a 3.11 0.15 -0.07 e03 Crush2 2 BCD1087a 3.08 0.14 0.49 8 ISSR548a 5.31 0.17 -0.48 a See Methods, designations of each trait b Flanking markers within the significance threshold at each border of the QTL range in the most significant experiments c Peak marker is the marker closest to the peak LOD score if QTL covered more than two loci. d Positive value of additive effect (Add) means the increased effect for the QTL was caused by the E. pilosa (30-5) allele e See the legend of Table 1, designations of each experiment Table 3: QTLs detected by composite interval mapping in the RIL population from the cross 'E. tef × E. pilosa (30-5)' (Continued) BMC Plant Biology 2007, 7:30 http://www.biomedcentral.com/1471-2229/7/30 Page 7 of 13 (page number not for citation purposes) Molecular linkage map with positions of QTLs for 19 traits on tef RIL population; E. tef × E. pilosa (30-5)Figure 1 Molecular linkage map with positions of QTLs for 19 traits on tef RIL population; E. tef × E. pilosa (30-5). The genetic distance in centimorgans (cM) is given on the left at the top. Six linkage groups are not presented because they did not contain significant QTLs. QTLs with the increasing effect contributed by E. pilosa (30-5) are in boldface. RZ154 TCD230a TB1 RZ909a RZ274 A1 TCD134b RZ387 TCD182b CSU60a RZ519a BCD207 TCD327a BCD349 BCD1087c BCD944b RZ467b RZ490 TCD99a CDO1160 TCD45** TCD99b CNLT119 KSUM152 RM159 1 ISSR842g BCD1087b* BCD1087a* ISSR836a* CNL78a CNL53 ISSR547 CDO78* CNLT146-T04a* BCD880 PALa* RZ876 RZ962c ISSR811c RM106 TCD273 2 TCD35 RM170a CDO20 TCD248* TCD95* ISSR549a CSU38 PRSC1_022 RM124a RZ444a RZ909b CNLT130 RZ444b 3 CNLT49a CNLT49b ISSR548b* RM170b RM176 TCD308 ISSR549b CNLT85 ISSR842b ISSR841b TCD219 6 ISSR811b ISSR840a CNL78b ISSR841c RM124b CNLT145 RZ698a KSUM195* inf14* TCD230b 7 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 Inter2 PedL Lodg Crush2 GY GY SB PSWt MT PWt SB CulmL CD1 CD2 PanL PH Ninter PedL PSWt GY SB CulmL Inter2 GY 100sw CulmL GY SB PanL SB PSWt GY SB CulmL PedL PanL PH Inter2 ISSR840b** DupW124 18 PSWt GY RZ395 RM134 ISSR836b* 16 GY TCD503 RZ698b** RZ519b 19 PWt PSWt RZ588** RM142 20 PWt PSWt GY SB PanL PH lfm256 TCD424 21 GY PedL PALb ISSR810 TCD227b* TCD316 TCD111 TCD227a ISSR548a CDO38 TCD323 ISSR812 8 ISSR842h KSUM22 RM110a** RM110b CNLT62 CNLT112 9 DupW4 ISSR842e RZ166 TCD415 TCD397a 11 MD Lodg PWt PSWt GY SB Lodg CulmL PanL PH Dia Crush2 PedL TCD52 CNLT78 CNLT41a CNLT41b ISSR842c TCD327b TCD327c 10 PWt PSWt SB Ninter Inter2 100sw SB PedL SB CulmL RM124c RZ467a RZ69* RZ251* CNLT65 13 Inter1 Inter2 HD PSWt CulmL CD1 PanL QTLs with unlinked loci; BCD944: Lodg, PedL CNLT12: PedL, Inter2 CNLT17: Inter1 CNLT127-T04: 100sw CNLT142-T03: PedL DupW216: PedL KSUM222: 100sw, CulmL RZ961: Inter1, Inter2 TCD134a: Lodg TCD182a: Lodg ISSR842d: PedL, PanL, Ninter BMC Plant Biology 2007, 7:30 http://www.biomedcentral.com/1471-2229/7/30 Page 8 of 13 (page number not for citation purposes) 1 st and 2 nd internode length (Intet1 and Inter2) Three and seven QTLs were identified for Inter1 and Inter2, respectively. These QTLs overlapped in LG13 where the R 2 was about 0.24, and longer internode length resulted from the E. pilosa (30-5)allele. The unlinked locus RZ961 was also associated with both of these traits. Crown diameter (Dia) Only one QTL, ISSR548a in LG8, was detected for Dia. This locus was associated with QTLs for nine different traits; PWt, PSWt, CulmL, PanL, PH, GY, SB, Lodg and Crush2 (Fig. 1). Most of these QTLs were unique to the DZBS location. Kaye Murri alleles increased crown diam- eter. Crushing strength at the 2 nd internodes (Crush2) Two QTLs were identified for Crush2. The traits of RPR and Crush were measured to evaluate the strength of culm in order to evaluate lodging resistance. However, QTLs for Crush2 (BCD1087a and ISSR548a) were not co-localized with QTLs for Lodg. RPR1, RPR2 and Crush1 did not show phenotypic variances among lines thus, QTL analy- ses were not available. Discussion Single marker analysis (SMR) detects associations between individual markers and traits; therefore, it does not require a genetic map to be applied. In this study we used SMR for a preliminary test of significance of all pol- ymorphic markers. For the loci that mapped into linkage groups [10], composite interval mapping (CIM) could be applied for detection and mapping of QTLs. Permutation tests were conducted to establish significant thresholds for CIM, reducing the chance of reporting false QTLs. In addi- tion, multiple-trait analysis (MT-CIM) was used to ana- lyze QTL over experiments, for detection of loci that consistently affected the phenotype across environments. The significant QTLs identified by all three analyses in common are presented herein (Table 3). Tef improvement has relied mostly on mass selection from landraces for the development of new varieties. The grain yield of tef has risen from 3,425 to 4,599 kg/ha over 35 years of breeding [11]. The average rate of yield increase per year for the period of 1960 to 1995 was esti- mated at 27.16 kg/ha (0.79%), using linear regression of mean grain yield of cultivars on year of release. This gain is similar to rates reported for spring barley, oat and spring durum wheat in Ethiopia [11]. However, the national average grain yield of tef is still about 0.8 t/ha [1] and is not competitive with that of other major grain crops. Grain yield was significantly correlated with all traits except PedL (Table 2). The associations of GY with HD, MD, PWt, PSWt, 100sw, SB, CulmL, CD1, CD2, PanL, PH, Inter1, Inter2 and Crush2 indicated that later maturing, taller, more vigorous, and larger plants resulted in more grain yield. Tefera et al. [7,8] showed most yield and yield related traits had high broad-sense heritability (H) in the population used in this study, and moderate to high H values were obtained in a population derived from an intra-specific cross. As expected, improvement of yield potential in tef has been associated with an increase of biomass yield and yield components. Among the 99 QTLs identified, 12 GY QTLs were detected in nine different linkage groups (Fig. 1). The map positions of the QTLs for yield related traits and SB on the same chromosomes over- lapped, thus supporting the significant phenotypic corre- lations (p < 0.001) (Table 2). Several chromosomal regions were associated with more than two traits indicating either linkage or pleiotropic effect. Clusters of QTLs (more than five QTLs) for various traits were identified on LG2, 3, 7, 8, 10, 13 and 20 (Fig. 1). Previous studies in cereal crops such as rice and wheat have also shown a clustering of agronomic QTLs [12-15]. The same chromosome region on LG21 was associated with positive and negative QTL alleles from E. tef for GY and PedL, respectively (Fig. 1), although the correlation between those two traits was non-significant (Table 2). The PedL QTL showed a similar relationship on LG10 with those of 100sw and SB which are yield related com- ponents. The association of two positive QTL effects in the same chromosomal region was reported for studies Table 4: Significant two-way interactions between marker loci determined using Epistat program. Trait Marker1 Marker2 MC-test Name Chr. Name Chr. GY CNL53 2 TCD227a 8 0.0028 GY lfm256 21 CNLT85 6 0.013 GY lfm256 21 RZ588 20 0.0031 GY TCD95 3 lfm256 21 0.0168 GY TCD95 3 TCD227a 8 0.0007 SB ISSR549a 3 CNLT78 10 0.0024 SB ISSR549a 3 ISSR841b 6 0.0019 SB ISSR549a 3 ISSR842e 11 0.0013 SB RM176 6 CNLT145 7 0.0016 SB RM176 6 ISSR549a 3 0.0005 SB RM176 6 ISSR549b 3 0.0027 SB RM176 6 ISSR842e 11 0.0001 SB RM176 6 RZ588 20 0.0046 SB RZ962c 2 ISSR842e 11 0.0001 SB TCD95 3 CDO38 8 0.0046 PedL CNLT12 un BCD944a un 0.0047 PedL CNLT12 un CNLT145 7 0.0003 PanL RM176 6 RZ588 20 0.0069 Inter2 CNLT145 7 RZ961 un 0.0021 Inter2 CNLT145 7 ISSR549a 3 0.0011 * Monte Carlo simulation to evaluate significance of interaction BMC Plant Biology 2007, 7:30 http://www.biomedcentral.com/1471-2229/7/30 Page 9 of 13 (page number not for citation purposes) involving O. rufipogon in rice [13,16]. The allele of O. rufipogon had a beneficial effect where the increasing effect for grain yield was linked to decreasing effect for plant height [13]. However, in some cases beneficial QTLs from O. rufipogon were associated with undesirable QTLs. For example, a QTL increasing panicle length QTL was in the same region as a QTL increasing the proportion of broken grains [16]. Where associations of desirable and undesira- ble agronomic QTLs are in the same chromosomal regions, careful selection would be needed to avoid unde- sirable characteristics in the derived lines. Epistasis is part of the genetic architecture of grain yield and other agronomic traits. Gene interaction has also been reported for a few phenotypic traits of tef [17-19] thus, it is not surprising to detect it for more complex quantitative characters in this study [20]. An analysis to identify the potential epistatic interactions between QTLs identified 20 marker loci resulting in 15 two-way interac- tions (Table 4). GY QTLs had five two-way interactions and TCD95 and lfm256 were actively involved in the epistasis. The most interesting interaction was between TCD95 on LG3, and TCD227a on LG8, for GY QTLs, because this was shown for SB QTL interaction as well (Fig. 1 and Table 4). In addition, QTLs on LG3 for GY and SB were detected in all three agro-ecology zones where agronomic traits were measured for this study. Likewise, the GY QTL (CNL53) on LG2 was detected across all three agro-ecologies and had significant interaction with TCD227a in LG8. Therefore, to improve grain yield, these three QTLs may need to be selected together. Genotype and environment interaction could influence the ability to detect QTLs, even though tef displays versa- tile agro-ecological adoption with good resilience to both low and high moisture stress. Individual QTLs were not consistently detected across environments, and inconsist- ent QTL detection has been observed and attributed to QTL × environment interaction, which has been com- monly observed in other grain yield QTL studies in cereal crops. Out of 12 GY QTLs, only two QTLs (LG2 and 3) were consistent across three agro-ecology zones. Three QTLs were detected in two agro-ecological zones: on LG7 (zones C2-1 and C3-3), LG8 (zones C2-2 and C3-3) and LG16 (zones C2-1 and C2-2). Even though, five GY QTLs were detected in multiple agro-ecology zones, there were no QTLs significant in all locations. The traits HD and MD as yield component traits are known to be sensitive to alti- tude because of day length. However, the HD and MD QTLs did not show discernible differences among differ- ent altitudes in this study. Assefa et al [21] demonstrated the diversity of yield related traits using 36 different germ- plasm populations collected from northern and central regions in Ethiopia corresponding to the same agro-ecol- ogy zones in this study. Regional differences in various traits of tef germplasm have been reported but altitude gradient regimes had no significant influence in affecting diversity levels in tef germplasm populations. Similar results were found in Ethiopian wheat, barley and sor- ghum germplasm [21]. Different soil types probably influenced QTL detection in this study. Two soil types were used in Debre Zeit: light soil (DZLS, Andosol, e04) and black soil (DZBS, Vertisol, e03 and e11). Plants were more vigorous and tall in the loamy Andosols, compared to the heavy textured Vertisol, even though the rainfall amount and temperature are the same for both soil types (Hailu Tefera, personal commu- nication). The QTLs for PWt, PSWt, and Ninter were iden- tified only at DZLS (e04), but the QTLs for 100sw, Lodg, PanL, and Inter2 were identified only at DZBS, 1999 (e03) (Table 3). Since those experiments were conducted at very similar conditions, it is likely that soil type was the major factor interacting with the QTLs. Teklu and Tefera [11] conducted a yield potential experiment in which 10 agro- nomic traits were examined for 11 tef varieties on two soil types. The most significant (p < 0.05) variety and soil type interactions were found for plant height and panicle length. Among four PH QTLs in this study, two were detected on LG7 (DZLS, e04) and LG8 (DZBS, e03) each. However, three QTLs for PanL were identified only in DZBS (e03), not in DZLS (Table 3). The environmentally sensitive QTLs for yield and yield components detected in this study clearly illustrate the importance of determining if QTLs by environment interactions are due to changes in magnitude or are crossover interactions before using MAS to select for QTLs. Identifying and selecting the proper allele at QTLs with crossover interactions requires careful evaluation in target environments. Inappropriate allele identification or selection could result in the indirect selection of QTL alleles with detrimental effects in some target environments. Low grain yield of tef is partly due to the low basic produc- tivity of currently available cultivars, together with suscep- tibility to lodging which has been the most serious agronomic problem. Lodging index showed positive and highly significant (p < 0.001) correlations with PSWt, 100sw, GY, SB and negative correlations with PedL thus, high yielding RILs tended to lodge (Table 2). Two of the Lodg QTLs, on LG8, were associated with PH, GY and yield related traits, and the other three QTLs were inde- pendent of yield related traits (Fig. 1). The positive corre- lation of lodging with yield and other important yield component traits indicates that improvement of lodging resistance in tef will be a challenging issue for a breeder. Of five Lodg QTLs, all alleles causing more lodging were from the tall, high yielding and more lodging resistant parent, Kaye Murri compared to E. pilosa (30-5) (lodging score 65.13 vs 81.50) (Table 1). This results from the unu- BMC Plant Biology 2007, 7:30 http://www.biomedcentral.com/1471-2229/7/30 Page 10 of 13 (page number not for citation purposes) sual patterns of correlations of several traits differentiating the cultivated and wild parents of this cross. The weak or non-significant correlations of Lodg with CD1, CD2, PedL, PanL, PH, Inter1, RPR1, RPR2, Crush1, and Crush2 were counterintuitive. On the other hand, CulmL Ninter, and Inter2, were positively correlated while Dia was nega- tively correlated with Lodg as would be expected. The lack of significance of the negative correlation coefficients with RPR and Crush traits can be attributed to the small number of replicates and environments as well as the dif- ficulty in measuring those traits. However, field observa- tions of the wild and cultivated parent suggest that the very thin culms, small crown diameter, and weak straw of the wild parent, rather than plant height, are the traits contributing most to its lodging susceptibility. Several studies have found that QTLs for lodging and plant height are linked or located in the same chromosomal regions and could be used as indirect selection parameters for bar- ley [22], rice [23], wheat [12], maize [24] and Italian rye- grass [25]. However, a reduction in plant height to improve lodging resistance may reduce the photosyn- thetic capacity of a canopy. In addition, the susceptibility to lodging differed among cultivars with similar plant height in wheat and rice [26,27]. Other factors such as stem cellulose or lignin content are related to stem rigidity [28] but were not measured in this study. One of the lignin biosynthesis genes, PAL (Phenylalanine ammonia- lyase from rice, X16099) co-localized with Lodg QTL in LG8 (Fig. 1) suggesting that it may be a candidate gene for this trait. The development of inter-specific populations is one strategy to broaden the genetic diversity of cultivated crops and to identify QTLs associated with beneficial traits, such as yield, grain quality and disease resistance [29]. E. pilosa (30-5) alleles had an agronomically benefi- cial effect on 27 out of the 99 (27.3%) QTLs detected in the present study, including HD, PWt, PSWt, GY, SB, CD1, PedL, PanL, PH, Inter1, Inter, and Crush2. This propor- tion is similar to that reported by Septiningsih et al [30], where 33% of the alleles from the wild O. rufipogon pre- sented favorable effects compared to O. sativa alleles. However, it is lower compared to the 53% reported by Thomson et al [15], with the same species. There were two QTLs identified on LG18 and LG20 with an increase in yield from the E. pilosa (30-5) alleles (Figure 1). The QTL on LG18 was not linked to any known undesirable QTLs and the E. pilosa (30-5) allele would be directly useful for developing breeding materials. However, the GY QTL interval (less than 10 cM) in LG20 was associated with a large increase in plant height, resulting in lodging. The GY QTL in LG20 may still be useful if the negative linkage can be broken or counteracted by other QTL reducing plant height. If markers can be successfully used to reduce link- age drag, the positive QTLs from E. pilosa (30-5) will be potentially useful for improving cultivated tef. Therefore, this study suggests that E. pilosa (30-5), and possibly other wild accessions, could be useful for diversifying the culti- vated tef germplasm pool. Conclusion The primary objective of this study was to determine the number and location of QTLs for important agronomic traits in tef. An inter-specific population was used to map 99 QTLs for 19 traits across 15 linkage groups. The inter- actions of genotypes and environments among QTLs were reported here to evaluate alleles for target breeding envi- ronments. The results of this QTL study are a first step towards the design of a marker-assisted selection program for tef improvement. Methods Mapping population construction Two hundred recombinant inbred lines (RILs) derived from individual F2 plants of the cross E. tef cv. Kaye Murri and E. pilosa (30-5) were developed using single seed descent method at the Debre Zeit Agricultural Research Center (DZARC), Ethiopia. The cultivar Kaye Murri is characteristically later maturing, thick culmed, tall in stat- ure, has a compact panicle structure, red lemma and white seed color. E. pilosa is early maturing, thin culmed, much shorter in stature, and has a loose panicle structure, exten- sive seed shattering, white lemma and dark red/brown seed color. These lines were phenotyped under field con- dition at three locations in Ethiopia in 1999. Of the 200 RILs, 181 lines survived across the three locations to gen- erate phenotypic data. Moreover, some lines which showed mechanical contamination were further elimi- nated and 162 RILs were considered for a subsequent phe- notyping in 2000. Ninety four RILs were used for construction of the linkage map of E tef cv. Kaye Murri × E pilosa (30-5) [10] and those RILs were used for QTL anal- yses reported in this study. Field trials Twenty-two traits were evaluated at eight different loca- tions in Ethiopia during the two-year period. In 1999, 200 RILs were planted in a randomized complete block design (RCBD) with four replicates at three locations (Akaki, Ale- mtena, and Debre Zeit Black Soil). Because of missing plots only 181 lines survived and were common across the three locations. In 2000, 162 lines were planted in a RCBD with two replicates at each of eight locations (Akaki, Alemtena, Debre Zeit Black Soil, Debre Zeit Light Soil, Denbi, Melkasa, Chefe and Holetta). The detailed information on field practices such as size of pots, polli- nation, fertilizer application etc. was described in Tefera et al. [7]. The eight locations were chosen based on their rep- resentation of the three major agro-ecosystems of tef in Ethiopia [31]. The humid zone (C1) in the Western [...]... linkage map for tef [Eragrostis tef (Zucc.) Trotter] Theor Appl Genet 2006, 113:1093-1102 Teklu Y, Tefera H: Genetic improvement in grain yield potential and associated agronomic traits of tef (Eragrostis tef) Euphytica 2005, 141:247-254 Borner A, Schumann E, Furste A, Coster H, Leithold B, Roder M, Weber W: Mapping of quantitative trait loci determining agronomic important characters in hexaploid wheat... length in cm last node and the bottom of the panicle (13) Panicle length (PanL): in cm from the base of the panicle to the tip (14) Plant height (PH): determined as the combined total of the culm length and panicle length (15) Number of internodes (Ninter): the total number of internodes on the plant (16 and 17) 1st Internode Length (Inter1) and 2nd Internode length (Inter2): measured as the length in. .. lines of Eragrostis tef × E pilosa J Genet and Breed 2003, 57:21-30 Tefera H, Assefa K, Hundera F, Kefyalew T, Teferra T: Heritability and genetic advance in recombinant inbred lines of tef (Eragrostis tef) Euphytica 2003, 131:91-96 Paran I, Zamir D: Quantitative traits in plants: beyond the QTL Trends Genet 2003, 19:303-306 Yu J-K, Kantety RV, Graznak E, Benscher D, Tefera H, Sorrells ME: A genetic linkage... morphological and agronomic traits in tef (Eragrostis tef) J Genet and Breed 2002, 56:353-358 Ketema S: Tef (Eragrostis tef) : Breeding, agronomy, genetic resources, utilization, and role in Ethiopian agriculture Institute of Agricultural Research, Addis Abeba, Ethiopia; 1993 Wartson L, Dallwitz WJ: The grass genera of the world Wallingford, Oxon, UK: CAB International; 1992 Ingram AL, Doyle JJ: The origin and... mapping of yield and yield related QTLs from an Indian accession of Oryza rufipogon BMC Genetics 2005, 6:33 McCartney CA, Somers DJ, Humphreys DG, Lukow O, Ames N, Noll J, Cloutier S, McCallum BD: Mapping quantitative trait loci controlling agronomic traits in the spring wheat cross RL4452 × 'AC Domain' Genome 2005, 48:870-883 Thomson MJ, Tai TH, McClung AM, Lai X-H, Hinga ME, Lobos KB, Xu Y, Martinez... Biology 2007, 7:30 regions of Ethiopia has a tef- growing period of more than 150 days and a growing season rainfall of more than 850 mm The wet semi-arid (C2) in the Central parts of the country is subdivided into two minor areas, high altitude (C2-1) more than 1900 masl and low altitude (C2-2) with 1700–1900 masl These areas receive a growing season rainfall of 450–850 mm and the growing period is between... JW: Inheritance of phenotypic traits in Tef: Seed color The Journal of Heredity 1989, 80:65-67 Berhe T, Nelson LA, Morris MR, Schmidt JW: Inheritance of phenotypic traits in Tef: Panicle form The Journal of Heredity 1989, 80:67-70 Bonaldo MF, Lennon G, Soares MB: Normalization and subtraction: two approaches to facilitate gene discovery Genome Res 1996, 6(9):791-806 Tefera H, Peat WE: Genetics of grain... describes the lodging index as the sum of the product of each scale of lodging (0–5) and its percentage divided by five (9) Culm length (CulmL): length in centimeters (cm) from the crown to the base of the panicle (10 and11) Culm Diameter at the 1st Internode (CD1) and Culm Diameter at the 2nd Internode (CD2): width in cm at the middle of the first and second basal internode, respectively, using a caliper... evaluated for 22 traits during the 1999 and 2000 growing seasons (Table 1) Ten plants per line were randomly selected at physiological maturity, and the following measurements were taken: (1) Days to heading (HD): number of days from planting to 50% of the plants in the plot showed panicle emergence (2) Days to maturity (MD): number of days from planting to the day when 50% of the plants in the plot reached... evolution of Eragrostis tef (Poaceae) and related polyploids: Evidence from unclear waxy and plastid rps 16 Am J Botany 2003, 90(1):116-122 Ayele M, Dolezel J, Van Duren M, Brunner H, Zapata-Arias FJ: Flow cytometric analysis of nuclear genome of the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter] Genetics 1996, 98:211-215 Tefera H, Assefa K, Belay G: Evaluation of interspecific recombinant inbred lines . 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. BioMed Central Page 1 of 13 (page number not for citation purposes) BMC Plant Biology Open Access Research article QTL mapping of agronomic traits in tef [Eragrostis tef (Zucc) Trotter] Ju-Kyung. diameter of 1 st internode b culm diameter of 2 nd internode c measurement of penetration strength in 1 st internode rind d measurement of penetration strength in 2 nd internode rind e measurement
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