Tef (Eragrostis tef) Recombinant Inbred Line Variety Development for High Potential Areas of Ethiopia

The national average yield of Tef is low at 1.75 t ha. This is partially due to lack of high yielding Tef genotypes for different Tef growing areas. Therefore, the present study was designed to develop high yielding, and desirable quality of improved Tef varieties suitable for high and optimum potential farming systems. Eight recombinant inbred lines (RILs) developed from a cross of DZ-01-353 x kaymurri plus two checks were laid out in a randomized complete block design using four replications in multi-environments for two years (2013 and 2014) to see the effect of genotypes, environments and GEI. ANOVA from additive main effect and multiplicative interaction (AMMI) for grain yield revealed highly significant (p<0.01) effect for genotypes, environments, and genotype by environment interaction (GEI. The effect of environment, genotypes and genotype by environment interaction accounted for 81.49, 3.98 and 14.15% of the total sum squares, respectively. A large sum of squares for environments indicated that the test environments were diverse with large differences among environmental means which causing most of the variation in grain yield. Therefore, results of combined data analysis across locations and over the years showed that variety DZCr429 (RIL 125)/Negus/ performed better and stable across five locations over two years among tested genotypes. Thus, variety Negus was identified and released as best promising Tef variety for production in high and optimum potential tef growing areas in the country. This variety should be used in similar agro ecologies to increase grain yield productivity and ensure food security in the country.


Introduction
Tef is the major Ethiopian cereal grown on 3.02 million hectares annually [4], and serving as staple food grain for over 73 million people in the country. It constitutes 30% of the total area allocated to cereals and contributes more than 20% of the total cereals production [4].
Tef hybridization began following the discovery of Tef flower opening time and consequent to that the artificial surgical binocular-aided hand emasculation and pollination technique by [24] The average annual genetic gain in tef grain yield was estimated as 0.8% from 1970 until 1995 [27] and o.58% from 1970 until 2012 [7] under lodging controlled and uncontrolled conditions respectively. Tef varieties developed through hybridization showed a yield advantage of 9.5% over those developed through direct selection from farmers' variety. In Tef improvement effort grain yield constituted the highest priority [15] Yield is a complex quantitative trait often affected by genotype, environment and genotype by environment interaction (GEI). The differential responses of genotypes across environments occur because of differences in expression of different sets or the same set of genes in different environments [18, 8 & 10]. GEI complicates selection of any superior genotype across environments because it reduces the association between phenotypic and genotypic values [6].
Yield stability usually refers to a genotype's ability to produce high or low yield consistently across a wide range of environments [2]. For grain yield stability analysis, a genotype having minimum cultivar superiority value is considered the most stable genotypes [17].
Nevertheless, the national yield per unit area (at 1.75 tha -1 ) still remains low, some of the factors contributing to low yield of Tef are; lack of outstanding cultivars and lodging, both biotic and abiotic pressures. Despite low average national yield, the national Tef research program developed 49 improved Tef varieties [20] yet, from the released tef varieties achieved the potential yield of the crop. It shows there is higher difference between the potential of Tef and the actual yield called yield gap which is less than half. This is due to the limitation of well performing and stable tef genotypes in multi environments of Tef growing areas in the Country. In order to alleviate these problems, currently the breeding program is focused on the development of varieties with high yield and good kernel quality. The segregating generations are handled by modified bulk method, i.e., raising bulk families in F3-F4 generations that are derived from individual F2 plants, keeping in view the need to generate more variability through hybridization. Thus, the objective of the present study was to develop high yielding and stable white seeded recombinants inbreed Tef variety (ies) to the farming community.

Inbred Line Development
Hybridization/crossing between DZ-01-353 x kaymurri (RIL 125) was made in 2003. The purpose was to develop stable, high yielding; and farmers and consumers preferred Tef varieties for the high rainfall and optimum moisture (high potential) areas of the country. In other words, it was targeted at developing varieties with high yielding potential and better quality than the improved contemporary standard check varieties Quncho [14] and Dagim [23]. DZ-01-355 was selected as maternal parent for its high yielding ability and wide adaptability. Likewise, Kaymurri was selected as a parent for its extra white seed color, relatively large kernel size, thick culm and vigorous growth habit. After a successful crossing rapid generation advancement up to two to three generations per year was made using off-season irrigation facilities. After homogeneity attained at F7 selection was made and transferred to observation nursery trial. Genotypes showed uniform and desirable traits selected for preliminary variety trial and from the preliminary variety trial those eight recombinant inbred lines showed good performance selected for multi-location trial. Among tested genotypes, variety Negus [DZ-01-353 x kaymurri (RIL 125)] was selected by its performance and tested for variety verification trial in 2017 and then the national variety release technical committee approved it. Finally, Variety Negus was developed as a recombinant inbred line through an F2 derived single-seed descent method; and following series of multi-environment yield tests in various major Tef growing areas of the country.

Genotypes, Testing Site and Design
The field experiment was tested at five locations, Debrezeit light soil, Debrezeit black soil, Minjar, Holota and Adadimariyam The trial was conducted using a RCB design with four replications throughout the testing sites. The description of testing sites is presented in Table 1. The trial was evaluated on the plot size of 4 m 2 with ten rows of 2 m length throughout all trial sites and 1.5 m between replication, 1 m between plot, 0.2 m between rows distances were maintained. The varieties were assigned to plots at random within each replication based on the randomization table made in computer. As per the research recommendations of 15kgha -1 , equivalent to 6 g plot -1 of seeds was disseminated along the surface of each row by hand drilling. 60 kg P 2 O 5 and 40N per hectare for light soil, 60 kg N and 60 kg P 2 O 5 per hectare for black soil fertilizer was applied, respectively, all at planting while urea was applied two times, the first application two weeks after sowing and top dressed at tillering stage. Hand weeding was made three times during the crop growth stage. A variety verification trial was conducted at Minjar, Debre Zeit, Holeta and Adet on the trial station and in eight farmers' field during 2016/17 main production season.

Data Recorded
Data on agronomic yield and yield related traits were collected both on plot and individual plant base. Data on days to heading or panicle emergence using the sowing date as a reference, lodging index, grain and biomass yield were taken on plot bases. Days of heading and maturity were taken when each plot attained 50% heading (panicle emergency) and 90% physiological maturity respectively, and days were calculated beginning from the date of sowing. Lodging index was assessed using the method [3] by considering assessments of both the lodging degree or the angle of leaning on 0 (completely upright) -5 (completely flat on the ground) and the severity as the percentage of the plot stand manifesting each of the 0-5 degrees of lodging. Then, lodging index for each plot was taken as the product sum of the degree of leaning and the respective per cent severity divided by five. Grain yield of each plot was measured on clean, sun dried seed and the measured grain yield value (g) was converted to kilogram per hectare for data analysis. Plant height (cm), and panicle length (cm) were taken on the five individual samples of plants which were randomly taken from the central rows of each plot, and the averages of five sample plants were as used for analysis.

Data Analysis
Data from individual locations combined over two years and location by years were analyzed by using SAS version 9.0 (2002) software [21]. The analysis of variance for grain yield and yield-related traits for each location and over two years was analyzed by using a randomized complete block design Factorial ANOVA model [9]. The combined analysis of variance across the location was done in order to determine the differences between genotypes across location, over two years and their interaction. Bartlett's test, [13] was used to assess the homogeneity of error variances prior to doing combine analysis over location and years and variance effect were considered as significant and highly significant at P< 0.05 and P< 0.01, respectively. GEA-R (2015) version 2.0 was used for the stability analysis. Mean comparison using Least Significant Difference (LSD) was performed to explain the significant differences among means of genotypes and environments (location x year).

Analysis of Variance
ANOVA from additive main effect and multiplicative interaction (AMMI) for grain yield revealed highly significant (p<0.01) effect for genotypes, environments, and genotype by environment interaction (GEI) ( Table 2). The effect of environment, genotypes and genotype by environment interaction accounted for 81.49, 3.98 and 14.15% of the total sum squares ( Table 2), respectively. A large sum of squares for environments indicated that the test environments were diverse with large differences among environmental means which causing most of the variation in grain yield. This might be due to the presence of variation in temperature, rainfall, soil type, soil fertility, and moisture availability. The AMMI analysis also showed that the first interaction principal component (PC1) and second interaction principal component (PC2) explained 44.14 and 22.42 of the interaction sum squares, respectively. The significant interaction indicated that the genotypes respond differently across the different environments. The significant variability of Tef genotypes in the present study are in line with the previous findings [1,10,16,5,25]. As indicated in Table 3, the average grain yield across location over two years ranged from 1547±110 kgha -1 (RIL 89) at L2 (Debrezeit light soil) to 3208±264 kgha -1 (RIL 125) at L1 (Minjar (Table 3). Moreover, performances of genotypes were not consistent across five locations over two years. For instance, at L1 genotype RIL 125, at L2 genotype RIL 29, at L3 genotype RIL 125, at L4 genotype RIL 87 and at L5 genotype RIL 125 were the top ranking genotypes with mean grain yield of 3208±264, 2142±192, 2645±143, 3100±330 and 2795±147, respectively. Thus, such inconsistent in yield ranking from location to location indicated the presence of possible cross over GEI as described by [10,5].

Combined Analysis of Variance and Mean Performance of Genotypes Across Locations over Two Years
Combined analysis of variance also showed highly significant difference (<.01) effect of location and genotypes for days to heading, days to maturity, days to grain filling, plant height, panicle length. Alike, year showed significant effect in all traits recorded except days to maturity and plant height. Variability among Tef genotypes for different traits across locations/environment was reported by [10,12,26].
As indicated in Table 4 and Figure 1, the interaction of genotype by year by location was highly significant (p<0.01) for grain yield and significant (P <0.05) effect for days to heading and above ground biomass. Likewise, year by location were highly significant for all recorded traits. Similarly genotype by year was significant for plant height and panicle length. In contrast, the interaction of genotype by year was not significant for days to maturity, grain filling period, lodging index, grain yield and above ground biomass. This significant effect due to genotype, location, year and their interaction effect indicated that the genotypes, years and locations were divergent to show considerable variation in Tef traits. Therefore, the significance of GEI indicated that the relative performances of the genotypes were not consistent across the test locations and years that had different effects on the yield potential of the tef genotypes. This result is in agreement with the previous reports [5, 11 & 22] for yield related traits. Among the tested genotypes, RIL 125 was the highest yielder genotype with the mean grain yield of 2758 kgha -1 followed by genotype RIL 87 (2658 kgha -1 ) and genotype RIL 29 (2638 kg ha −1 ) respectively, whereas the lowest mean grain yield (2342 kg ha -1 ) and 2376 kkgha -1 were registered from Genotype RIL 89 and a local check ( Table 4). As indicated in the same Table 4, four genotypes scored highest grain yield over the grand mean (i.e., 2522 kg ha -1 ) and four candidate genotypes scored mean grain yield above the standard check variety Quncho. This finding is in agreement with the previous study with [5,11,27] who reported in yield variation among Tef genotype. The candidate genotype, Genotype RIL 125, was statistically high yielder than the other genotypes and showed 11.7% and 16.1% yield advantage over the standard check Quncho (2470 kgha -1 ) and local check (2376kgha -1 ), respectively. Therefore, this genotype has been verified in 2017 and visited by the national variety releasing technical committee. Accordingly, genotype RIL 125 has been officially released for its high yielding, medium maturity, very white grain color, and high adaptability in the high potential Tef growing areas of Ethiopia.
Variety Negus is white seeded, broad adaptability, medium maturity, high yielding Tef variety with grain yield advantage of 11.7% and 16.1% over the standard check (Quncho) and local check, respectively. Negus takes 50 days to head and 105 days to mature (Table 4). It is 94 cm tall in height with 37 cm panicle length (Tables 4). It has got yellowish lemma color, red anther color, loose panicle form and very white seed color. Negus has gained immense farmers' attention due to its yielding potential and stable performance, very white caryopsis color and good straw yield (straw yield is no less important than grain yield) at participatory variety selected Table 3. Grain yield (kgha -1 ) of ten Tef genotypes across locations over two years (2013)(2014)

Stability Analysis
Mean grain yield performance and its stability 10 Tef genotypes across five locations over two years are shown in Table 5 and Figure 2. The mean grain yield value of genotypes averaged over location by year indicated that genotype RIL 125 had the highest (2758 kgha -1 ), genotype RIL 89 (2343 kgha -1 ) and local variety (2376 kgha -1 ) the lowest grain yield, respectively. Genotype superiority with the small measured value indicates the more stable genotypes. Therefore, from the present study, genotype RIL 125 was the most stable and genotype RIL 89 was the most unstable genotypes, respectively. The comparison of variety Negus with the standard check variety Qunco has shown in Figure   3. From this Figure 3 the performance of Variety Negus was superior to standard variety Quncho in tested sites.

Conclusion and Recommendation
Genotype by location by year interaction has a key effect on crop variety development by complicating the release of varieties across challenging environments (location x year). Analysis of variance for every five locations and combined over two years showed significant differences among Tef genotypes, locations, years and year by location interaction (GEI) for grain yield and most of the yield-related traits. Likewise the three way interaction Genotype by location by year reveal significant difference for days to heading and above ground biomass and highly significant difference for grain yield. The significant interaction effects indicated the inconsistent performance of genotypes across the tested locations and seasons. Among the tested genotypes, RIL 125, RIL 87, RIL 29 and RIL 113 had mean grain yield above the overall mean grain yield of evaluated Tef genotypes. However, only the candidate genotype RIL 125 (Negus) had mean grain yield above the standard check variety Quncho.
Considering the 10 environments data (location x years) and field performance evaluation during the variety verification trial, the national variety releasing committee has approved the official release of candidate genotype, DZ-01-353 x kaymurri (RIL 125 ), with the vernacular name of "Negus" for high, medium and similar agro ecologies.