High-Throughput Discovery of Mutations in Tef Semi-Dwarfing Genes by Next-Generation Sequencing Analysis

Abstract

Tef (Eragrostis tef) is a major cereal crop in Ethiopia. Lodging is the primary constraint to increasing productivity in this allotetraploid species, accounting for losses of 15-45% in yield each year. As a first step toward identifying semi-dwarf varieties that might have improved lodging resistance, an 6· fosmid library was constructed and used to identify both homeologues of the dw3 semi-dwarfing gene of Sorghum bicolor. An EMS mutagenized population, consisting of 21,210 tef plants, was planted and leaf materials were collected into 23 superpools. Two dwarfing candidate genes, homeologues of dw3 of sorghum and rht1 of wheat, were sequenced directly from each superpool with 454 technology, and 120 candidate mutations were identified. Out of 10 candidates tested, six independent mutations were validated by Sanger sequencing, including two predicted detrimental mutations in both dw3 homeologues with a potential to improve lodging resistance in tef through further breeding. This study demonstrates that highthroughput sequencing can identify potentially valuable mutations in under-studied plant species like tef and has provided mutant lines that can now be combined and tested in breeding programs for improved lodging resistance.T EF [Eragrostis tef (Zucc.) Trotter] is an allotetraploid (2n = 4X = 40) species that is native to Ethiopia. Tef belongs to the grass subfamily Chloridoideae, a lineage that has received very little research attention. Tef has an estimated genome size of 713-733 Mbp (Ayele et al. 1996), which is 60% larger than the rice genome.As a staple food in Ethiopia, covering more acreage than any other crop in that nation, tef possesses several advantageous characteristics, including excellent storage properties and a very nutritious seed with excellent protein and mineral composition (Stallknecht et al. 1993). In addition, tef plants grow well under extreme environmental conditions such as drought and water-logging. However, compared to other crops, the average yield of tef is quite low, at an average of 700 kg ha 21 (Central Statistics Authority 2005). Tef has a tall and tender stem that is susceptible to lodging caused by wind and rain. As a consequence, the yield of tef is reduced 15-45% each year, depending on the weather and the variety. Furthermore, an increased incidence of lodging is associated with fertilizer application. Using QTL to improve lodging resistance indicated a positive correlation between lodging and yield, such that the most lodging-resistant progeny also had the lowest yield (Yu et al. 2007). This is probably because such traits as larger seed size and higher seed number create a heavier panicle that leads to a greater likelihood of lodging.In other cereal crops (Peng et al. 1999;Sasaki et al. 2002;Multani et al. 2003), semi-dwarf varieties have been found to provide both lodging resistance and higher yields. As shown in the earliest studies (Quinby and Karper 1954;Jennings 1964;Walcott and Laing 1976;Foster and Rutger 1978), semi-dwarf varieties have both strong s...

Semi-dwarf tef lines for high seed yield and lodging tolerance in Central Ethiopia

“…Lodging, wider uses of low yielding cultivars, drought and different other stresses are the major factors contributing to the low productivity of tef . Lodging affects both the grain yield and quality of tef, and is found to reduce grain yield by approximately 15 to 45% (Ketema, 1993;Zhu et al, 2012), depending on the weather condition and inherent nature of the variety. Hence, lodging is a crucial problem to address as long as tef production and research is concerned.…”

Perspectives on the Application of Next-generation Sequencing to the Improvement of Africa’s Staple Food Crops

“…The use of multidimensional pooling of DNA samples enables screening of DNA pools for multiple independent mutations in any target gene using NGS, which provides a cost-effective assay. This has led to the discovery of rare mutations in rice and wheat, termed TILLING by sequencing [92], tef [93], and in animals [94]. Different sample pooling schemes for NGS, which further enhance the power of NGS in processing multiple samples in parallel have been developed [95].…”

Dwarfing of Breadfruit (Artocarpus altilis) Trees: Opportunities and Challenges

“…In these species, most genes are represented by multiple homoeologous copies, and phenotypic effects in a single mutant may be masked by wild-type homoeologues present in another genome. This not only raises doubts as to the effectiveness of the facile screen of plant height for the outcome of mutagenesis, but also implies that in some cases, combined mutations by developing double or triple mutants through genetic crosses is required [123].…”

“…These include orthologs of the wheat with reduced height-1 rht gene and the rice semi-dwarf sd1 gene, both encoding DELLA proteins, an important components of GA signalling, and those of sd1 encoding key enzymes in the GA biosynthesis pathway [25,29]. Using this high throughput platform, Zhu et al recently performed screen at these dwarfing candidate genes to identify semi-dwarf mutants in Tef (Eragrostis tef), an under-studied species [123]. Following sequenced with Roche 454 sequencing technology, they identified several mutant lines with mutation in the target regions from an EMS-mutagenized population consisting of 21,210 individuals [123].…”

“…Using this high throughput platform, Zhu et al recently performed screen at these dwarfing candidate genes to identify semi-dwarf mutants in Tef (Eragrostis tef), an under-studied species [123]. Following sequenced with Roche 454 sequencing technology, they identified several mutant lines with mutation in the target regions from an EMS-mutagenized population consisting of 21,210 individuals [123].…”