Ethiopia is the second largest sorghum producer in Africa, after the Sudan and first among countries that have contributed many germplasm collections to the world collections of sorghum at both International Crop Research Institute for Semi-Arid Tropics (ICRISAT) and Griffin by National Plant Germplasm System (NPGS) (Demeke, 2013). It is one of the most important staple cereal crops after tef [Eragrostis tef (Zucc.) Trotter.] and maize (Zea mays L.) and holds third largest share of total cereal production with tef, maize, sorghum and wheat (Triticum aestivum L.) accounting for about 24.0, 16.8, 14.6, and 13%, respectively. Being an indigenous crop to Ethiopia, it is cultivated in almost all regions by subsistence farmers for various uses including as food and feed, house and fence construction, and prepare local beverages (CSA, 2014).

Though it was difficult to determine when and where sorghum domestication occurred, different studies suggested Ethiopia as a center of origin of sorghum due to the wide variation of the crop (McGuire, 2008; Vavilov, 1951). These also enable Ethiopian sorghum landraces as a source for an important agronomic trait including resistance to pest, sorghum midge (Contarinia sorghicola), and high lysine and protein contents (Fetene et al., 2011; Council, 1996). Sorghum had five basic races; namely bicolor, caudatum, durra, guinea and kafir. The entire races were differentiated morphologically based on their inflorescence, grain and glumes (Harlan and Wet, 1972). Clarissa et al. (2013) also described the geographic pattern of distribution of each race appears following the topography and climate variation present in Ethiopia. All the basic sorghum races except kafir also reported in Ethiopia. Accordingly, sorghum race durra is the main crop of the eastern highland region and mid elevation terrace of the north, while caudatum race is grown primarily in hot, dry valleys and lowland savannas in the south and west of Ethiopia. The intermediate race durra-bicolor predominates in the southwestern highland region, where cooler temperature and rain are higher than eastern and northern region. In contrast, bicolor and guinea races represent a very small part of Ethiopian sorghum diversity and both are mostly found in the Rift Valley region (House, 1985).

The diversity studies involving Ethiopian germplasms indicated the presence of huge genetic and morphological variations within their regions of origin and adaptation zones. Gebrekidan (1981) classified sorghum adaptation zones as: lowland (<1600 m above sea level (masl)), intermediate (1600-1900 masl) and highland (>1900 masl) in Ethiopia (Gebrekidan, 1981). Qualitative and quantitative studies, in addition to RAPD, AFLP and SSR markers utilized by different scholars also revealed the same amounts of variations among their collections (Ayana and Bekele, 1998; Geleta et al., 2006; Cuevas and Prom, 2013). SSR marker studies from Eritrean, Eastern Kenya, Benin and Zambia collections also showed presence of wide genetic diversity of sorghum bicolor in Africa mainly in Eastern regions (Tesfamichael et al., 2014; Catherine et al., 2016; Antoine et al., 2015;  Ng'uni et al., 2011). The existence of imbalanced sorghum races in the sample collections of the different studies from Ethiopia might contribute to the overall observed genetic variation (Cuevas and Prom, 2013). Ethiopian Biodiversity Institute (EBI) collected large numbers of farmers’ landraces though they do not have a racial category. Antoine et al. (2015) also recommended research on genetic diversity to integrate both botanical races and morpho-physiological characteristics of the crops for better preservation of sorghum genetic resources. In addition, morphological studies involving germplasms from Ethiopia and Eritrea show the greatest share of variation observed were carried by their panicle compactness and shape, which is 31% (Ayana and Bekele, 1998). Therefore, racially partitioned diversity studies among founding major basic races and representative of the whole collections of Ethiopian adaptation zones were lacking. Hence, the present study aimed at analyzing the genetic variation within basic Ethiopian sorghum races.

Germplasm collections

The accessions used for this study were landrace accessions collected by EBI. A total of 107 sorghum landrace accessions were selected based on phonological evaluation of inflorescence and spiklet types at their maturity time in order to define the racial classifications in 2015/2016 cropping season at Arsi Negelle Research Station based on their passport data. The materials were received and planted along with other germplasm by Melkassa Sorghum Improvement Program. In addition to the difference in head morphology, geographical distribution of the sorghum races across the country were considered for selection of their adaptation zones. All basic sorghum races except kafir and from the intermediate types, widely distributed durra-bicolor were included. The selected 107 sorghum landrace collections were grouped based on their source of origins into 12 populations, which each contained 9 landrace accessions for DNA extraction and genotyping study.

DNA extraction and PCR amplification

The seeds of collected sorghum genotypes were planted at National Agricultural Biotechnology Research Center (NABRC), Holetta, on seedling tray in greenhouse for germination. Genomic DNA was extracted from 2-week old seedlings using fresh leaves according to Xin et al. (2003) utilizing only two ordinary buffers that made the genomic DNA available for PCR amplification reactions. Approximately 50 mm2 single leaf sample per landraces was harvested to PCR plates for their DNA extraction. The two buffers include: Buffer A made from 100 mM NaOH and 2% Tween 20, which are made fresh from their stock solutions (10M NaOH and 20% Tween 20) and Buffer B consisting of 100 mM Tris-HCl and 2 mM EDTA, whose pH set to 2.0. Once the buffers are ready, the genomic DNA was extracted with the following procedures: (1) Approximately 30 mm2 leaf tissue transferred to 96-well plates; (2) 50 µL buffer A added and incubated for 10 min at 95°C in thermo cycler; (3) 50 µL buffer B was added and mixed at moderate speed; (4) Aliquot PCR mixture to 96-well plates at a reaction volume of 20 µL/well; (5) and finally transfer approximately 1.5 µL DNA from the crude DNA plates to PCR plates with a 96-pin applicator (Figure 1).

Twelve polymorphic SSR primer pairs (Table 1) were selected for genotyping the selected 107 sorghum landraces. PCR amplification was carried out in 20 µL reaction volume containing 1.5 µL crude genomic DNA, 2.25 µl PCR buffer with MgCl2 (17.5 mM), 1.8 µl of dNTPs (10 mM), 0.45 µl each of forward and reverse primers (10 mM), 0.133 µl of Taq Polymerase (5U), 0.1% BSA (Bovine Serum Albumin) (w/v) and 1% PVP (w/v). The amplifications were carried out with thermo cycler programmed for initial denaturation at 94°C for 15 min, the second denaturation at 94°C for 30 s, annealing at 50°C for 1 min, extension at 72°C for 2-min, final elongation at 72°C for 20 min and holding temperature at 4°C until conclusion. PCR products were analyzed by loading the 3 µl PCR products along with a 3 µL loading dye mixed with Gel Red (at a ratio of 1000:1) using 3.5% agarose gel electrophoresis run with 100 V for 3 h along with DNA Ladder (500 bp bioline Hyperladder V).  

Marker polymorphism

A total of 110 alleles were separated using 12 SSR markers (Table 2). The number of alleles per marker ranged from 5 (xtxp298 and xtxp258) to 18 (xtxp211) with an average of 9.2 alleles per locus. Xtxp298 and xtxp012 hold the lowest and highest number of genotypes (6 and 19, respectively) with an average of 15.8 genotypes per locus. The allele frequencies varied from 0.005 for marker xtxp285 to 0.648 for xtxp312 with an overall average frequency of 0.109. The mean number of alleles per locus observed in this study was higher than similar SSR studies with accessions from North Eastern Benin, 7 (Antoine et al., 2015), Zambia, 4.4 (Ng'uni et al., 2011), Eastern Kenya, 5.05 (Catherine et al., 2016), Eritrea, 4.8 (Tesfamichael et al., 2014) and Egypt, 7.3 (El-Awady et al., 2008), and Ethiopian collections in combination with other countries (Agrama and Tuinstra, 2003). However, it is lower than Cuevas and Prom (2013) population structure and diversity study for 137 Ethiopian germplasm conserved at USDA-ARS National Plant Germplasm System, that is 14 per locus. Out of the total 110 alleles, specifically 42 alleles had frequencies below 0.05 (rare alleles), 22 alleles had a frequency within 0.05 to 0.10 (common alleles) while the rest 46 alleles had frequency higher than 0.10 becoming an abundant allele. While across all races average number of frequencies ranged from 7.17 in intermediate durra-bicolor to 4.75 in race bicolor whereas mean gene diversity ranged from 0.32 (bicolor) to 0.22 (durra). Their mean number of alleles within the different races ranged from 7.17 (durra-bicolor) to 4.75 (bicolor). Likewise, their gene diversity ranged from 0.77 for durra-bicolor to 0.70 for durra, guinea and bicolor (not shown).

In general, the racial classification among S. bicolor could be used for in-situ and ex-situ conservation and genetic dissimilarity with their respective agronomic characteristics favors the future crops germplasm breeding programs. In this regard, Ethiopian sorghum races were structured into four major clusters according to their racial difference except for race bicolor and guinea. Bicolor found being scattered within other groups. The greatest genetic distance found between bicolor and caudatum while between the Ethiopian zones, central and south Tigray and east Harerghe. There was also a huge variation observed among the populations of both racial classification and ecological zones (61.38 and 55.17%, respectively). The greater mean number of alleles per locus (9.2) and PIC value of 0.76 in the present study also indicate the presence of high genetic diversity among Ethiopian sorghum collections and the discriminatory power of the selected markers. There is also a moderate levels of genetic differentiation among races (FST= 0.07) and Ethiopian sorghum producing zones (FST=0.13). Hence, racial groups could also be used as representation of the germplasm collection along with the commonly known diverse agro-ecological and zonal collections and their adaptation zones.

In line with the present study, the future research areas should include molecular studies along with the morphological components using markers linked to specific agronomic traits to enable the use of racial groupings within sorghums in its breeding areas. In addition, classification of the national sorghum germplasm collections according to their race was also needed since the Ethiopian landraces have been used as the source of important traits.

The authors have not declared any conflict of interests.

The authors thank the technical staff at National Agricultural Biotechnology Research Center, Holetta for generously helping technically throughout the time in their laboratory and Melkassa management staff’s in easing the work with financial issues.