Genetic Relationship between Sweet Grain Sorghum and the Other Sorghum Types Cultivated in Burkina Faso Assessed with Nuclear Microsatellite Markers SSRs ()
1. Introduction
Sorghum is a very diverse species. In Burkina Faso, different types of sorghum (grain sorghum, sweet grain sorghum, sweet stalk sorghum, dyer sorghum.) are cultivated with associated agricultural practices. Several studies conducted on grain sorghum [1] [2] [3], sweet stalk sorghum [4], and sweet grain sorghum [5] [6], respectively, revealed the existence of genetic diversity within these sorghums. The co-evolution of crop diversity and local agricultural practices ensure their maintenance in agroecosystems [7] [8] [9]. In fact, multiple cropping of sorghum in the same field is a common agricultural practice in our countries, especially with the reduction of arable land. Sweet grain sorghum is generally cultivated in hut fields and sometimes in bush fields in association with other types of sorghum or cereals such as corn. This farmer management method ensures the maintenance of genetic variability in the cultivated plants, but also promotes significant gene flow between local cultivars and their close relatives [10]. Moreover, the different types of sorghum present significant inter-fertilization possibilities due to allogamy, which can reach 30% [11], and to their evolution often in the same geographical areas and in the same environments. Inter- and intra-fertilization of sorghum types in the context of selection and development of sweet grain sorghum raises the problem of its conservation and stability in its environment.
However, the genetic relationship between grain sorghum, sweet grain sorghum, and sweet stalk sorghum is not well known, which could constitute a constraint to the rational exploitation of the potential of each type of sorghum. Good knowledge of the relationships between the different sorghum cultivated would make it possible to consider improving one type of sorghum with another type while maintaining the characteristics of interest of the latter. Thus, the present study aims to further knowledge of the specific relationships among these sorghum. This includes 1) assessing the genetic diversity of each type of sorghum and 2) determining the genetic relationship between sweet grain sorghum and the other types of sorghum grown in Burkina Faso precisely grain sorghum and sweet stalk sorghum using microsatellite markers.
2. Material and Methods
2.1. Plant Material
The plant material consists of 34 sorghum accessions including 10-sweet grain sorghum, 10-sweet stalk sorghum, and 14-grain sorghum. Twenty-four accessions were obtained from the genebank of the “Laboratoire Biosciences” of “Université Joseph KI-ZERBO” and 10-grain sorghum accessions were received from the INERA Saria genebank (Burkina Faso).
2.2. Methods
2.2.1. DNA Extraction
Seeds of the 34 sorghum accessions were sown in pots in September 2018. These pots were stored in the experimental garden of our research Institute “Unité de Formation et de Recherche en Sciences de la Vie et de la Terre” of “Université Joseph KI-ZERBO”. For each accession, 100 mg of young fresh leaves (14-days-old) were collected and finely ground in 750 µl of pure water using a mortar. The grindings of each sample obtained were centrifuged at 10,000 RPM (rotations per minute) for 10 mins. At the end of this operation, the supernatant was removed and 200 µl of DNAzol was added to the pellet. The tubes containing the DNAzol and the pellet were then shaken to completely recover the pellet in the lysis buffer. The collected extracts were then, incubated at 65˚C in a water bath for 2 h. At the end of the incubation, the obtained extracts were cooled for a few minutes at room temperature of the laboratory and then centrifuged at 10,000 RPM for 15 mins. The supernatant was collected in eppendorf tubes and stored at −20˚C.
2.2.2. Characteristics of the SSR Markers Used
Fifteen microsatellite markers were used in this study. The distribution of these markers covers seven of the 10 chromosomes of the sorghum genome. These markers were chosen for their polymorphism revealed in previous studies of sorghum genetic diversity in Niger [12] [13], Mali [14], Burkina Faso [15] [16] [17], and from a global collection [18]. The characteristics of the molecular markers are presented in Table 1.
2.2.3. Amplification PCR
PCRs were performed in a final volume of 20 μl containing 1 μl of 3' primer (forward primer) at 0.2 µM, 1 μl of 5' primer at 0.2 µM (reverse primer), 9 µl of ultrapure water, 4 µl of PCR premix, and 5 μl of accession DNA. The PCR premix consisted of 0.4 µl of 10 mM concentration dNTPs, 2 µl of 10 X Buffer, and 0.4 µl of Taq polymerase containing 2U.
PCR amplification was performed following a program consisting of an initial denaturation phase at 94˚C for 4 mins, followed by a series of 35 cycles and a final extension at 72˚C for 4 mins. Each cycle included a denaturation phase at 94˚C for 45 s, a hybridization phase at the temperature (T m˚C) of each primer for 1 min, and an extension at 72˚C for 1 min 30 s.
2.2.4. Electrophoretic Migration and Band Reading
The amplification products were then subjected to electrophoresis at 100 V on a 2% agarose gel prepared with a 1x TBE solution. The migration time was 2 h in 0.5x Tris Borate EDTA buffer (TBE). Deposits were made in the presence of a molecular weight marker ranging in size from 50 bp to 1500 bp. The gel was then immersed in a 5% Bromide Ethidium (BET) solution used as a fluorescent developer for 15 mins. It was then rinsed with distilled water for 3 mins before being read under ultraviolet light from a model UVDI-254 trans-illuminator topped with a 10 mega pixel camera. Bands were identified on the basis of their position on the gel. Thus, a binary coding was used with 1 in case of presence and 0 in case of absence of band for each individual and for each primer tested.
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Table 1. List of microsatellite primers used and loci revealed.
Tm: Hybridization Temperature, Repeated motive: G: Guanine, C: Cytosine, A: Adénine; T: Thymine.
3. Statistical Analysis of Molecular Data
The assessment of genetic diversity using microsatellite markers (SSR) was carried out at three levels: intra-population diversity, inter-population diversity and the general structure of the collection.
Analysis intra-type genetic diversity of sorghum
To assess genetic diversity within each sorghum type, the Genalex software was used to estimate genetic diversity parameters such as the polymorphism rate (P), the average number of alleles per locus (A), total number of alleles per sorghum type (At), the number of effective alleles (Ae), the number of distinct or private alleles (AP), the expected heterozygosity (He) or Nei's gene diversity index (D), the observed heterozygosity (Ho), the Shannon diversity index (I) [19], and polymorphic information content (PIC) [20].
Analysis inter-type genetic diversity of sorghum
For the comparison of subpopulations defined according to sorghum types, the index of genetic differentiation between subpopulations (Fst) and the minimum Nei distance between pairs of genetic groups were calculated using Genetix software and Fstat software. Structuring was also obtained using the same software. The genetic similarity between sorghum types was tested by correspondence factor analysis (AFC).
4. Results
4.1. Level of Diversity of Nuclear SSR Markers Tested
The results of the genetic diversity assessment of the three cultivated sorghum types using the 15 polymorphic microsatellite markers are reported in Table 2. A total of 43 alleles were identified with numbers ranging from 2 (Xtxp40, Xcup02, Xcup11, Xcup63, Xcup62) to 3 (Xtxp145, Xtxp295, SbAGB02, Xtxp114, gpsb1123, Xtxp10, Xcup14, Xtxp57, Sb6-84, Xcup53) per locus. The number of effective alleles ranged from 2.68 (Xtxp295) to 1.13 (Xtxp114) with an average of 1.63 alleles. The expected unbiased heterozygosity (He) ranged from 0.20 for the sb6-84 locus to 0.50 for the SBAGB02 locus with an average of 0.43. As for the observed heterozygosity (Ho), its value ranged from 0 to 0.53 with an average of 0.14. Seven loci namely Xtxp40, Xtxp145, Xcup02, Xcup11, Xcup63, Xtxp114, Xcup62 showed zero observed heterozygosity values. The Shannon diversity index (I) ranged from 0.26 for the Xtxp114 primer to 0.98 for Xtxp295 with an average of 0.61. The potential for polymorphism information (PIC) ranged from 0.12 (Xtxp114) to 0.63 (Xtxp295). Figure 1 shows the migration profile obtained with the Xcup14 marker for 34 individuals
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Table 2. Diversity parameters of the 15 tested nuclear markers.
A: number of alleles per locus; Ae: effective number of alleles; Ho: observed heterozygosity; He: expected heterozygosity; PIC: Polymorphism Information Content; P: polymorphism rate; I: allelic Shannon diversity index.
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Figure 1. Migration profile obtained with the Xcup14 marker for 34 individuals. Legend: M: DNA Marker.
4.2. Genetic Diversity of Sorghum Accessions
4.2.1. Comparison of Genetic Diversity Parameters of Sorghum Types
The results in Table 3 revealed a low number of private alleles for sweet grain sorghum, sweet stalk sorghum and grain sorghum of 0.06 and 0.13, respectively. The level of diversity was significant within the different sorghum types with a total of 18 alleles detected for sweet grain sorghum and 22 alleles for sweet stalk sorghum and grain sorghum, respectively. All sorghum types showed higher expected heterozygosity (He) than observed. However, sweet grain sorghum had the lowest values of observed heterozygosity (Ho = 0.17), expected heterozygosity (He = 0.35), Shannon diversity index (0.5), and marker PIC (Polymorphism Information Content) (PIC = 0.31). Sweet stalk sorghum and grain sorghum on the other hand expressed similar or close values for the same diversity parameters.
4.2.2. Differentiation and Genetic Distance among Sorghum Types
The minimum Nei distance results (Table 4) showed a low level of differentiation among the three sorghum types studied. The highest Nei distance was observed between sweet grain sorghum and sweet stalk sorghum (0.12). For the genetic differentiation index by sorghum type pair, it was significant only between sweet grain sorghum and sweet stalk sorghum.
4.2.3. Structuring of the Diversity of All Accessions
The results of the spatial representation of the accessions structure through Correspondence Factorial Analysis (Figure 2) revealed a distribution of the accessions of the three types of sorghum into two large groups according to their genetic similarity in orthogonal space and according to the alleles present. Thus, a weak differentiation is observed between grain sorghum and the two other types of sorghum. The first group (G1) is made up of grain sorghum and sweet stalk sorghum accessions and the second group (G2) of grain and sweet grain sorghums.
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Table 3. Intra-type diversity level of sorghum using SSR microsatellite markers.
At: total number of alleles per sorghum type; Ae: effective number of alleles; Ho: observed heterozygosity; Ap: number of private alleles; He: expected heterozygosity; PIC: Polymorphism Information Content; I: allelic Shannon diversity index; P: polymorphism rate.
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Table 4. Inter-type genetic differentiation of sorghum using SSRs.
Legend: The upper diagonal in bold is the comparison of Fst by pair of sorghum types and the lower diagonal is the minimum distance of Nei; ns: not significant.
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Figure 2. Spatial representation of the structuring of sorghum accessions in a 1/2 factorial design. Legend: Sweet stalk sorghum accessions, Sweet grain sorghum accessions, Grain sorghum accessions.
5. Discussion
The low number of private alleles identified could be related to the gene flow that exists between these sorghum types because despite its preferentially self-pollinated mode of reproduction, sorghum has a relatively high rate of outcrossing ranging from 3% to 31% [11]. Indeed, all five races of the bicolor subspecies can easily cross with each other as well as with wild sorghums of the bicolor species and have fertile hybrids [21]. The three types of sorghum could thus share a common genetic heritage. Similar results were also reported by [22].
The different sorghum variants encountered are thought to be the result of spontaneous mutations in sorghum. Indeed, the mutation occurs approximately every hundred million base pairs in every generation regardless of the living organism especially in eukaryotes [23]. Thus, these mutations have contributed significantly to the processes of evolution, speciation, domestication, and adaptation to environments and agricultural practices of cultivated plant species. These mutations may have been selected naturally or by man according to the objectives sought, which leads to a slow evolution of the genome over time. The genetic diversity of sorghum is therefore the result of a long process of evolution and previous selection within the species.
Sweet grain sorghum showed the lowest expected and observed heterozygosity values. This could be explained by the spatial isolation practiced by the farmers. Indeed, sweet grain sorghum is mostly cultivated in the hut fields, unlike grain sorghum cultivation in the bush fields. This farmer management method would result in a lower intra-type genetic diversity, resulting in a higher homozygosity compared to sorghum grown in bush fields where several types coexist. Previous studies by [24] on early maize varieties in Burkina Faso and [25] on late and early millet varieties in Niger reported similar results.
The low genetic differentiation observed between sweet grain sorghum and grain sorghum would suggest a greater closeness between these two types of sorghum due to their selection criterion based mainly on grain. Thus, sweet grain sorghum would be grain sorghum selected for the taste quality of the grain at a doughty stage of their development and for the earliness of their cycle. Indeed, at the dough stage, sugar is present in the form of fructose, glucose, sucrose etc [26]. These sugars are converted during maturation into starch which is the main reserve form of sugar in grains in cereals [27] [28]. The accumulated starch retains very little sweetness in mature grain. Work on sweet corn or maize has shown that the recessive su allele is responsible for the sweetness trait in homozygotes of the susu genotype. This allele would allow an accumulation of phytoglycogen instead of the traditional starch [29] [30]. This could be the case for sweet grain sorghum. Therefore, the sweet grain trait is due to the expression of the recessive su allele carried on chromosome 4 and results in high sugar production. The gene flow between grain sorghum and sweet grain sorghum could then evolve towards a loss of the sweetness of the grain in the latter.
The significant differentiation index recorded between sweet grain sorghum and sweet stalk sorghum would be of genetic origin. Indeed, these two types of sorghum express the very sweet character but in different accumulation organs. In sweet grain sorghum, the starch biomass is accumulated in the grains and in sweet stalk sorghum, and the saccharide biomass is located in the stem. This divergence of accumulation organs would be closely related to the expression of genes on the morphology of conductive tissues, which is expressed by a better development of the xylem in sweet stalk sorghum than in the other types of sorghum. Similar observations were reported by [31] on two sorghum varieties (grain sorghum and sweet stalk sorghum). A process of change in expression of genes responsible for the sweetness trait could be responsible for this divergence in sugar accumulation in these sorghum. This phenomenon would be common in eukaryotic organisms and would often give rise to new phenotypes. These changes have been frequently used as indirect indicators of functional gene divergence. [32] previously reported this divergence process in plants.
Moreover, this differentiation could be due to cultivation practices associated with the peasant management of sorghum cultivation. Indeed, it is very frequent and even common to encounter grain sorghum-sweet grain sorghum or grain sorghum-sweet stalk sorghum associations in the farming environment. This is not the case for the sweet grain sorghum-sweet stalk sorghum association. Thus, the latter, which could share few genes with each other, would have a common gene pool with grain sorghum, hence the structuring of the 34 accessions into two groups, the first consisting of grain sorghum and sweet grain sorghum, and the second consisting of grain sorghum and sweet stalk sorghum. These results could be explained by the fact that when two groups of initially identical individuals are isolated and maintained in isolation for a sufficient time, differences will develop and accumulate over time between the two groups. If no homogenization process between the two groups is present (exchange of alleles), after a certain time, the differences will be sufficient for the two groups to be no longer compatible or interbreeding [33].
The three sorghum types are thus not well individualized genetic entities despite their distinct names, morphological differences, and differences in organs of interest for each cultivated type. Similar results were reported by [34] on rainfed and transplanted sorghum in the Lake Chad basin. In addition, some authors have shown that differences within a species can also be due to the influence of the environment [35].
The genetic proximity of the crops has led to the possibility of gene flow between sorghums. In the case of related species, management of gene flow is important for maintaining stability, production potential, and even quality of crop products. Management of crops with cross-fertilization potential involves space-time methods such as staggered sowing dates, crop separation, or choice of associations
6. Conclusion
The nuclear microsatellite markers used in this study were quite informative and revealed a little divergence between sorghum types within the collection. Thus, despite belonging to three morphologically well-differentiated sorghum groups, sweet grain sorghum and grain sorghum remain very similar genetically. The sorghum type factor is therefore not a very reliable criterion for characterizing genetic diversity. Differentiation between sorghum types could also be due to physiological or biochemical factors. The results obtained show the need for further study by sequencing the genome of the different types of sorghum cultivated.
Data Availability
The data supporting the findings of this study are available on request from the corresponding author.
Acknowledgements
We are grateful to the International Foundation for Science for the financial support we received during this study.