Main differences between grain and sweet sorghum.
Sorghum is one of the main cereal crops, its consumption is large, since it provides grain, fiber and biofuel. Likewise, its genome, with only 10 diploid chromosomes, makes it an attractive model for research and genetic improvement. Sorghum is the most studied C4 plant of its genus; several lines have been developed under three main characteristics: grain, forage and sugar biomass. Compared to other crops, sweet sorghum possesses high levels of highly fermentable sugars in the stem. Also, it has the ability of producing high production yields in marginal lands. These characteristics make it and attractive crop for the generation of biofuels. Molecular markers associated to several resistances and tolerances to biotic and abiotic factors have been described in literature. These allow the development of high-density linkage maps, which, along with the rising availability of sorghum genomes, will accelerate the identification of markers and the integration of the complete genome sequence. This will facilitate the selection of traits related to biofuels and the marker-assisted genetic improvement. Most of the information presented in this review is focused in Sorghum bicolor (L.) Moench. However, from the bioenergetics perspective, it is limited to sweet sorghum, which represents a promising opportunity for further studies.
- genetic differences
- trait sequencing
- post-transcriptional regulation
Sweet sorghum is a natural variant of the common grain sorghum, with high content of sugars in the stem. It is frequently regarded as a “smart crop”, since it can produce food as well as fuel. Currently, there are no records of commercial production of sweet sorghum in México, since it is a crop of recent introduction in the country. The first appearance of this crop in México was in the INIFAP Campo Experimental Río Bravo in the year of 2006, with the varieties Dale, Topper 76–6, Theis and M-81E . Since then, the INIFAP genetic improvement program for sorghum, has searched for varieties which can adapt to the arid and semi-arid regions of the northeast or the country. These conditions are met by the variety RB Cañero, which has been tested in different environments, where it has shown a remarkable development and a high potential to produce bioethanol sugars .
Despite sweet sorghum is still a new crop in Mexico, the productive potential and the area of exploitation are highly promising. In 2010, INIFAP estimated the area available in México for sweet sorghum production, being the northeast region suitable with more than 4.38 million hectares . The latter, pictures México as a country with great expectative of self-sufficiency in the generation of its own biofuel. The use of biofuels, being a cleaner and more efficient way of producing energy, would help to reduce pollution and greenhouse gases, which destabilize climate . Pursuing this goal, biotechnology is an effective tool to develop methods which can optimize the bioethanol production from sweet sorghum. This review presents some of the biotechnological advances, as well as the current state of the genetic and molecular studies in sorghum, related to new routes to achieve an efficient generation of biofuels from this crop. This knowledge makes clear the necessity for effort and economic investment on this field, to reach self-sufficiency in the generation of energy sources in the country.
2. Sorghum as a research model
Sorghum possesses a small genome, which makes it attractive as a model organism for studies focused on understanding the structure, function and evolution of the cereals’ genome. Sorghum is a tropical crop with a typical C4 photosynthesis, which uses a complex specialized biochemical and morphological system for carbon assimilation when it is exposed to elevated temperatures. This is a unique feature among the species of the same family it belongs to.
Like in other crops, sorghum is compared to other Poaceae of agricultural relevance such as maize (
Sorghum represents an excellent model for research, since linkage mapping methodologies have been successfully implemented on it and possesses a wide mating system by self-pollination, which tends to preserve the association relationships for longer time periods compared to the self-pollination of cereals like maize, which facilitates the development of pure lines. Also, its genome sequence is available in several databases .
3. Differences between sweet sorghum and grain sorghum
Sweet sorghum plant produces sugars which can be directly fermented, together with its ability to produce high biomass volumes under adverse conditions, this crop is consider ideal for the generation of bioethanol of first and second generation. Also, its cultivation is suitable for marginal lands, avoiding competence for land with other food crops [11, 12, 13]. However, the genetics underlying these traits have been barely studied. The genetic differences between sweet and grain sorghum consist on a series of variations in the sequence and alterations of the genetic structure. The variations at sequence level are usually identified by single-nucleotide polymorphisms (SNPs), association sequences, genetic diversity and domestication [14, 15, 16].
Sweet sorghum has been found on different races , which challenges its origin, selection and genetics. This also suggest high genetic variability between sweet and grain sorghum, which could be exploited for genetic improvement of sweet sorghum. Currently the BTx623 grain sorghum genome sequence is available , which provides a genomic base for comparative studies of the genome. Regardless this achievement, it is still difficult to access the information related to the hidden variability among genomes of the same species. Zheng et al.,  studied the resequencing of the two sweet and one grain sorghum genomes, with the aim of identify polymorphism patterns of the sequences and structural variations, using BTx623 as a reference genome. This study allowed the identification of great differences in the number of SNPs, indels, copy number variations and structural variations (SV) among these genomes. The comparison of this genetic variation defined potential genomic regions and metabolic pathways associated to sweet sorghum and traits such as sugar production. Table 1 presents phenotypic and genotypic differences between grain and sweet sorghum.
|Trait||Grain sorghum||Sweet Sorghum|
|Biomass (g plant−1)||67||605–1096|
|Spike (dry weight, g) (g plant−1)||24.2||60–80|
|Roots (dry weight, g) (g plant−1)||15.1||68–88|
|Stem (dry weight, g) (g plant−1)||27||164|
|Line BTx623 (grain)||Indels||16,781 (7,977 genes)|
|vs.||SVs||1,847 (2,071 genes)|
|Line Keller (sweet)||Protein functional divergence||563(SNP), 287(Indels), 69 (SV)|
4. Sorghum genetic mapping
Building a linkage map is the fundamental step required for a detailed study of genetic improvement of crops by marker-assisted selection. Mapping of sorghum genome based on DNA markers started in the 90’s, and nowadays there are several genetic maps available. It is important to mention that sorghum, particularly
The first genetic maps built where based on DNA analogy tests based on corn genome Binelli
Kong et al.  mapped a RIL population with 31 SSR polymorphic
Apart from these linkage maps, integrated maps have also been built. An integrated linkage map of SSRs and AFLPs from sorghum was reported by Kong
Recently, a high genetic density map was published by Ji
Another method used is the comparative genome mapping. This particular method is interesting for geneticists and evolutionary biologists to elucidate the mechanisms determining chromosome’s evolution. Comparative genome mapping provides a powerful technique to study the way and the time where chromosomal evolution occurs . This approach involves the use of molecular markers, such as RFLPs, to map the genomes of two species for a group of markers in common (
Until 2015, more than 850
Regardless of the multiple QTLs already reported, very few studies have been done with the aim of genetically improving these traits. In one of these, a quantitative gene (dw3), orthologous to branchytic 2 (br2) from corn, was cloned with the intention of reducing plant height. This gene is a P-glycoprotein which modules auxin transport in maize stems . Another group of researchers cloned and sequenced, from the cultivar dulce Rio, homologous genes of the sucrose transporter proteins (SUTs), which were compared to the published sequence of BTX623 grain sorghum variety. It was possible to identify six SUTs in BTx623, along with nine differences in the amino acids sequence of SbSUT5 between the two varieties. Two of the five remaining SUTs exhibited unique variations in the amino acids sequences of SbSUT1 and SbSUT2, whereas the rest shared identical sequences. It was also proven that in a mutant of
5. Genome sequencing and sorghum functional genomics
Massive sequencing of the line BTx623 is nowadays completed and approximately 10.5 million of reads (8X coverage) have been deposited in the NCBI database. In the preliminary assembly, more than 97% of the genes codifying for proteins (Expressed Sequence Tag, EST) in sorghum were found in 250 large contigs. The majority was able to be joined, ordered and oriented using genetic and physical maps to reconstruct the full chromosomes. The preliminary alignment assembly for the sorghum sequence was based on methyl-filtrated sequences. Also, the assembly for sorghum, maize, sugar cane transcripts, as well as
The spatial structure of the genes in sorghum is represented by approximately 125,000 ESTs, which have been grouped in 22,000 unigenes, representing more than the 20 diverse libraries of different genotypes . Around 50,000 methyl-filtrated reads, which provide an estimated coverage of 1X  have been assembled into contigs. Another representative strategy is the cloning and direct sequencing (Cot-Base cloning), which was used in sorghum in 2001 for the first time . This method offers the potential to cover and increase this coverage more than could be achieved with ESTs and methyl-filtrated reads as demonstrated in maize.
The progress in transcriptomes’ characterization has been parallel to the identification of differential genes expressing in response to biotic and abiotic factors, as well as to damage caused by insects, dehydration, high salt concentration, abscisic acid , methyl-jasmonate, salicylic acid and amino cyclopropane carboxylic acid .
6. Post-transcriptional regulation by miRNAs in sorghum
The micro-RNAs (miRNAs) are small RNA molecules of approximately 21 nucleotides, which play an important role in the post-transcriptional genetic regulation inhibiting the translation of the messenger RNAs (mRNAs) by blocking translation machinery or by excision of the mRNAs . In plants, the majority of miRNAs promote the degradation of mRNA targets by perfect or almost perfect mating of the complimentary RNA strands . miRNAs intervene in a variety of biological processes, such as development and identity of organs, metabolism and stress responses . A substantial number of miRNAs has been identified in different plants, and recently the number of studies in sorghum has been increasing with respect to the identification of miRNAs and their target genes.
Recently, Katiyar et al.  showed the importance of studying miRNAs and other RNA molecules using RNA sequencing from the libraries created from genotypes of a variety tolerant to drought (M35–1) and one susceptible. These varieties were cultivated in controlled conditions as well as in drought stress. After sequencing the RNA profiles generated, it was possible to identify 96 miRNAs regulated by the stressed caused by drought conditions. This represents new perspectives for the genetic engineering regarding the potential of miRNAs to improve drought resistance as well as other types of abiotic stresses.
Following the same research line, in 2016, Hamza et al., used 8 deregulated miRNAs by abiotic stress in 11 elite varieties of sorghum under low water availability and drought . This study showed that the miRNAs miR396, miR393, miR397-5p, miR166, miR167 and miR168 have a significative deregulation, being sbi-miR396 and sbi-miRNA398 the ones with higher overexpression for all the genotypes. This same research group has studied the effects of drought and salinity in the miRNAs profiles generated in
Other important trait to improve sweet sorghum is sugar accumulation, which has been already studied by Yu et al. , who propose mir-271 as a specific miRNA of the Rio sweet sorghum variety, related to cellulose synthesis and sugar accumulation. A full detailed list with most of the relevant miRNAs for the genetic improvement of sorghum in biofuels production was published by Dhaka et al. .
7. Transformation and reverse genetic in sorghum
Methods for sorghum transformation have been available since the beginning of the 90’s, initially by protoplasts  and cell culture , and subsequently
One of the main arguments against the use of transgenics is the use of selection markers such as herbicide tolerance and the fact that they stay inside the genome of the transformed plant. The main issue is the possibility of the cultivar’s pollen to pollinate related weeds and therefore the resistance is inherited to undesired plants. Against this problematic, there are several efforts to generate marker-free transgenics. An example is presented by Lu
In other hand, sorghum offers the opportunity to complete what has been previously described in the reverse genetics of rice and maize, providing to the genetic and familiar studies, those genes which are hard to manipulate in these crops. This allows the directed functional analysis to specify the genes in sorghum related to traits such as hydric stress and the production of certain sugars by genetic association. To accelerate the specific direct identification of genes, mutant lines using ethyl methane sulfonate (EMS) have been created. For the genotype BTx623 there are 1,600 M3 annotations, individual pedigrees, which was characterized . Currently, each of the inspected M3 lines is distinguishable from the original stock and some have multiple mutant phenotypes. The additional M2 mutants are available for the scientific community for the production of thousands of M3 additional lines.
Until few years ago, even with the genome sequencing technology for the elite line BTx623, the genetic sources and sorghum germplasm where limited, making hard the functional validation of the sequenced genes. In 2016, 4,600 M4 mutant pedigrees where created by EMS mutagenizing of BTx623 seed, which were later transformed in lines by single-seed descendant method . The sequencing of 256 mutant lines revealed more than 1.8 million of induced canonical mutations, affecting 95% of the sorghum genome.
The studies here presented represent an introduction to the current state of sorghum genomics. Regardless these advances have contributed relevant achievements to what it is known about genetic diversity of this species, it is still necessary to develop further studies, which its aim is focused in sweet sorghum. However, the knowledge acquired in grain sorghum and other related species, constitute an important molecular base to continue developing research studies which allow to know sweet sorghum and its unreported genomic regions. In them could lie the key for the increased production of sugars, lignin and other traits of interest such as tolerance to new plague’s appearances such as yellow aphids and/or diseases. It is also necessary to develop genetic maps which allow the localization of genetic codifying regions to certain traits of agronomic interest. Regarding molecular studies in Mexico, there are no reports of genetic maps or genomics performed in sweet sorghum. This represents an opportunity to develop research lines which allow to generate the country’s own sweet sorghum genotypes carrying tolerances to adverse biotic and abiotic conditions predominant in the country. This would allow the growth on its production and of its sub products, focusing in alternative environment friendly energy sources.