Studies by Embrapa and partners have developed three methodologies for early selection of cassava clones with waxy starch. These works are part of the Company's effort to obtain a variety of cassava with this special type of starch that can be produced on a large scale in Brazil. Cassava starch is basically composed of two types of polysaccharides: amylose and amylopectin. Typically, commercial varieties have between 19% and 27% amylose in the starch composition. Waxy starch contains a maximum of 5% amylose, a characteristic of great interest mainly to the food industry, due to the lower starch retrogradation (loss of water after cooling the finished product). Therefore, waxy cassava should bring benefits associated with better quality and shelf life of products derived from the industrialization of refrigerated and frozen foods that have waxy starch in their composition.
These works, recently published in international journals, arose from the need to accelerate the process of developing waxy clones from Embrapa's cassava genetic improvement program. The main objective is to optimize the process of selecting clones with this characteristic of interest, taking only those that present waxy starch to the field.
“The methodologies bring broad advantages to the traditional methods of evaluating waxy starch in cassava, which, despite being accurate, are carried out late”, points out agronomist Eder Jorge Oliveira, researcher at Embrapa Mandioca e Fruticultura (BA), author of the work together with a team of students from the Federal University of Recôncavo da Bahia (UFRB), led by Cátia Dias do Carmo, main author of the studies, which are part of her doctoral thesis, supervised by Oliveira. Hernán Ceballos, breeder at the Cassava Program at the International Center for Tropical Agriculture (Ciat), in Colombia, is co-author of two of the works.
Oliveira lists the potential impacts of these methodologies: reduction of the area required for planting clones by at least 75%, possibility of carrying out crosses already at the seedling stage (before the field stages), optimization of the costs of the genetic improvement program, guarantee of traceability of future cultivars with waxy starch for the purposes of protecting intellectual property rights and, finally, optimizing the time needed to develop cassava cultivars with waxy starch.
Carmo evaluates the results achieved. “They represent important advances in the early identification of genotypes with waxy starch in cassava. It is gratifying for us, who work with agricultural sciences, to have the opportunity to develop technologies and methods with direct applicability. It is a double contribution: you have scientific articles with the role of disseminating and contributing to knowledge on a certain subject and methods with potential use within breeding programs”, adds the author.
Conventionally, the assessment of the waxy phenotype is done at the time of harvesting the plants using the iodine test on the roots. Based on this test, it is possible to visually separate the color of the roots whether they are waxy or not. Although this methodology is accurate, it requires a lot of time, as the crop cycle lasts, on average, one year.
The researcher explains that, when you cross a waxy source with a non-waxy source, all the seeds derived from this crossing have the non-waxy phenotype. They have the waxy starch allele (specific variation of the gene that will determine how a given characteristic will be expressed in the individual), but it is not expressed because the inheritance of resistance is homozygous recessive.
“With this, it is possible to self-fertilize or carry out a new cycle of crossing between F1s plants to generate seeds that have segregation for waxy starch. This second generation leads to an expected segregation of three to one, with three parts having non-waxy, therefore unwanted, phenotypes and only one part, 25%, with a waxy phenotype. After crossing, it is still necessary to plant the seeds in a greenhouse, obtain the plants and transplant them to the field for conventional evaluation of the phenotype only at the end of the crop cycle. Basically, we assess whether they are waxy or not waxy at the time of harvest, when we do the iodine test, which takes a lot of time”, explains Oliveira.
Two of the methodologies are based on molecular analyses, with very high accuracy (precision). The objective of the first study “Associative mapping for waxy starch in cassava”, published in the journal Euphytica, specialized in plant breeding, was to evaluate genome-wide association studies to detect variants related to waxy starch and identify alternative genes or genomic regions responsible for feature. A total of 351 accessions belonging to the Active Cassava Germplasm Bank of
Embrapa Cassava and Fruticulture, along with 31 waxy starch hybrids from crosses with the source AM206-5 (first natural waxy cassava genotype discovered by Ciat), were analyzed.
The first step, then, was to develop a methodology to locate the gene. “It’s an analysis that doesn’t necessarily go straight to the gene. It looks for a location on the chromosome that may be associated with the phenotype. Depending on the number of markers distributed throughout the genome, it is possible to detect, more or less easily, the genes of interest. That's the strategy. We identified a region on chromosome 2 that very clearly differentiates waxy from non-waxy, and, relatively close to this locus controlling this characteristic, we have the gene [GBSS1] that conditions this waxy phenotype in other species. Therefore, this version of this gene also exists in cassava”, says the researcher. Oliveira says that this methodology was a precursor to the following, more refined work on gene marking.
In the second study, entitled “Identification and validation of point mutations associated with the waxy phenotype in cassava”, published in BMC Plant Biology, a series of point mutations were identified that show the difference between the two types of clones. “We took a group of waxy and non-waxy genotypes [89 in total], sequenced the GBSS1 gene from these clones and aligned them with DNA sequences deposited in the National Center for Biotechnology Information (NCBI) database, a global database that brings together data from genome sequencing. We thus discovered regions that are common to waxy and non-waxy clones and, above all, regions that are different, several mutations that show differences between waxy and non-waxy clones”, describes the researcher.
Once this was done, Oliveira informs that primers (initial indicators) were designed to try to amplify these regions and check whether there was the possibility of molecularly separating the clones with a certain mutation from others. A specific mutation was thus identified, and a series of markers were designed to check whether it was possible to separate the waxy and non-waxy phenotypes. A type of marker was developed for this type of analysis, called KASP (Kompetitive Allele Specific PCR).
“The KASP strategy allows us to very clearly differentiate these two situations, with a great advantage: in addition to identifying those 25% of clones that are already waxy, we are able to select clones that are heterozygous [whose alleles for a given characteristic are different]. If, by chance, we want to move forward with any of the heterozygous clones for later self-fertilization, we can also separate these individuals. This brings a great methodological advantage to the breeding program, by also separating what is heterozygous, that is, with at least one waxy allele, which can also proceed to a second crossing cycle”, he states.
Oliveira emphasizes that any breeding program that uses this marker will be able to separate these types of genotypes with great accuracy (98%). And he adds that there is the future prospect of including a series of other characteristics currently available for genotyping on this platform. “A kind of chip for all the characteristics so that, in the seedling phase, we can segregate all those individuals that we identify with the greatest potential to become a variety.”
The third methodology, described in the work “Identification of cassava genotypes with waxy starch using near-infrared spectroscopy”, published in Crop Science, no longer involves genomic analysis but rather leaf analysis, using near-infrared spectroscopy (NIRS) — a method that uses the interaction between matter and electromagnetic radiation — and can be carried out immediately after seed germination, while still in a greenhouse.
According to the researcher, any organic compound that absorbs energy differently in this NIRS range can be used in the construction of phenotype classification models. “Starch is basically made up of amylose and amylopectin. In the case of common starch, you have a range of 19% to 27% amylose. The remainder is amylopectin. In waxy, you have practically 100% amylopectin and 0% amylose. Therefore, there is a very clear difference between the starch produced by waxy clones compared to non-waxy clones, and this variation can be easily captured via NIRS.”
Oliveira explains that starch synthesis takes place in the leaves. It is then transported to the roots. Therefore, there is a way to capture this form of starch already in the leaf. The samples were then standardized and used for NIRS reading. The work consisted of trying to create a model to classify whether the material is waxy or not waxy. According to the researcher, five different models were used and the two that showed the greatest efficiency were chosen, with classification accuracy above 84%. “This means that I will be correct in saying which are waxy or not waxy in 84% of the samples that I read with NIRS, simply by analyzing the leaves, early on, while still in a greenhouse.”
The main advantage of this strategy in relation to genomic analysis, according to Oliveira, is the reduced cost. It is necessary, as he highlights, to practically harvest the leaf, macerate it, prepare it, make readings and predictions. While in genomic analysis there is an expense with reagents, equipment maintenance, service costs if the material is sent for genotyping in another region of the country or even abroad. "Despite having less accuracy than the KASP analysis, the NIR analysis has this great advantage", he points out.
An important advance was achieved by Ciat, which identified the cassava gene responsible for waxy starch. Embrapa was the only Brazilian institution that received this material and is now seeking to incorporate the production of waxy starch into a national variety. The prospects for using waxy cassava starch are promising, as it has different properties from waxy corn, being able to reach new market niches and allowing the creation of products with unique specificities.
Ciat researcher Hernán Ceballos says that there is an interesting connection between cassava and corn when it comes to the waxy mutation. According to him, it was in corn that waxy starch was first identified, in China, but it was not explored for decades after its discovery. During World War II, cassava starch from Thailand could no longer be imported into the United States. Ceballos highlights that the specific applications and uses of each type of starch are so important that American scientists began looking for an alternative to replace cassava starch, which could no longer be imported.
It was then discovered that waxy corn shares many of the properties of native wild-type cassava starch. Thus, the waxy corn industry emerged, still important today in countries where cassava cannot be cultivated. “This story illustrates the importance and impact that different types of starch have on functional properties and, ultimately, industrial uses. As the waxy mutation was identified, important characteristics emerged, and are still being discovered, that make it very useful for specific uses. Waxy cassava starch will be particularly useful for Brazilian markets”, adds Ceballos.
Ceballos also mentions the importance of the Ciat/Embrapa partnership. “Both institutions benefited greatly from the exchange of germplasm, experiences and ideas. We dream together and work hard to make those dreams come true. Brazil is a key country in cassava production and a leader in the mechanization of cultural practices. Ciat learned a lot from Embrapa when it comes to mechanization and industrial production of cassava starch. In fact, it was in a Brazilian cassava starch factory that, for the first time, the idea of looking for waxy cassava came up,” he says.
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