Molecular Plant Breeding 2024, Vol.15, No.2, 63-69 http://genbreedpublisher.com/index.php/mpb 65 RNAi produced starch containing up to 50% amylose (Zhou et al., 2020). Three mutants with long fragment deletions in the second exon of SBE2 showed higher amylose (up to 56% in apparent amylose content) and resistant starch (up to 35%), and also resulted in starch viscosity with a higher pasting temperature and peak time (Luo et al., 2022). Simultaneous suppression of both SBE1 and SBE2 endowed cassava with a reduced degree of polymerization of 6–13 chains in amylopectin (Utsumi et al., 2022b). GWD1-RNAi cassava plants not only showed both retarded plant and storage root growth, had excess starch accumulation in leaves, and also led to changes in physico-chemical properties of transient and storage starch (Zhou et al., 2017). MeSSII-RNAi cassava had an increase in amylose content and presented alterations in starch physicochemical properties in the storage roots (He et al., 2022). In fact, engineering cassava as well as testing in the field are still in its infancy (Koehorst-van Putten et al., 2012; Zambrano et al., 2022). 5 Quantitative Trait Loci (QTL) Controlling Starch Yield and Properties Starch yield and properties are very close but different traits, which are associated with QTLs. The QTLs could be used for identification of key target genes of interest and for selection of cassava germplasms of desirable traits for breeding. So far, research on QTL controlling starch yield and properties has not been as extensive as one might think. Fifteen QTLs associated with starch pasting viscosity were identified by using 100 lines of an F1 mapping population from a cross between two cassava cultivars Huay Bong 60 and Hanatee (Thanyasiriwat et al., 2014). Total 115 QTLs controlling starch yield and properties on starch content, amylose content, pasting temperature, thermal and retrogradation, and textural property were reported from 2005-2018 (Tappiban et al., 2019), with candidate genes. Five QTLs for starch content were identified with 2 cassava cultivars of CI-732 (high dry matter content and starch content) and MNga-1 (low dry matter content and starch content) by simple interval mapping (Prasannakumari et al., 2021). With a panel of 276 cassava genotypes by using the genome-wide association study (GWAS), 21 starch pasting property-related QTLs were recently found (Phumichai et al., 2022). 6 Challenges in Engineering Cassava Starch Yield and Starch Properties Cassava improvement either through conventional cross-breeding or by engineering biotechnologies faces more rigorous challenges (Otun et al., 2023). After entering the era of omics, many new and powerful genetic engineering technologies have emerged and are constantly being improved, such as CRISPR/Cas9 for gene editing, and RNAi and virus-induced gene silencing (VIGS) for suppressing gene expression. Each technology has its own pros and cons. For all these technologies, the basic principle and requirement is high specificity and precision (Senthil-Kumar and Mysore, 2011; Ma et al., 2014; Rössner et al., 2022). However, although not all, unexpected off-target phenomena and non-specific events are also commonly reported. The engineering strategies based on Agrobacterium-mediated overexpression (Utsumi et al., 2022a), CRISPR/Cas9, RNAi, and VIGS have been used for cassava improvement and gene function identification research. The challenges are, but not limited to, as follows. It is currently not very clear about the chromosomal ploidy and heterozygosity for the vast majority of cassava cultivars. Cassava materials resulting from natural outcrosses are preferentially retained in the long-term production and breeding process because larger and much more vigorous cassava materials from outcrosses are more favored by farmers. Therefore, it can be speculated that most of the cultivars/elite variety should be heterozygous polyploids. However, such heterozygosity results in wide and unpredictable diversity of phenotypes that breeders are interested in but farmers dislike in propagation (Ceballos et al., 2004). The heterozygosity makes it very likely that some key starch biosynthesis genes are in a heterozygous state. For MeSSI gene, there are 5 heterozygous loci in coding regions in 44 cassava accessions, and 1 heterozygous locus is in non-coding region in 44 cassava accessions (Vasconcelos et al., 2016). With regard to MeGBSS1 gene, only one copy is in cassava genome (Tappiban et al., 2019), however, there existed 1 heterozygous locus in coding regions in 87 cassava accessions, and 5 heterozygous loci were present in non-coding regions in 84 cassava accessions (Vasconcelos et al., 2016). The MeSBE gene had 1 heterozygous locus in non-coding regions in 280 cassava accessions (Vasconcelos et al., 2016). In addition, expression of genes encoding starch biosynthesis enzymes, such as AGPases, shows changes with tissues and growth stages of cassava (Tappiban et al., 2019). All these factors
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