**4.3 Image-based phenotyping**

Digital image analysis allows the extraction of information regarding root color based on the strong correlation that exists between digital and virtual data [55]. Imaging techniques possess high resolutions, which permit the visualization of the sample from several dimensions and generating multiple data. Image-based phenotyping is used to quantify complex plant characters such as growth pattern, photosynthetic abilities, yield, tolerance to biotic and abiotic stress, both in controlled environments and in the open field. Plants imaging aims to measure a character quantitatively through the interaction that takes place between light and the plant such as reflection, absorption, and transmission of sent photons of which all plant cells and tissue possess specific wavelength for light reflection, absorption, and transmission. Since the presence of carotenoid is linked with the intensity of yellow color, it is taken that this type of phenotyping is ideal for the quantification of root carotenoid content. There are different aspects to image-based phenotyping, and they include thermal infrared imaging, imaging spectroscopy, fluorescence imaging, visible imaging, laser imaging, and hyperspectral imaging [55]. The advantages of imaging techniques include the following:

I.It is time saving.


#### **4.4 iCheck Carotene**

This is a portable device consisting of two components, namely the measuring unit (iCheck™ Carotene) and the disposable reagent vial (iEx™) where the reaction is performed. The disposable reagent vial contains 2 mL of a mixture of reagents, which is needed for carrying out the reaction. The iCheck Carotene is very portable weighing about 250 g with dimensions (200 mm x 104 mm x 40 mm) making it easily transportable. It uses rechargeable batteries, which can be used to take up to about 400 measurements, which saves automatically and can be retrieved at will as a text file with the use of a USB cable. The iCheck Carotene is a rapid screening method, which is cost-effective, user-friendly, simple, and inexpensive. It does not require highly skilled and specialized personnel for its operation, neither does it need an expensive laboratory setup with equipment and specified chemicals; therefore, it is suitable for the quantification of a large number of samples within a short period of time with accurate results especially where there are no labs available, and there is a large number of cassava genotypes to be screened [56].

#### **4.5 High-performance liquid chromatography (HPLC)**

This is an advanced form of liquid chromatography, which is used in the separation, identification, and quantification of components in a mixture of molecules encountered in chemical and biological systems. It is associated with high reproducibility, ease of selection, manipulation, and high rate of recovery [57]. Its working principle involves a solution of the sample being injected into a column of a porous material (stationary phase) while a liquid (mobile phase) is pumped at high pressure into the column. The sample separates based on the differences in the rates of migration through the column, which results from the partitioning of the sample between the stationary and the mobile phase [57, 58].

In cassava phenotyping, HPLC is used in the separation and quantification of individual carotenoids, which are different in their provitamin A activity. Although it has high reproducibility, its analysis is expensive, costing 50–70 US dollars per sample with very low throughput. It is time-consuming, labor-intensive, and requires a highly sophisticated laboratory setup with highly skilled personnel and strictly adhered quality control regiment [57].

#### **4.6 Ultraviolet–visible (UV–vis) spectrophotometer**

The UV–Visible Spectrophotometer is a type of spectrophotometer, principle of which is based on the absorption of ultraviolet light or visible light by chemical compounds, and this results in the production of distinct spectra. It is a device that precisely measures electromagnetic energy at specific wavelengths of lights. UV–visible spectrophotometer uses light over the ultraviolet range of (185–400 nm) and visible range (400–700 nm) of the electromagnetic radiation spectrum. Carotenoids concentration, for example, is determined spectrophotometrically by measuring the absorbance (also referred to as optical density) of the extract at various wavelengths. The absorption spectrum of β-carotene (carotenoids) peaks between 450 and 475 nm. UV spectrophotometer has been mostly used to quantify carotenoids in cassava and other plants. Jaramillo et al. observed that spectrophotometer reading gave a higher quantity of total carotenoids content (30.0 μg/g) compared with the use of iCheck devise (24.7 μg/g). Other authors have also quantified carotenoids in cassava using the spectrophotometer [5, 12, 15, 57]. The major throwback with the use of this instrument is that it is cumbersome and time-consuming with low throughput especially when dealing with large breeding populations.

#### **5. Marker-assisted selection of carotenoid-rich cassava**

Over the years, conventional breeding has been augmented by various innovative molecular marker-aided techniques. Genetic differences that exist between individual species and organisms represent a genetic marker. Generally, they do not represent the target genes themselves but act as "signposts" or "landmarks" representing DNA along chromosomes. The first marker technologies involved the use of biochemical markers such as isozymes and allozymes. These gave way to the first-generation DNA markers such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR). Advances in sequencing technology enhanced the use of DNA-sequencing based markers such as SSR and SNP, giving rise to automated high-throughput genotyping [59]. For a genetic marker to be useful, the marker locus has to show experimentally detectable variation among individuals [15, 60]. The variation can be due to single-nucleotide polymorphisms or deletions/insertions or major chromosomal changes. Molecular genetic markers can be used to study the diversity of the observable variation at population or species level [59]. They can also be used to map genomes, identify regions of the genome controlling a trait, and follow a segment of interest of the genome in a plant breeding scheme [59–61].

Molecular markers are usually utilized in a breeding program to facilitate and speed up the selection process as such, carotenoids are boosted through markerassisted selection (MAS) on target genes [62]. Some of the applications of molecular markers such as RFLP, AFLP, RAPD, SSR in cassava include taxonomical studies, understanding the phylogenetic relationships in the genus, confirmation of ploidy, genetic diversity assessment, and genetic mapping studies in cassava [59], making MAS a reality for application in breeding programs [63]. SSRs have also been used to select for carotenoids in cassava [64]. The reduced cost of the new technologies increases the discovery and utilization of new set of molecular markers that is amenable for the high-throughput genotyping [65].

Recently, single-nucleotide polymorphism (SNP) markers are increasingly being used for genotyping to study gene function. SNPs work as molecular markers that help locate genes associated with a trait and are used for genotype sequencing. SNPs may play a direct role in a trait and affect gene function if they occur within a gene or in a regulatory coding region and thus serve as molecular markers. These markers can be applied in the following: genetic architecture detection, association studies, conservation genetics, genetic diversity, and are fast becoming the marker system of choice in marker assisted plant breeding programs. Some genotyping methods that can specifically genotype an SNP affecting a trait in a collection of population include the use of KASP (competitive allele-specific polymerase chain reaction (PCR) markers, especially for a small number of SNPs [65, 66]. It utilizes a unique form of competitive allele-specific PCR combined with a novel, homogeneous, fluorescencebased reporting system for the identification and measurement of genetic variation occurring at the nucleotide level to detect single-nucleotide polymorphisms (SNPs) or inserts and deletions (InDels) [36, 38]. KASP chemistry provides a versatile choice that can be applied to small- and large-scale projects. It is suitable for use on a variety of equipment platforms and provides flexibility in terms of the number of SNPs and


*\* MAF–major allele frequency, Het–heterozygosity, PIC–polymorphic information content, Chromameter b\*, PSY2– Phytoene synthase2 gene, lcyE–Lycopene epsilon cylase gene, Pulpcol–pulp-color score, TC SPEC–total carotenoid by spectrophotometer, TC–iCheck total carotenoid by iCheck Fluoro, TBC–Total β-carotene. Source: Table 2 [12].*

#### **Table 2.**

*Summary results of validated SNP markers on cassava breeding collection.*

the number of samples able to be analyzed. To facilitate the selection of carotenoidrich cassava genotypes, six KASP SNP markers were designed on candidate genes and validated on 650 elite cassava accessions of which PSY2\_572 explained most of the phenotypic variation (R2 = 0.75) in root pulp color (**Table 2**) [12].

Most recent advances in next-generation sequencing technologies have enabled the use of genome-wide SNP markers for genomic selection. The genomic selection tool is believed to significantly increase the efficiency of breeding by increasing the speed and accuracy of selection in a breeding program by predicting the genetic value of individuals at an early selection stage [67]. Genomic selection models have also been implemented by [68], to fast-track the improvement of provitamin A carotenoids in cassava using a total of 23,431 single-nucleotide polymorphic markers.
