CMD Evaluation, DNA Extraction & Genotyping

Selection for resistance to cassava mosaic disease in African cassava germplasm using single nucleotide polymorphism markers

Field experiment and CMD evaluation

A population comprising 145 cassava genotypes was evaluated for CMD incidence and severity at the Teaching and Research Field of the Department of Agronomy, University of Ibadan, Nigeria, during two cropping seasons (2019/2020 and 2020/2021). The seeds of the genotypes were derived from open-pollinated crosses and were collected from five female parents (IITA-TMS011368; IITA-TMS011371; IITA-TMS011412; IITA-TMS070593 and IITA-TMS070539).

To minimise experimental error related to the large experimental field size, the genotypes were divided into four sets. The trial was laid out using a randomised complete block design with two replications per set. The experimental site was cleared and ridges were made with a row spacing of 3 m between sets. A total of 20 cuttings (25–30 cm long) from matured stems of each genotype were planted in a plot size of 20 m2 at a spacing of 1 m x 1 m. The experimental location was chosen because of the high disease pressure by cassava mosaic virus.9 Moreover, the early stage (first 6 months) of the plants corresponded with the period of high whitefly activity, resulting in a significant risk of disease exposure. Variety Data on CMD incidence and severity scores were collected at 1, 3, and 5 months after planting. The incidence was recorded as the ratio of the number of plants with symptoms to the total number of plants per plot. The severity of CMD was measured on a scale of 1 to 5, with 1 denoting no symptoms, 2 denoting mild chlorotic areas on most leaves, with the remaining parts of the leaves and leaflets appearing green and healthy, 3 denoting a pronounced mosaic pattern on most leaves, with distortion of the lower one-third of most leaves, 4 denoting severe mosaic pattern on most leaves, and 5 denoting very severe mosaic symptoms on all leaves, often accompanied by stunting of the plant.

DNA extraction

A modified Dellaporta approach was used to extract genomic DNA from freeze-dried leaf samples.20 The DNA quantification and purity were checked using a NanoDrop spectrophotometer (ND-8000, Thermo Fisher Scientific, USA). The absorbance at a wavelength of 260/280 nm ranging from 1.80 to 2.0 indicates that the DNA solution was free of contaminants. The quality of the DNA samples was checked on 1% agarose gel (Sunrise 96, Biometra, Göttingen, Germany) and bands were viewed on a gel documentation system (Labnet ENDURO GDS Gel Documentation System Aplegen) incorporated with an ultraviolet transilluminator.

Kompetitive allele-specific PCR genotyping

Markers S12_7926132 and S14_4626854 derived from Rabbi et al.18 were used to screen the cassava genotypes for resistant and susceptible alleles. The sequences of the specific forward and common reverse primers used for the Kompetitive allele-specific PCR (KASP) genotyping were extracted from Ige et al.21 and are presented in Table 1. A positive (IITA-TMS-IBA070593) and two negative (IITA-TMS-IBA30572 and IITA-TMS-IBA30555) control varieties were included in the genotyping as well as a no-template control of 5 µL of distilled water. The KASP reaction mix consisted of 5 µL of DNA at a concentration of 50 ng and 5 µL of the prepared genotyping mix 5 µL (2 × KASP master mix) and 0.14 µL primer mix. The PCR cycling was performed as follows: hot-start activation at 94 °C for 15 min followed by 10 touchdown cycles (20 s at 94 °C; touchdown at 61 °C to 55 °C initially and dropping by 0.6 °C per cycle for 60 s), followed by 26 additional cycles (20 s at 94 °C; 55 °C for 20 s). The KASP genotyping reactions were run on the Roche Light Cycler 480-II at the Biosciences Unit, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria.

The SNPs were called using KlusterCaller software (LGC, Biosearch Technologies, USA) and visualised based on the fluorescence signal using the SNPviewer software (LGC, Biosearch Technologies), where data on SNP allele calls were visualised graphically. Fluorophores FAM and HEX plotted on the x- and y-axes, respectively, allowed the distinction of the assayed genotypes.

Article TitleSelection for resistance to cassava mosaic disease in African cassava germplasm using single nucleotide polymorphism markers

Published
January 27, 2022
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Abstract

Cassava mosaic disease (CMD) is one of the main constraints that hamper cassava production. Breeding for varieties that are CMD resistant is a major aim in cassava breeding programmes. However, the use of the conventional approach has its limitations, including a lengthy growth cycle and a low multiplication rate of planting materials. To increase breeding efficiency as well as genetic gain of traits, SNP markers can be used to screen and identify resistant genotypes. The objective of this study was to predict the performance of 145 cassava genotypes from open-pollinated crosses for CMD resistance using molecular markers. Two SNP markers (S127926132 and S14_4626854), previously converted into Kompetitive allele-specific PCR (KASP) assays, as well as CMD incidence and severity scores, were used for selection. About 76% of the genotypes were revealed to be resistant to CMD based on phenotypic scores, while over 24% of the total population were found to be susceptible. Significant effects were observed for alleles associated with marker S12_7926132 while the other marker had non-significant effects. The predictive accuracy (true positives and true negatives) of the major _CMD2 locus on chromosome 12 was 77% in the population used in this study. Our study provides insight into the potential use of marker-assisted selection for CMD resistance in cassava breeding programmes.


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