How the International Crops Research Institute for the Semi-Arid Tropics used KASP to advance genotyping solutions for sorghum breeding

Sorghum [Sorghum bicolor (L.) Moench] is a diploid cereal crop used for food, drink (beer), livestock feed, fuel and as an environmentally friendly building material. Grown on an estimated 40 million hectares globally, it is classed as the 5^th most grown cereal crop. Sorghum plays a critical role in food security in the semi-arid tropics, particularly in Africa, where it is second only to maize (Zea mays L.) by area of cultivation. In these regions, smallholders depend on it for food and to generate a subsistence income.¹  

The main five varietal groups within cultivated sorghum are morphologically distinct. The abundant genetic variation has enabled sorghum to adapt to various agroecologies, climates and environments, and positioned it as a key resource for crop improvement. Several genetic studies have shown significant genetic variation in grain yield and quality, resistance to Colletotrichum sublineolum and Striga hermonthica, as well as resistance to abiotic stresses¹.

ICRISAT 

The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) is the premier drylands agricultural research institute, dedicated to uplifting smallholder farmers and ensuring food security in semi-arid tropics. They are a partner in the Consultative Group for International Agricultural Research (CGIAR) the largest not-for-profit organisation in the world dedicated to research and improvement of agricultural resources.  

ICRISAT mandated crops are sorghum, millets (pearl millet, finger millet and small millets), teff, chickpea, pigeonpea and groundnut. Its scientific team in collaboration with national partners used marker-assisted breeding to create the world’s first pigeonpea hybrid, downy mildew resistant pearl millet variety (H77/833-2) and early-maturing groundnut. The institute has also led the development of Africa’s first biofortified pearl millet. One of their long-term goals is to make genotyping a routine part of the variety development process available to breeders in the semi-arid tropics at a price the market can afford. The technology of choice for this would be relatively straightforward to engage with without the need for large upfront investment. After all, there is no point in having a useful tractor if you can’t afford the gas. 

ICRISAT’s sorghum genotyping  

ICRISAT’s sorghum breeding programme in Eastern and Southern Africa (ESA) and India is using the crop’s rich genetic diversity to improve varieties. It has developed hybrids for five major market segments. The same programme also generates sorghum genetic stock that supports the national breeding programmes in ESA. The Institute aimed to create a repository of validated genotyping markers available to all breeders through ICRISATS’ ‘Excellence in Breeding’ platform. 

KASP 

ICRISAT uses competitive allele specific PCR (KASP) for routine end-point genotyping of their mandated crops. Damaris A. Odeny, Principal Scientist, Genomics, Pre-Breeding & Bioinformatics is actively involved in ICRISAT-ESA and India sorghum improvement program by developing and validating advanced breeding tools. Damaris elected to use KASP due to its genotyping ability, availability at a sustainable market cost, ease of use and simplicity of development. 

Validating KASP markers for abiotic stress resistance, agroecology and hybridity confirmation 

The team at ICRISAT set out to validate 49 KASP markers for quality control (QC) in sorghum. They selected more than 700 lines from the ICRISAT-ESA’s breeding programme, attributed with target traits of drought tolerance (DL), low temperature tolerance (LT) and with proven performances in target agroecologies - dry lowland (DL) or sub-humid (SH) areas. They also created an outgroup using sixteen genotypes from Burkina Faso. For hybridity confirmation, parental lines and the putative F₁s were also included. All were planted at the World Agroforestry Center (ICRAF) in Nairobi, Kenya¹.  

The breeding lines were initially genotyped using 49 KASP assays, 46 of which were found informative. Hybridity testing was done on a total of 39 F₁ plants derived from a few targeted crosses. The F₁ plants alongside their parents were genotyped using 10 markers that were selected as follows: three SNPs from the top 10 most informative, three SNPs from those ranked between 11 and 20, and four trait-associated markers¹. 

Overall informativeness and chromosomal distribution of the QC SNP markers 

Polymorphism information content (PIC), minor allele frequency (MAF) and observed heterozygosity (Ho) (Table 1) were used to rank the best performing markers. Genotypes with more than 40% missing markers were excluded as were three monomorphic SNPs. 

Table 1. Summary of marker informativeness based on different categories.

The 46 markers were well distributed across the linkage group 5 of the genetic map¹. 
A positive correlation was recorded between cluster PIC values and the genetic variation in each of the clusters. 

Table 2.  Principal component plots 

Principal component (PC) plots using different marker rankings were scored across the entire germplasm set to show how informative they were. The top 10 most informative markers explained 45.4% of genetic variation. (while the markers ranked 11-20 and 21-30 explained 39.3% and 30.1% of genetic variation respectively)¹. 

Discriminant analysis of principal components (DAPC) using the top 30 markers identified three distinct breeding clusters from five major clusters. 

Table 3.  DAPC

DAPC output revealed five groupings, of which three were distinct. Membership of each cluster is defined on the right-hand side of the figure¹.  

The research validated the KASP QC marker panel for sorghum which is able to distinguish between breeding lines and provide hybridity confirmation. The scientists conducting the research expect the markers to work across sorghum germplasm globally. 

Cost-effectiveness and ease of use 

By making these validated, cost-effective, easy-to-use KASP makers available to a wider breeding community, the researchers see a real opportunity for sorghum breeders to add these genotyping tools to their breeding programmes to deliver a significant step forward to improving opportunities for genetic gain. 

The wide range of these markers, including those linked to traits like stay-green, as well as parentage and hybridity testing, and their use across global pools of breeding lines will allow accurate comparisons between lines. As sorghum has a huge genetic diversity, the opportunity to use these markers to build a wealth of genetic knowledge can significantly assist breeders in their journey to create genetic gain in sorghum breeding lines.¹  

ICRISAT’s work on sorghum genotyping will continue as they delve deeper into the species’ genome, including understanding the full biology of the Stay-green trait. This work will deliver a greater understanding and a bank of knowledge on the genetics of sorghum, a key global crop. 

Stay-green trait 

Sorghum has been successfully cultivated in arid saline-alkali land, semi-arid land and deserts³. This has been partly attributed to the crop’s ability to draw water from deeper soil depths due to root length and root angle³. Certain cultivars manage to stay green much longer even during extended periods of drought. This translates into better yield in comparison to non-stay-green types. 

Earlier research has identified genetic loci Stg1, Stg 2, Stg3 and Stg4 that contribute to the stay-green trait³. ICRISAT focused on the Stg3 loci as it is present in two main varieties used as donors of drought tolerance in ESA. Integration of SSR and SNP markers lead to subdivision of Stg3 into Stg3A and Stg3B. A few KASP markers were then selected from Stg3A and Stg3B for further validation². 

Validating Stay-green markers 

Seven hundred and twenty-five breeding lines from the ICRISAT-ESA breeding programme including 161 that had historical data on drought tolerance including two known sources of Stg3 were selected to validate the Stg3 KASP markers. Out of the ten KASP markers, 9 amplified across 718-745 genomes including 155 of the 161 confirmed drought-tolerant lines².  Four of these markers had co-segregated and had the same allelic profile across selected varieties. On further analysis and to simplify the process in terms of cost, the research showed that two of these markers can be used as an indicator to track these QTLs across the breeding cycle and validate the transfer of Stg3A and Stg3B into the breeding lines². 

Introgression of these new QTLs by marker assisted backcrossing has been successfully demonstrated by research carried out by the University of Agriculture India⁴. The team at ICRISAT are continuing to investigate the Stay-green trait as it will be a vital component of sorghum seed stock as the climate continues to evolve.  

To find out more about the work of ICRISAT and to read their research papers please visit ICRISAT.org. 

To find out more about KASP please visit KASP genotyping assays, PCR-based genotyping | LGC Biosearch Technologies. 

Further reading

• Explore the vast applications of KASP genotyping for plant breeding programmes 
• KASP Celebrating 21 years of game changing genotyping 
• ICRISAT powerpoint on stay green trait 

Watch

• Sorghum breeding: Understanding drought tolerance and validating Stay-green KASP markers 

References 

1. Gimode DM, Ochieng G, Deshpande S, Manyasa EO, Kondombo CP, Mikwa EO, Avosa MO, Kunguni JS, Ngugi K, Sheunda P, Jumbo MB, Odeny DA. Validation of sorghum quality control (QC) markers across African breeding lines. Plant Genome. 2024 Feb 26:e20438. doi: 10.1002/tpg2.20438. Validation of sorghum quality control (QC) markers across African breeding lines - PubMed (nih.gov) 
2. Mwamahonje, A., Eleblu, J. S. Y., Ofori, K., Deshpande, S., Feyissa, T., & Tongoona, P. (2021). Drought tolerance and application of marker-assisted selection in sorghum. Biology, 10(12), 1249. Biology | Free Full-Text | Drought Tolerance and Application of Marker-Assisted Selection in Sorghum (mdpi.com) 
3. Hongxiang Z., Yingying D., Xianmin Di., Na S.,Molecular mechanisms of stress resistance in sorghum: Implications for crop improvement strategies, Journal of Integrative Agriculture Volume 23, Issue 3, 2024,Pages 741-768, https://doi.org/10.1016/j.jia.2023.12.023. 
4. Priyanka, S., G. Girish, R. Lokesha, B. V. Tembhurne, Amaregouda Patil, and Ayyanagouda Patil. 2023. “Introgression of Stay Green Quantitative Trait Locus (QTLS) into Elite Sorghum Variety by MABC”. International Journal of Environment and Climate Change 13 (10):999-1016. https://doi.org/10.9734/ijecc/2023/v13i102747
    

Tables 1-3 Gimode DM, Ochieng G, Deshpande S, Manyasa EO, Kondombo CP, Mikwa EO, Avosa MO, Kunguni JS, Ngugi K, Sheunda P, Jumbo MB, Odeny DA. Validation of sorghum quality control (QC) markers across African breeding lines. Plant Genome. 2024 Feb 26:e20438. doi: 10.1002/tpg2.20438. Validation of sorghum quality control (QC) markers across African breeding lines - PubMed (nih.gov)