Delivering better cultivars, faster, is a continuous challenge for plant breeders. Genetic tools and discoveries have transformed breeding strategies, but even once suitable trait-markers are identified, turning these into routine, reliable selection tools can still be difficult.
For quantitative traits such as pear flesh firmness, which change as the fruit develops and ripens, traditional phenotypic selection is slow, labour-intensive and complicated by seasonal variation.
However, when breeders switch to using molecular markers techniques for selection, several practical issues can occur:
- QTLs and associated traits can look promising
in discovery populations but prove weak or may be inconsistent in elite breeding germplasm. - Firmness phenotyping requires fruit at a specific maturity stage, specialised equipment and destructive tests, limiting how early and how many individuals can be screened.
- Converting a known single nucleotide polymorphism (SNP) into a robust, high-throughput genotyping assay that fits existing lab and data workflows takes time and expertise.
- Limited budgets and staff mean molecular tools must be low cost per sample and have a clear impact on selection decisions to justify routine use.
Scientists at Shanghai Academy of Agricultural Sciences addressed these pain points in their work on pear flesh firmness in Pyrus pyrifolia.1 Starting with the identification of a flesh firmness-related QTL, they moved to a single, dominant SNP with a clear effect on firmness, before converting that SNP into a KASP assay that can be deployed at scale in their breeding programme. They added transcriptome data to place the QTL in biological context, strengthening its association with flesh firmness-related genes. Below, we explore how they achieved this.
Defining contrasting parents and the mapping population
The researchers selected two Pyrus pyrifolia cultivars with contrasting firmness – the softer ‘Zaoshengxinshui’ cultivar, and firmer ‘Qiushui’ cultivar. They planted ninety-two progeny from crosses involving contrasting phenotypes and measured flesh firmness of parents and offspring at 100 days after blossom over two years using a texture analyser.
They observed similar firmness ranges in both years, with ‘Qiushui’ firmer than ‘Zaoshengxinshui’ and offspring spanning 1.5-5.8 N/cm², indicating a stable, quantitative trait.
QTL mapping
The research team constructed a genetic map based on previous research2 and the genome of Pyrus pyrifolia ‘Cuiguan’ was used as the reference genome for pear.
With the offspring phenotype data from the two years, the team performed interval mapping to detect putative QTLs for flesh firmness-related traits. They grouped the markers into linkage groups (LG) using a single-linkage clustering algorithm, taking the independent logarithm of odds score as a distance measure.
They found a consistent QTL peak on LG14 at 139.857 cM, which showed the highest mean logarithm of odds value across years – this was designated qFirmness‑LG14.
Identification of a trait-associated SNP 
The team examined five markers that were found at the
qFirmness‑LG14 peak and focused on Marker1512129. This marker contained alleles ‘lm × ll’ from the parents, with the flesh firmness related to the ‘m’ allele.
The site carries a C/T SNP. They found that the C allele was associated with high firmness, behaved dominantly, and increased average firmness by 21.4% compared with genotypes lacking the C allele at that position.
Designing a KASP assay
The researchers submitted sequences of Marker1512129 (C/T SNP) to LGC Biosearch Technologies to order a KASP™ Assay Mix for endpoint genotyping analysis. They tested the KASP assay on sixteen offspring and obtained genotype calls consistent with re-sequencing data, demonstrating that the assay is robust and effective.
Adding biological context with transcriptomics
The research team integrated transcriptomic data to better understand how the qFirmness‑LG14 region might influence the structure and composition of the fruit cell wall and the activity of enzymes and regulators involved in cell wall modification.
They performed RNA-seq on ‘Qiushui’ and ‘Zaoshengxinshui’ at five time points and established that 75 days after blossom was a key stage for firmness determination. They studied genes within the 462 kb genomic interval around qFirmness‑LG14 on chromosome 13 that were differentially expressed between the two cultivars and had the potential to be candidates for firmness regulation, based on annotation data.
They found that a xyloglucan endotransglucosylase 1 gene (PpXTH1) was upregulated in ‘Qiushui’ and two transcription factors (PpHY5 and PpERF113) were upregulated in ‘Zaoshengxinshui’, which could act as negative regulators of high flesh firmness.
They also found many cell wall-related genes (e.g. Pectate lyase, Pectin acetylesterase, Pectin methylesterase, and 4-coumarate-CoA ligase) and transcription factors (e.g. ERF, WRKY) that are all potential upstream and downstream genes related to flesh firmness.
This combination of QTL mapping with transcriptomics narrows down plausible causal genes and provides biological context for marker development.
Check out how other researchers have used KASP in these peer-reviewed papers.
Integrating KASP genotyping into breeding programmes
The firmness-associated QTL qFirmness‑LG14 and the associated KASP marker provide a practical, robust assay to select for more desirable flesh firmness, rather than relying solely on late, variable phenotyping.
As new QTLs for firmness and additional target traits are identified in pears and other crops, building panels of KASP assays can support multi-trait selection. These panels can enable earlier screening of seedlings at higher throughput and with greater confidence in marker performance.
KASP genotyping technology has expansive applications in modern plant breeding programmes, with flexibility for service-based genotyping or in-house use, thanks to its compatibility with existing lab infrastructure.
For expert support converting your own SNPs into KASP assays for routine use in your breeding pipelines, contact us and try KASP for free.
Further reading
- Explore the vast applications of KASP genotyping for plant breeding programmes
- The comprehensive ebook: Overcoming agricultural challenges with KASP genotyping chemistry
References
- Jiang S, Zhang J, Wang X, et al. Identification of Candidate Genes Associated with Flesh Firmness by Combining QTL Mapping and Transcriptome Profiling in Pyrus pyrifolia. Int J Mol Sci. 2024. 22;25(21):11347. doi:10.3390/ijms252111347
- Jiang S, Luo J, Wang X, et al. QTL mapping and transcriptome analysis to identify genes associated with green/russet peel in Pyrus pyrifolia. Sci Hortic. 2022. 293: 110714. doi:10.1016/j.scienta.2021.110714
