Sequencing vs. amplification for the estimation of allele dosages in sugarcane (Saccharum spp.).

Premise: Detecting single-nucleotide polymorphisms (SNPs) in a cost-effective way is fundamental in any plant breeding pipeline. Here, we compare three genotyping techniques for their ability to reproduce the allele dosage of SNPs of interest in sugarcane (Saccharum spp.). Methods: To identify a reproducible technique to estimate allele dosage for the validation of SNP markers, the correlation between Flex-Seq, kompetitive allele-specific PCR (KASP), and genotyping-by-sequencing and restriction site-associated DNA sequencing (GBS+RADseq) was determined for a set of 76 SNPs. To find alternative methodologies for allele dosage estimation, the KASP and Flex-Seq techniques were compared for the same set of SNPs. For the three techniques, a population of 53 genotypes from the diverse sugarcane panel of the Centro de Investigación de la Caña de Azúcar (Cenicaña), Colombia, was selected. Results: The average Pearson correlation coefficients between GBS+RADseq and Flex-Seq, GBS+RADseq and KASP, and Flex-Seq and KASP were 0.62 ± 0.27, 0.38 ± 0.27, and 0.38 ± 0.30, respectively. Discussion: Flex-Seq reproduced the allele dosages determined using GBS+RADseq with good levels of precision because of its depth of sequencing and ability to target specific positions in the genome. Additionally, Flex-Seq outperformed KASP by allowing the conversion of a higher number of SNPs and a more accurate estimation of the allele dosage. Flex-Seq has therefore become the genotyping methodology of choice for marker validation at Cenicaña.

Premisa: Detectar polimorfismos de un único nucleótido (SNP) de forma costo­efectiva es fundamental en cualquier programa de mejoramiento genético. En este artículo nosotros comparamos tres técnicas de genotipado para medir su habilidad en reproducir las dosis alélicas de SNPs de interés en caña de azúcar (Saccharum spp.). Métodos: Para identificar una técnica reproducible para la estimación de dosis alélicas durante los pasos de validación de marcadores, la correlación entre Flex­Seq, kompetitive allele­specific PCR (KASP), y genotyping­by­sequencing and restriction site­associated DNA sequencing (GBS+RADseq) fue determinada para un set de 76 SNPs. Para identificar metodologías alternativas en la estimación de las dosis alélicas, las tecnologías KASP y Flex­Seq fueron comparadas para el mismo grupo de SNPs. Para las tres técnicas, una población de 53 genotipos fue seleccionados de la población diversa de caña de azúcar del Centro de Investigación de la Caña de Azúcar (Cenicaña), Colombia. Resultados: El promedio del coeficiente de correlación de Pearson entre GBS+RADseq y Flex­Seq, GBS+RADseq y KASP, y Flex­Seq y KASP fue de 0.62 ± 0.27, 0.38 ± 0.27, y 0.38 ± 0.30, respectivamente. Discusión: Flex­Seq reprodujo las dosis alélicas determinadas usando GBS+RADseq con buenos niveles de precisión debido a su profundidad de secuenciación y habilidad de secuenciar posiciones especificas en el genoma. Adicionalmente, Flex­Seq superó a KASP al permitir la conversión de un número mayor de SNPs y al estimar las dosis alélicas de forma más precisa. Flex­Seq por tanto se convierte en la metodología de genotipado de elección para la validación de marcadores en Cenicaña.

Genetic association analysis in sugarcane (Saccharum spp.) for sucrose accumulation in humid environments in Colombia.

BACKGROUND: Sucrose accumulation in sugarcane is affected by several environmental and genetic factors, with plant moisture being of critical importance for its role in the synthesis and transport of sugars within the cane stalks, affecting the sucrose concentration. In general, rainfall and high soil humidity during the ripening stage promote plant growth, increasing the fresh weight and decreasing the sucrose yield in the humid region of Colombia. Therefore, this study aimed to identify markers associated with sucrose accumulation or production in the humid environment of Colombia through a genome-wide association study (GWAS). RESULTS: Sucrose concentration measurements were taken in 220 genotypes from the Cenicaña's diverse panel at 10 (early maturity) and 13 (normal maturity) months after planting. For early maturity data was collected during plant cane and first ratoon, while at normal maturity it was during plant cane, first, and second ratoon. A total of 137,890 SNPs were selected after sequencing the 220 genotypes through GBS, RADSeq, and whole-genome sequencing. After GWAS analysis, a total of 77 markers were significantly associated with sucrose concentration at both ages, but only 39 were close to candidate genes previously reported for sucrose accumulation and/or production. Among the candidate genes, 18 were highlighted because they were involved in sucrose hydrolysis (SUS6, CIN3, CINV1, CINV2), sugar transport (i.e., MST1, MST2, PLT5, SUT4, ERD6 like), phosphorylation processes (TPS genes), glycolysis (PFP-ALPHA, HXK3, PHI1), and transcription factors (ERF12, ERF112). Similarly, 64 genes were associated with glycosyltransferases, glycosidases, and hormones. CONCLUSIONS: These results provide new insights into the molecular mechanisms involved in sucrose accumulation in sugarcane and contribute with important genomic resources for future research in the humid environments of Colombia. Similarly, the markers identified will be validated for their potential application within Cenicaña's breeding program to assist the development of breeding populations.