Desarrollo de marcadores moleculares tipo KASP (“Kompetitive Allele Specific PCR”) útiles para la selección en programas de mejora

Resumen

Wheat is one of the three most important crops for human feed.

The content and composition of endosperm seed storage proteins are the main responsible for wheat quality.

These proteins are mainly glutenins and gliadins.

In wheat (especially in bread wheat, Triticum aestivum), glutenins are the most important determinants of end-use quality.

Up to now, sodium dodecyl sulphate–polyacrylamide gel
electrophoresis (SDS-PAGE) of grain proteins is the most widely method used for the identification of glutenins, which can be classified as high molecular weight glutenins (HMWGS) and low molecular weight glutenins (LMW-GS).

HMW-GS are highly diverse and are encoded by Glu 1-1 and Glu-1-2 genes, located at Glu-A1, Glu-B1 and Glu-D1 loci, for which several alleles that influence the quality of dough have been identified and characterized.

The interpretation of HWM glutenin subunits by the SDS-PAGE method is not always accurate due to the small differences between the electrophoretic mobility of protein bands.

Moreover, this method is time-consuming and not suitable for high-throughput analysing. The solution to this problem would be to classify the HMW-GS allelic variants by using molecular DNA markers.

The objective of this work was to develop and validate a set of molecular markers for the main HMW-GS that could assist in the characterization of this key determinants of wheat quality.

A collection of 30 selected varieties, 22 of bread wheat and 8 of durum wheat, representing a great allelic diversity was chosen, including at least two varieties carrying the same allele.

The genotype of the varieties was verified by SDS-PAGE from flour extracted from the seeds prior to the DNA extraction. The sequences of the different alleles of the Glu-A1, Glu-B1 and Glu-D1 loci were obtained from the databases and bibliography and were further analysed with the Geneious software for the identification of polymorphic regions suitable for the design of KASP molecular markers. In the present work, thirteen KASP molecular markers were designed, ten markers designed from SNPs polymorphisms (Single Nucleotide Polymorphism) and three from InDels polymorphisms (Insertion/Deletion).

The KASP markers designed from SNPs worked as expected. The markers for the Glu-A1 and Glu-D1 loci discriminated among the different subunits properly, while for the Glu-B1 gene there were some misclassifications.

KASP markers designed from Indels did not work as expected, suggesting that this type of polymorphisms is not the best option for KASP design, at least in these complex loci.

In this work, some KASP markers for HMW-GS related to good and bad end-use quality have been developed in wheat, and they are ready to be tested in a large set of germplasm in order to evaluate their potential use in high-throughput screening in breeding programs.