Validação de marcadores SSR-Seq para Eugenia klotzschiana O.BERG. (Myrtaceae)

Abstract

Eugenia klotzschiana O. Berg. (Myrtaceae) is popularly known as the Cerrado pear. The species is listed in the group of priority plants for research in the publication “Plants for the Future of the Central-West Region”. The Cerrado pear can be consumed in natura or in the production of jellies and sweets, presenting high nutritional value. Molecular markers are useful tools for the characterization of plant genetic resources. Among the available molecular markers, microsatellites or Simple Sequence Repeats (SSRs) are useful in the investigation of the genetic structure for population studies in plants such as E. klotzschiana, aiding in their conservation and sustainable use. The objective of this study was to establish a base protocol for genotyping by sequencing (GBS) of SSR markers and to make available polymorphic and informative loci for the species E. klotzschiana. Thus, 22 pairs of primers previously developed for the species were used to test genotyping in different multiplex systems, in order to verify the influence of the quantity of primers amplified simultaneously in the detection of alleles. DNA was extracted from the leaf tissue of 37 individuals collected in the municipality of Senador Canedo-GO using the CTAB 2% protocol. To verify the quantity and integrity of the extraction, horizontal electrophoresis was performed in 1% agarose gel. After confirmation of amplification, the primers were tested in different multiplex PCR (mPCR) systems, containing between 5 and 22 pairs of primers per system. The multiplexes were assembled in three tests, with different quantities of primers in the mPCR systems. In test 1 (T1), four multiplexes were assembled with a minimum of five and a maximum of six primers each; in test 2 (T2), two mPCRs were assembled with half the quantity of primers each; in test 3 (T3), a single multiplex was assembled with all primers amplified simultaneously. In order to obtain a better amplification pattern, different primer concentrations were tested for mPCRs. From the validation of the library pool at 4nM, the 500-cycle Miseq v2 cartridge was prepared for sequencing on the MiSeq Illumina® platform. Sequence quality analysis was performed in FastQC and allele calling using the SSR-GBS pipeline. Even with primer concentration adjustment, T1 and T2 presented amplification patterns with more intense bands, when compared to the T3 test in which all 22 primers were used in a single PCR reaction. The analyses suggest that there was great success in microsatellite genotyping by sequencing in E. klotzschiana. Allele calling during sequencing obtained a low base calling error rate, with an average Phred value of 38. As expected, there was a difference in allele calling between tests, with an increase in lost data in test 3. The assignment of alleles that were called the same for the same loci between the T1 and T2 tests was above 90%, with the exception of a single locus that obtained 70% similarity. As the T3 test had a higher rate of genotyping errors that required manual checking (MC), had few reads (PR) and/or were left without amplification (NA), this obtained a lower similarity between the loci evaluated. Finally, the sampled population was predominantly made up of clonal individuals. The results obtained demonstrate the effectiveness of genotyping by sequencing (GBS) for the species E. klotzschiana, providing valuable information for its genetic characterization. These data may contribute to future conservation strategies and sustainable use of the Cerrado pear.