Identification of zygotic and nucellar seedlings in Citrus limon: the search for molecular markers

All Spanish lemon cultivars are polyembryonic. Thus, when a breeding program in lemon tree (Citrus limon) is carried out by hybridization, the identification of zygotic seedlings with molecular markers is a critical stage. The aim of this study was to obtain a group of molecular markers able to identify zygotic plants belonging to a cross of two Spanish lemon cultivars 'Verna 51' and 'Fino 49'. Fourteen SSRs markers developed for different Citrus species and sixteen SSRs markers used in unrelated species were tested but neither trial was successful. Similarly, SNPs developed for other lemon tree cultivars were also tested but none of them proved useful for our study. In light of these results, it was decided to look for specific SNP type molecular markers for 'Verna 51' and 'Fino 49'. Samples were collected from both cultivars, the DNA was extracted, and two libraries constructed were sequenced with Illumina technology. After running the bioinformatics discrimination process on the nearly 5 million “SNP” type variants initially detected, 35 homozygous variants were selected that differed between both cultivars: 25 SNPs from coding regions and 10 SNPs from non-coding regions. Primers to amplify these SNPs were designed and tested in the laboratory. Four of these SNPs, two from coding regions and two from non-coding regions, successfully distinguished the hybrids from the nucellar plants. In order to get more molecular markers of this type, the raw data obtained from the sequencing of 'Verna 51' and 'Fino 49' were used to perform a new SNP calling. Thirty six new molecular markers were detected in homozygosis. They will be tested at our laboratory to confirm their suitability for detecting hybrid plants.