AUTHOR=Palumbo Fabio , Qi Peng , Pinto Vitor Batista , Devos Katrien M. , Barcaccia Gianni 

TITLE=Construction of the First SNP-Based Linkage Map Using Genotyping-by-Sequencing and Mapping of the Male-Sterility Gene in Leaf Chicory

JOURNAL=Frontiers in Plant Science

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2019.00276

DOI=10.3389/fpls.2019.00276

ISSN=1664-462X

ABSTRACT=<p>We report the first high-density linkage map construction through genotyping-by-sequencing (GBS) in leaf chicory (<italic>Cichorium intybus</italic> subsp. <italic>intybus</italic> var. <italic>foliosum</italic>, 2<italic>n</italic> = 2<italic>x</italic> = 18) and the SNP-based fine mapping of the linkage group region carrying a recessive gene responsible for male-sterility (<italic>ms1</italic>). An experimental BC<sub>1</sub> population, segregating for the male sterility trait, was specifically generated and 198 progeny plants were preliminary screened through a multiplexed SSR genotyping analysis for the identification of microsatellite markers linked to the <italic>ms1</italic> locus. Two backbone SSR markers belonging to linkage group 4 of the available <italic>Cichorium</italic> consensus map were found genetically associated to the <italic>ms1</italic> gene at 5.8 and 12.1 cM apart. A GBS strategy was then used to produce a high-density SNP-based linkage map, containing 727 genomic loci organized into 9 linkage groups and spanning a total length of 1,413 cM. 13 SNPs proved to be tightly linked to the <italic>ms1</italic> locus based on a subset of 44 progeny plants analyzed. The map position of these markers was further validated by sequence-specific PCR experiments using an additional set of 64 progeny plants, enabling to verify that four of them fully co-segregated with male-sterility. A mesosynteny analysis revealed that 10 genomic DNA sequences encompassing the 13 selected SNPs of chicory mapped in a peripheral region of chromosome 5 of lettuce (<italic>Lactuca sativa</italic> L.) spanning about 18 Mbp. Since a MYB103-like gene, encoding for a transcription factor involved in callose dissolution of tetrads and exine development of microspores, was found located in the same chromosomal region, this orthologous was chosen as candidate for male-sterility. The amplification and sequencing of its CDS using accessions with contrasting phenotypes/genotypes (i.e., 4 male sterile mutants, <italic>ms1ms1</italic>, and 4 male fertile inbreds, <italic>Ms1Ms1</italic>) enabled to detect an INDEL of 4 nucleotides in its second exon, responsible for an anticipated stop codon in the male sterile mutants. This polymorphism was subsequently validated through allele-specific PCR assays and found to fully co-segregate with male-sterility, using 64 progeny plants of the same mapping BC<sub>1</sub> population. Overall, our molecular data could be practically exploited for genotyping plant materials and for marker-assisted breeding schemes in leaf chicory.</p>