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. 2019 Mar 11;10:276.
doi: 10.3389/fpls.2019.00276. eCollection 2019.

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

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

Fabio Palumbo et al. Front Plant Sci. .
Free PMC article

Abstract

We report the first high-density linkage map construction through genotyping-by-sequencing (GBS) in leaf chicory (Cichorium intybus subsp. intybus var. foliosum, 2n = 2x = 18) and the SNP-based fine mapping of the linkage group region carrying a recessive gene responsible for male-sterility (ms1). An experimental BC1 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 ms1 locus. Two backbone SSR markers belonging to linkage group 4 of the available Cichorium consensus map were found genetically associated to the ms1 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 ms1 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 (Lactuca sativa 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, ms1ms1, and 4 male fertile inbreds, Ms1Ms1) 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 BC1 population. Overall, our molecular data could be practically exploited for genotyping plant materials and for marker-assisted breeding schemes in leaf chicory.

Keywords: Cichorium intybus; genetic linkage map; genotyping-by-sequencing (GBS); male sterility; ms1 locus; single nucleotide polymorphism (SNP) markers; transcription factor MYB103.

Figures

FIGURE 1
FIGURE 1
Schematic representation of the breeding populations developed for mapping the gene responsible for male-sterility in Cichorium intybus. A male-sterile mutant plant (genotype msms), belonging to a cultivated population of radicchio was crossed with a male-fertile plant (genotype MsMs) selected from a spontaneous accession of wild chicory. An F1 male-fertile plant heterozygous at the male-sterility locus (genotype Msms) was selfed and an F2 male-sterile plant (genotype msms) of the segregating population was then backcrossed as seed parent to a sister F1 male-fertile plant (genotype Msms) used as pollen donor. Individuals of the BC1 population were phenotyped by visual observations of anthers (in vivo screening for the presence/absence of pollen) and cytological investigations (in vitro staining of pollen using acetocarmine and DAPI solutions; for detailed information on pollen viability analysis please see Barcaccia and Tiozzo Caenazzo, 2012). At flowering, anthers were preliminary screened for the absence vs. presence of pollen and microscopy was then used to validate the sterile vs. fertile phenotype (mutants were characterized by shapeless, smaller and shrunken microspores as compared to the wild-type ones).
FIGURE 2
FIGURE 2
Molecular markers mapped on linkage group 4 and found associated to the male sterility locus (ms1) of C. intybus. (A) Linkage group 4 constructed from Cadalen et al. (2010). (B) Genetic distances, expressed in cM, among three SSR markers (in red), one CAPS marker derived from the MADS-box region (in blue) and the male-sterility locus, whose estimates were calculated using a BC1 population (198 samples) segregating 1:1 for the male-sterility trait. The SSR markers were retrieved from Ghedina et al. (2015) while the CAPS marker was developed in the present work. (C) Linkage group 9 [corresponding to linkage group 4 of Cadalen et al. (2010)] constructed using the data from the Genotyping-By-Sequencing (GBS) approach and based on 44 samples of the BC1 population mentioned above. In red are reported the two SSR already used for the first SSR-approach, in bold are listed the 13 SNP showing ≤3 recombinants with the ms1 locus. (D) Chromosomal region around the ms1, considering the recombinant data of the aforesaid 13 SNP and a total number of 108 BC1 samples.
FIGURE 3
FIGURE 3
The first SNP-based linkage map in C. intybus. Tags underlined represent those mapped sequences showing a significant match (BLASTN, similarity >90%, E-value < 1E-50) with contigs from the first genome draft of chicory (Galla et al., 2016) that in turn aligned against protein from the TAIR database (BLASTX, E-value < 1E-5). The correspondence between each underlined tag and the best TAIR match is reported in Supplementary Table S3. The male-sterility locus (ms1) was assessed by recording the target locus as a putative gene fully co-segregating with the trait. Four SSR markers, M4.10b, M4.12 [from linkage group 4 of Cadalen et al. (2010)], M2.6 and M2.4 [from linkage group 2 of Cadalen et al. (2010)] were used to genotype the same samples employed for the SNP-based map and integrated in the linkage map. M4.10b and M4.12 were chosen because co-segregating with the ms1 (Barcaccia and Tiozzo Caenazzo, 2012, 2014), M2.6 and M2.4 were selected because were found to be associated with the sporophytic self-incompatibility (SSI) locus by Gonthier et al. (2013).
FIGURE 4
FIGURE 4
Analysis of MYB103 in C. intybus. (A) Micro-mesosynteny between a peripheral region of chromosome 5 of L. sativa and a DNA region of 9.9 cM around ms1 locus of C. intybus, suggested a possible involvement of MYB103 in the male sterility condition. (B) The alignment of eight MYB103 sequences – four sequenced from male fertile (wt) unrelated accessions and four from male sterile accessions (ms), highlighted an insertion of four bases in the second exon of the latter group. The insertion introduced a pre-mature stop codon and the predicted protein resulted 123 amino acids shorter (i.e., 198 amino acids long rather than 321 long for the wt protein). (C) Example of the AS-PCR profiles generated in the male sterile mutants (ms), male fertile wild-type (wt) parents and a representative subset of 8 BC1 progeny plants (segregating 1 Msms : 1 msms) with the allele-specific primers for MYB103.

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