v Grape ripening and anthocyanin accumulation Phenylalanine - PowerPoint PPT Presentation

Carolina Royo , Maite Rodríguez-Lorenzo, Pablo Carbonell-Bejerano, Nuria Mauri, Félix Cibríain, Julián Suberviola, Ana Sagüés, Javier Ibáñez, José M. Martínez-Zapater v

Grape ripening and anthocyanin accumulation Phenylalanine Chromosome 2 MybA2 MybA1 Anthocyanidins Black allele MybA1 UFGT Black allele MybA2 Anthocyanins MybA2 MybA1 White allele Black allele Berry growth Chromosome 2 Sugars MybA2 MybA1 White allele Anthocyanins White allele MybA2 MybA1 White allele Loss of functional allele Carolina Royo - ICVV

Somatic Variation Background periclinal chimera SAM (Adapted from Walker et al 2006)

Loss Of Heterozygosity around color locus on chr2 Background Garnacha Tinta Garnacha Roja Garnacha Blanca (Adapted from Vezzulli et al 2012 , Migliaro et al 2014) Carolina Royo - ICVV

Tempranillo Tinto and Blanco Whole Genome Sequence Background A Structural rearrangement graph: Deletion-type Tandem duplication-type Tail-to-tail inverted Head-to-head inverted Unknown 2 CNV 0 -2 SNP 1 0.5 0 1 3 5 7 9 11 13 15 17 18.8 1 3 5 7 9 11 13 15 17 19 21 23 25 1 3 5 7 9 11 13 15 17 19 21 23 (Mb) LG 2 LG 5 LG 9 B Original chromosomes Rearranged chromosomes Inv T1 T2 T3 Inv T4 Inv T3 * LG 2 LG 2 T5 T2 ? T5 T4 T3 T5 T4 LG 5 LG 5 T1 ? T1 T2 ? LG 9 LG 9 Lost fragments Chromothripsis (Adapted from Carbonell-Bejerano et al 2017) Carolina Royo - ICVV

Tempranillo Blanco Affected Traits Background Rearranged chromosomes are gamete-lethal Fruit set Fruitset (%) 70 60 50 40 30 20 10 0 Alfaro Rincón R1 Corera Grajera Blanco Cenicero Nalda Albelda Azofra Fonzaleche Grajera Tinto Complex genome rearrangements cause side-effects in yield production Carolina Royo - ICVV

Tempranillo and Garnacha accessions Plant Materials Tempranillo Tinto Tempranillo Gris Tempranillo Blanco Garnacha Tinta Garnacha Roja Garnacha Blanca 6 9 1 5 2 60 GR GB GB 2 39 21 Carolina Royo - ICVV

SNP genotyping along chromosome 2 Heterozygous in Garnacha Tinta and Tempranillo Tinto Chromosome 2 LOH genotyping: Heterozygous in Garnacha Tinta 48 SNP chip: Heterozygous in Tempranillo Tinto Chromomose 2: MybA2 MybA1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mb SNP selection from: Lijavetzky et al., 2007, Carbonell-Bejerano et al., 2017, Laucou et al., 2018 M inor A llele F requency (MAF) ≈ 0.5 L oss O f H eterozygosity ( LOH ) delimits likely deleted fragments Carolina Royo - ICVV

Loss Of Heterozygosity in Tempranillo Gris Heterozygous in Garnacha Tinta and Tempranillo Tinto Chromosome 2 LOH genotyping: Heterozygous in Garnacha Tinta 48 SNP chip: Heterozygous in Tempranillo Tinto Chromomose 2: MybA2 MybA1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mb Tempranillo Blanco WGS 2.37 Mb Tempranillo Gris type 1 (1 accesion) 1.44 Mb Tempranillo Gris type 2 (1 accesion) 1.50 Mb Tempranillo Gris type 3 (3 accesion) 1.64 Mb Tempranillo Gris type 4 (4 accesion) 2.87Mb Results obtained in roots = L2 Carolina Royo - ICVV

Loss Of Heterozygosity in Tempranillo Gris Heterozygous in Garnacha Tinta and Tempranillo Tinto Chromosome 2 LOH genotyping: Heterozygous in Garnacha Tinta 48 SNP chip: Heterozygous in Tempranillo Tinto Chromomose 2: MybA2 MybA1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mb Tempranillo Blanco WGS Tempranillo Gris type 1 (1 accesion) Tempranillo Gris type 2 (1 accesion) Tempranillo Gris type 3 (3 accesion) Tempranillo Gris type 4 (4 accesion) Tempranillo Blanco regeneration through somatic embryogenesis from Tempranillo Gris type 1 Carolina Royo - ICVV

Loss Of Heterozygosity in Tempranillo and Garnacha Heterozygous in Garnacha Tinta and Tempranillo Tinto Chromosome 2 LOH genotyping: Heterozygous in Garnacha Tinta 48 SNP chip: Heterozygous in Tempranillo Tinto MybA2 MybA1 Chromomose 2: 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mb Tempranillo Blanco WGS 2.37 Mb Tempranillo Gris type 1 1.44 Mb Tempranillo Gris type 2 1.50 Mb Tempranillo Gris type 3 1.64 Mb Tempranillo Gris type 4 2.87Mb Garnacha Blanca type 1 ( 21 accessions and 2 Garnacha Roja ) 0.68 Mb Garnacha Blanca type 2 (39 accessions) 1Mb

Loss Of Heterozygosity in Garnacha Blanca Chromosome 2 LOH genotyping chip: Heterozygous in Garnacha Tinta and Tempranillo Tinto 48 SNP Heterozygous in Garnacha Tinta Heterozygous in Tempranillo Tinto Chromomose 2: MybA2 MybA1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mb 14123701 15130499 Garnacha Blanca type 1: WGS  GT 77 and GB 77 14661047 14983912 14123701 15130499 Garnacha Blanca type 2: WGS  GT 1608 and GB 1662 Selection of two pairs of Garnacha for W hole G emone S equencing ( WGS ) Carolina Royo - ICVV

Garnacha Whole Genome Sequence analysis in progress Loss Of Heterozygosity (LOH) Copy Number Variation (CNV): Structural variation as deletions, insertions, and duplications. Carolina Royo - ICVV

Garnacha WGS: Heterozygosity on chromosome 2 Garnacha Blanca type 1 Garnacha Blanca type 2 GT77 / GB77 GT1608 / GB1662 LOH LOH SNP number 0 5 Mb 10 Mb 15 Mb chr2 position 0 5 Mb 10 Mb 15 Mb MybA MybA SNP genotyping: chr2:14123701-14661047 chr2:14123701 -15130499 chr2:14983912-15130499 Carolina Royo - ICVV

Garnacha WGS: LOH and Copy Number Variation on chromosome 2 Garnacha Blanca type 1 Garnacha Blanca type 2 GT77 / GB77 GT1608 / GB1662 LOH LOH SNP number 0 5 Mb 10 Mb 15 Mb chr2 position 0 5 Mb 10 Mb 15 Mb MybA MybA GT77 / GB77 GT1608 / GB1662 Log 2 Ratio 0 5 Mb 10 Mb 15 Mb chr2 position 0 5 Mb 10 Mb 15 Mb

Conclusions 1. The genotyping tool that we design proved useful to delimit deletions on chromosome 2. 2. The loss of color is related to hemizygous deletions at the color locus in all the variants studied by whole-genome sequencing. 3. In Tempranillo, multiple independent SV types were detected, including minimal deletion lines suited for the development of improved clones of Tempranillo Blanco. 4. In Garnacha Blanca, the results indicate the likely presence of recurrent SV fragile sites. Carolina Royo - ICVV

Maite Rodríguez-Lorenzo Pablo Carbonell-Bejerano Nuria Mauri Javier Ibáñez José M. Martínez-Zapater Félix Cibríain Julián Suberviola Ana Sagüés Carolina Royo

Carolina Royo