MOLECULAR BREEDING OF TOMATO FOR FRUIT QUALITY AND SHELF LIFE

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R.SELVAKUMAR Ph.D Scholar

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Presented by R.SELVAKUMAR

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Molecular Breeding for Fruit Quality and Shelf Life in Tomato

Aim:

Aim Discuss the possibility of multi-trait indices for quality within the context of traditional breeding and genome wide selection Discuss opportunities for new cultivar/varieties/hybrids based on real or perceived health benefits

INTRODUCTION :

INTRODUCTION Solanum lycopersicum L. ( Peralta and Spooner, 2006 ), 2n=2X=24 Second most consumed vegetable after potato Poor Man’s Orange 15 to 25 mg/100 g of vit-C & 4 times the vit “A” content of orange juice ( Gould, 1971) Protective Food Age of Solanum ∼12 million years (My), but tomato ∼ 7My ( Nesbitt and Tanksley, 2002 ) ∼ 950 Mbp of DNA , ∼35000 genes , >75% heterochromatin & largely devoid of genes, Possible spreading routes of the tomato beginning in the 16th century ( Esquinas-Alcázar and Nuez, 1995 ).

QUALITY:

QUALITY Quality is a combination of visual stimuli (e.g. size, shape, colour) & sensory factors (e.g. sugar, acidity, taste) Degree of excellence Fruit Quality traits are quantitatively inherited Quality for fresh and processing tomato fruit FRESH TOMATO PROCESSING TOMATO 1. Fruit Size and Shape Sugar 2. Colour Total Solids 3. Flavour & Aroma pH 4. Nutritional Quality Total Acidity a. Vitamins Viscosity b. Antioxidants 5. Firmness/Shelf life 6. Organoleptic Quality

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Genetic Variability and Relationship Between Quality Traits “Lycopene” (5 times higher) & Ascorbic Acid S. cheesmaniae “Ascorbic acid” Cis-3-Hexanol “Carontenoids” Total Solids Sugar Flavonoids Volatile Compounds

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Relationships between various traits, components and technologies applicable in tomato fruit quality (Arnaud Bovy, Plant Research International, Wageningen, The Netherlands )

Molecular Breeding:

Molecular Breeding Marker Assisted Breeding : a. Genotyping and Creation of Molecular Maps b. QTL (Quantitative Trait Loci) mapping or Association Mapping : A region of genome that is associated with an effect on a quantitative trait Mapping Population: F 2 population, Recombinant Inbred Lines (RILs), Backcross Inbred Lines (BILs), Back Cross lines, Advanced Back Cross lines (ABCs), Introgression Lines (ILs), Linkage Map based on molecular markers c. Marker Assisted Selection (MAS) Marker Assisted Backcrossing (MABC) Marker Assisted Recurrent Selection (MARS) Genomic Selection

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Traits Wild Species Used Mapping population Genes/ QTL’s Chromosome Fruit size/ Fruit weight S. pimpinellifoilum F 2 fw 1.1 1 S. pimpinellifoilum F 2 fw 1.2 1 S. pimpinellifoilum F 2 fw 2.1 ( lc ) 2 S. pimpinellifoilum F 2 fw 2.2 2 S. pimpinellifoilum F 2 fw 3.1 3 S. pimpinellifoilum F 2 fw 11.3 ( f ) 11 S. pennellii BC 1 5 Q 2,4,8 S. chmielewskill BC 1, BC 2 6 Q 1,4,6,7,9,11 S. cheesmaniae F 2 , F 3 11Q 1,2,3,4,6,7,9,11,12 S. chessmaniae F 8 , RIL 13 Q 1,2,3,4,6,7,9,12 S. pennellii ILs 18Q Many chromosome S. pimpinellifolium BC 1 7 Q 1,2,8,11 S. pimpinellifolium BC 2 , BC 3 8 Q 2,3,4,5,7,9 S. peruvianum BC 3 , BC 4 10 Q 1,2,3,7,8,9, 10, 12 Molecular Breeding for Fruit Size

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f - large fruits ‘ f ‘flowers fw11.3 (∼37%; previously known as fasciated, f )

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Fruit shape Wild Species Used Mapping population Genes/ QTL’s Chromosome Bumpiness S. pimpinellifolium F 2 3 Q 8, 9, 11 Bell Pepper S. pimpinellifolium F 2 3 Q 2, 8 S. pennellii F 2 1 Q 2 Blossom End Blockiness S. pimpinellifolium F 2 1 Q 2 Elongated S. pimpinellifolium F 2 2 Q 6, 9 Heart Shaped S. pimpinellifolium F 2 4 Q 1,2,3,7 Pear Shaped S. pimpinellifolium F 2 2 Q 2, 10 Stem End Blociness S. pimpinellifolium F 2 6 Q 1,2,3,7,8,12 fs8.1- fresh market (round) and processing (blocky) tomatoes ovate - round to pear-shaped fruit Molecular Breeding for Fruit Shape

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a. Elongated fruits fs8.1 - elongated fruit shape B. Pear-shaped fruit fas FAS Long John Yellow stuffer Ovate fs8.1 Fruit Shape Mutants

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Increase in Tomato Locule Number Is Controlled by Two Single-Nucleotide Polymorphisms Located Near WUSCHEL Case Study: 1 (Lippman and Tanksley, 2001)

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( Munos et al., 2011 ) Fruit morphology of lines used B. Phenotypic effect of QTLs C. Expression Analysis of the two Candidate Gene 2.4±0.5 2.7±0.5 3.5±0.5 Cervil (C) - S.pimpinellifolium Levovil (L) - S. lycopersicum F 1- C X L CF12C- NILs CF13L-NILs P<0.001 P<0.001 (Acc. No. AJ538329) (Tomato Unigene No.SGN-U585584) (Tomato Unigene No.SGN-U593757)

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( Munos et al., 2011 ) Effect of lc on locule number ns-not significant P<0.001) Fine Mapping of lc QTL Ultra High resolution Mapping of lc QTL 1608bp

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Fruit colour Wild Species Used Mapping population Genes/ QTL’s Chromosome Crimson S. lycopersicum F 2 1 G (og c , cr) 6 High pigment 1 S. lycopersicum S. cheesmaniae F 2 1 G (hp-1) 2 High pigment 2 S. lycopersicum S. pennellii BC 1 1 G (hp-2) 1 Lycopene S. pimpinellifolium S. parviflorum S. lycopersicum S. pennellii BC 1 S 1 BC 3 RIL ILs 8 Q 5 Q 2 Q 2 Q 1,4,5,6,7,10,12 Orange S. pennellii BC 2 2 Q 11, 12 Yellow S. parviflorum BC 3 1 Q 12 Molecular Breeding for Fruit Colour Two major groups of pigments: Carotenoids & Chlorophylls Lycopene is the red pigment and also contains β-carotene, phytoene, phytofluene, ζ- carotene, γ- carotene, neorosporine and lutein

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Green striped - gs Green flesh Paler corolla color phytoene synthase (PSY1) Yellow Flesh ( yf ) COLOUR MUTANTS 13 fold Flavonol

NUTRITIONAL QUALITY:

NUTRITIONAL QUALITY Traits Wild Species Used Mapping population Genes/ QTL’s Chromosome Fruit antioxidant capacity S. pennellii ILs 5 Q 3, 6, 7, 10 Fruit Ascorbic acid S. pennellii ILs 6 Q 3, 5, 10. 12 Fruit colour- Carotenoids S. lycopersicum RIL 3 Q 2, 3, 8 Fruit colour- β -carotene S. cheesmaniae S. habarochaites S. pennellii F 2 , ILs 1 G (B) 6 S. parviflorum BC 3 6 Q 2, 4, 8, 9, 10,11 S. pennellii ILs 1 G (B) 6 S. pennellii ILs 2 Q 6 -Wealth of genetic variability within landraces and wild species for fruit nutritive value -Decreased risk of cardiovascular disease and certain cancers, RAPD and AFLP and SCAR and CAPS linked to the B genes have been identified ( Zhang and Stommel, 2000 )

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Tangerine- t Carotenoid isomerase - crtiso Crimson Lycopene beta cyclase (Cyc-B) lycopene beta cyclase (Cyc-B) Fruit Colour Mutants Intense Pigment

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Delta carotene- Del Double mutant Abg/+ hp1/hp1 (purple fruit with anthocyanin) Double mutant Aft/Aft hp2/hp2 (anthocyanin in fruit epidermis) Fruit Colour Mutants Aft - S. chilense Abg - S. lycopersicoides Atv - S. chessmaniae Flavnoids - S. pennellii var. puberulum

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Molecular Aspects of Anthocyanin fruit Tomato in Relation to high pigment-1 Case Study: 2 Table 1: Average relative anthocyanidin concentration in ripe fruits of LA1996 (Aft/Aft), Moneymaker (+/+) and their F 1 plants (Aft/+) Average relative flavonol concentration in ripe fruit of LA1996 (Aft/Aft), Moneymaker (+/+), and their F1 plants (Aft/+) Fold increase in transcription of key structural genes of the flavonoid biosynthetic enzymes and Ant1 in mature-green fruits harvested from homozygous and heterozygous Aft plants relative to red-fruited Moneymaker plants (+/+) (Maya sapir et al ., 2008) Table 2: Table 3: (g of fresh weight) ( g of fresh weight )

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Fig 1: Codominant polymorphisms between the Ant1 alleles originating from Solanum lycopersicum (Moneymaker, +/+), Solanum chilense (LA1996, Aft/Aft), and their F 1 hybrid (Aft/+). M is a 2-log DNA size ladder. Fig 2: Mapping of Ant1 to the tomato genome. PCR products obtained by amplification of DNA extracted from Solanum lycopersicum (M82), Solanum pennellii (Pen) and the 3 introgression lines spanning chromosome 10 (IL 10-1, 2, 3). M is a 2-log DNA size ladder. Map of the tomato chromosome 10 showing the 3 introgression segments The dotted line in IL 10-1 shows the expected location of the Ant1 gene. (Maya sapir et al ., 2008) Fig 3:

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Average relative concentrations of major anthocyanidins detected in ripe fruits of parental and F 3 genotypes Average relative concentrations of major flavonols detected in ripe fruits of parental and F 3 genotypes (Maya sapir et al ., 2008) Table 4: Table 5: 5, 19, and 33 fold increase of petunidin, malvidin & delphinidin respectively in the double mutants Inference: (g of fresh weight) (g of fresh weight)

Molecular Breeding for Fruit Flavour:

Molecular Breeding for Fruit Flavour More than 400 aroma volatiles have been identified in tomato, but only 15–20 are present in sufficient quantities to impact flavor ( Buttery, 1993; Baldwin et al ., 2000 ). Humans have c. 350 olfactory receptor genes Flavor=sugars + acids + 20 or more volatile chemicals + primary and secondary metabolites Volatile genes are two classes : those that encode enzymes responsible for synthesis of the end products & those encoding factors that regulate pathway output Molecular Assisted Flavour Breeding: Identification of genes encoding biosynthetic enzymes through the extensive genome and expressed sequence tag (EST) databases It contains over 3,30,000 ESTs assembled into 46, 849 unique sequences Combination of genome and transcriptome sequencing High-throughput molecular marker screening now makes breeding for flavor much more realistic.

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QTL discovery: S. pennellii (Tieman et al ., 2006b) or S. habrochaites (Mathieu et al ., 2008), locus at the bottom of chromosome 1 that specifically affects 2-methoxyphenol (Guaiacol). S. pennellii locus at the bottom of chromosome 4 is altered in a very large set of primary metabolites ( Schauer et al ., 2006 ) and flavor volatiles ( Tieman et al ., 2006b ). QTLs identified for altered in volatile emissions ( Saliba-Colombani et al ., 2001; Tieman et al ., 2006a )

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Map locations of QTLs with altered volatile emissions QTLs affected in volatile emissions ( Mathieu et al., 2009 ) Flavour compounds in tomato fruits: identification of loci and potential pathways affecting volatile composition Case Study: 3

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Carotenoids and apocarotenoid volatiles produced by LA3923 (A) Developmental series of fruits from control (LA4024, squares) and LA3923 (triangles) harvested at breaker (0) and subsequent days ( B) Emissions of 6-methyl-5-hepten-2-one (MHO) (red) and geranylacetone (blue) from fruits at the indicated stages of ripening C) Quantification of lycopene (red) and b-carotene (green) LA1777 is the S. habrochaites parent. the broken ordinate scales for values on the graphs shown in (B) and (C). Graphs show values 6SE. ( Mathieu et al., 2009 ) S. lycopersicum cv. E6203

Shelf Life:

Shelf Life Climacteric Fruit

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Fruit ripening Wild Species Used Mapping population Genes/ QTL’s Chromosome Alcobaca S. lycopersicum S. pimpinellifolium F 2 BC 1 Alc alc 10 10 Colourless non ripening S. lycopersicum S. chessmaniae F 2 Cnr 2 Never ripe S. lycopersicum S. chessmaniae F 2 Nr 9 Non ripening S. pennellii S. chessmaniae F 2 nor 10 Polygalacturonase S. pimpinellifolium BC 1 TOM 6 10 Ripening inhibitor S. pennellii S. chessmaniae F 2 rin 5 Uniform ripening S. pimpinellifolium BC 1 u 10 Fruit ripening S. lycopersicum S. pennellii S. pimpinellifolium S. pimpinellifolium S. peruvianum F 2 F 2 BC 1 BC 2 , BC 3 BC 3 , BC 4 2Q Many Loci 3 Q 4 Q 4Q 5,12 All 2,8,9 2,4,7,8 2,3,8,9 Molecular Breeding for Shelf Life

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rin nor Fruit Ripening Mutants

CONCLUSION :

CONCLUSION Developed and utilized the pre-designed phenotypes (fruit shape, size, and colour) Characterization and functional valitation of trait based genes Enhancement of protective food diversity for consumers (major and micro nutrients) Utilization of molecular markers for vegetable breeders

Future Thrust:

Future Thrust Development of SNP markers for high throughput genome analysis. Sequencing of tomato genome leads to development of additional sequence specific markers To streamline the use of QTLs Need to understand the potential negative association of flavour, metabolites with undesirable genes Need dissect the functional markers for specific traits Combination of traditional breeding protocols & marker assisted breeding

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