Multiplex PCR for Early Generation Identification of Tomato Segregants Carrying Ty-2, Ty-3 and Ph-3 Resistance Alleles Against Leaf Curl and Late Blight Diseases.

Deployment of different natural disease resistance alleles is the most sustainable and eco-friendly way for multiple disease management in tomato. Diagnostic molecular markers are indispensible in this effort as they offer early generation identification of resistance alleles in an environment-independent manner. Moreover, optimized multiplex polymerase chain reaction (PCR) for detecting different disease resistance alleles in a single reaction can speed-up the selection process with cost and labour-effectiveness. Here we report the optimized multiplex detection and stacking of leaf curl disease resistance alleles Ty-2 and Ty-3 along with late blight disease resistance allele Ph-3 in tomato genotypes and F2 segregants. The triplex assay could be replaced by a duplex assay (for Ty-2 and Ty-3 resistance alleles) followed by analysis at Ph-3 locus to achieve further cost-effectiveness. We identified two plants in F2 populations derived from the Arka Samrat (F1) x Kashi Chayan combination to carry the Ty-2, Ty-3 and Ph-3 resistance alleles in homozygous condition. Early generation genotyping also allowed us to identify a few morphologically better segregants, where further marker assisted selection (MAS) should identify superior multiple disease resistant lines. Thus we advocate the utility of multiplex PCR in MAS to address multiple disease resistance breeding in tomato.

Marker assisted early generation identification of root knot disease resistant orange tomato segregants with multiple desirable alleles.

Enhanced bioavailability of cis-isomers of lycopene, accumulated in orange-fruited tangerine mutant has broadened the scope of nutritional enrichment in tomato. At the same time, advancements in the field of marker assisted selection (MAS) have made the stacking of multiple desirable alleles through molecular breeding to develop superior tomato genotypes possible. Here we report seedling stage MAS from 146 F2 plants, to identify 3 superior performing, root knot disease resistant orange-fruited segregants. In the selected segregants, fruit weight ranged from 39.2 to 54.6 g, pericarp thickness ranged from 4.56 to 6.05 mm and total soluble solid content ranged from 3.65 to 4.87° Brix. Presence of parental diversity allowed identification of the other desirable alleles of the genes governing late blight and mosaic disease resistance, growth habit (determinate and indeterminate) as well as fruit elongation and firmness. Resistance to root knot disease of the selected 3 segregants was also validated through a unique method employing in vitro rooted stem cuttings subjected to artificial inoculation, where the resistant parent and the selected segregants developed no galls in comparison to ~ 24 galls developed in the susceptible parent. The selected segregants form the base for development of multiple disease resistant, nutritionally enriched orange-fruited determinate/indeterminate tomato lines with superior fruit quality. The study also highlights the utility of early generation MAS for detailed characterization of segregants, through which multiple desirable alleles can be precisely targeted and fixed to develop superior tomato genotypes. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01361-1.

Rapid genotyping in tomato by VPCR using agarose gel-resolvable InDel markers.

Insertion/deletion (InDel) markers are second most abundant polymerase chain reaction (PCR)-based molecular markers having enormous applications in genotyping and molecular breeding in different crops. Although standard polymerase chain reaction (PCR) for DNA amplification generally takes ~ 1.5 to 2 h, small amplicons can be effectively generated using dynamic heating and cooling through PCR with "V"-shaped thermal profile (VPCR) in ~ 15 to 20 min. Here, we evaluated the applicability of a partly modified VPCR method for amplifying InDels of tomato genome. Out of the 31 InDel markers tested in 15 diverse tomato genotypes, 29 markers resulted in sharp amplicons, where 26 markers were found to be polymorphic. Using this method, the individual DNA amplification reactions could be completed within ~ 30 min. The method was effective for primers varying in melting temperature (T m) and GC contents. Furthermore, the need for empirically determining suitable annealing temperature could be bypassed using this generalised thermal profile. Through our results, we advocate the use of this method of DNA amplification in other plants to achieve rapid genotyping using standard molecular biology equipments and procedures. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03499-x.

Marker assisted stacking of Ty3, Mi1.2 and Ph3 resistance alleles for leaf curl, root knot and late blight diseases in tomato.

Developing multiple disease resistance through naturally available host resistance alleles is a challenging as well as rewarding area of research. Availability of host resistance alleles and the reliability of their identification through diagnostic molecular markers have paved the way for stacking of these resistance alleles for developing important genetic resources in tomato. Here we report the marker assisted stacking of Ty3, Mi1.2 and Ph3 alleles, governing leaf curl, root knot and late blight disease resistance, respectively, in superior F4 segregants of tomato derived from two diverse parents (i.e., BRDT-1 and H-88-78-1). Marker assisted selection was applied only on morphologically superior segregants at F2 and F3 generations, which helped us in identifying suitable lines even from a relatively small population. The diagnostic values of the employed molecular markers advocate that the identified superior segregants, carrying all the three aforementioned resistance alleles in homozygous condition, are suitable to be explored as valuable genetic resources for developing multiple disease resistance through rapid introgression of these genes in different genetic background of tomato. Identification of suitable segregants derived from these lines should be promising for obtaining improved cultivars in near future. Nevertheless, these lines might be further explored to decipher the intrinsic details of host's resistance mechanism involving genetic interactions between different resistance factors. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01277-2.

Development and validation of the OVATE gene-based functional marker to assist fruit shape selection in tomato.

Fruit size, shape and colour are important determinants of fruit quality in tomato. Among the different genetic factors, the OVATE gene is a key regulator of fruit elongation in tomato. The loss-of-function recessive ovate allele results from a functional single nucleotide polymorphism (SNP) in the second exon of the gene to produce fruit elongation and variable fruit shapes in different genetic backgrounds. The mutation has also been associated with increased fruit firmness, a desirable trait for processing purpose of tomato. However, the recessive nature of this important mutant allele makes its identification and utilization in breeding programme difficult. Hence, we developed the OVATE gene-based functional marker using the tetra-primer amplification refractory mutation system (T-ARMS) strategy. The developed functional marker was capable of identifying the allelic status at OVATE locus in a co-dominant manner, using routine polymerase chain reaction (PCR) followed by standard agarose gel electrophoresis. Trait-marker association of the developed functional marker was validated in the F2 segregants bearing elongated and non-elongated fruits. Thus, the functional marker developed and validated in this study will assist the tomato breeders in identification and introgression of the desired allelic version of the OVATE gene in a time-, labour- and cost-effective manner. Moreover, identification of the allelic status at the OVATE locus will help in exploring its interacting partners and modifiers for detailed understanding of the fascinating genetics behind fruit shape variation in tomato. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03029-7.

Development of tetra-primer amplicon refractory mutation system (T-ARMS) strategy for identification of the dark green mutant allele in tomato.

The high pigment (hp) tomato mutants increase the fruit pigment content in tomato. The dark green (dg) tomato mutant belonging to this category contains the hp-2 dg recessive allele, resulting from a single nucleotide polymorphism (SNP) in the DEETIOLATED1 (DET1) gene. Naturally, identification of this functional SNP is crucial for introgression of this important mutant allele in suitable tomato cultivar(s). Hence, we developed a functional co-dominant marker for detecting the hp-2 dg mutant allele using the tetra-primer amplicon refractory mutation system (T-ARMS) strategy. Although the other available techniques for identification of SNPs require specialized costly equipments and reagents, our method allows the reliable identification of this functional SNP in time-, labour- and cost-effective manner, using standard molecular biology equipments and reagents. To the best of our knowledge, this is the first report of T-ARMS strategy for the detection of the dg mutant allele in tomato, which will definitely help in identification and introgression of this allele by the breeders, working even under limited fund and resources.

Skin colour, carotenogenesis and chlorophyll degradation mutant alleles: genetic orchestration behind the fruit colour variation in tomato.

The genetics underlying the fruit colour variation in tomato is an interesting area of both basic and applied research in plant biology. There are several factors, like phytohormones, environmental signals and epistatic interactions between genes, which modulate the ripe fruit colour in tomato. However, three aspects: genetic regulation of skin pigmentation, carotenoid biosynthesis and ripening-associated chlorophyll degradation in tomato fruits are of pivotal importance. Different genes along with their mutant alleles governing the aforementioned characters have been characterized in detail. Moreover, the interaction of these mutant alleles has been explored, which has paved the way for developing novel tomato genotypes with unique fruit colour and beneficial phytonutrient composition. In this article, we review the genes and the corresponding mutant alleles underlying the variation in tomato skin pigmentation, carotenoid biosynthesis and ripening-associated chlorophyll degradation. The possibility of generating novel fruit colour-variants using different combinations of these mutant alleles is documented. Furthermore, the involvement of some other mutant alleles (like those governing purple fruit colour and high fruit pigmentation), not belonging to the aforementioned three categories, are discussed in brief. The simplified representation of the assembled information in this article should not only help a broad range of readers in their basic understanding of this complex phenomenon but also trigger them for further exploration of the same. The article would be useful for genetic characterization of fruit colour-variants and molecular breeding for fruit colour improvement in tomato using the well-characterized mutant alleles.