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.

Tetra-Primer Amplification Refractory Mutation System (T-ARMS).

Single-nucleotide polymorphisms (SNPs), the most abundant genetic variation in the population, have become the molecular marker of choice. Generally, the efficient detection of SNPs requires specialized costly equipment. Although there are a few strategies for detecting SNPs through polymerase chain reaction, followed by restriction enzyme digestion and agarose gel electrophoresis, these methods are time-consuming and might be less diagnostic. Interestingly, the tetra primer amplification refractory mutation system (T-ARMS) strategy utilizes a pair of allele-specific primers in a single PCR for the diagnostic detection of SNPs in a codominant manner through standard agarose gel electrophoresis. The simplicity and robustness of the strategy have inspired the researchers to adopt this low-cost method of SNP detection in different crop plants. Here, we have described the principle, methods, and conditions for the T-ARMS strategy. The described methodology starts from the isolation of genomic DNA and ends with the post-PCR analysis of refractory amplicons in standard agarose gel electrophoresis. The limitations and future perspectives are also discussed. Taken together, T-ARMS evolves as a method of choice for low-cost SNP detection in plants.

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.

First Report of Agrobacterium rhizogenes-induced Hairy Root Formation in Selaginella bryopteris: a Pteridophyte Recalcitrant to Genetic Transformation

Abstract we report A. rhizogenes-induced hairy root formation in S. bryopteris, a medicinally and commercially important plant. A. rhizogenes strain LBA1334 co-cultivated with explants (root, rhizophore, stem portion near the root, and stem with intact fronds) for 24 and 48 h after transformation for induction of hairy roots. The induction of hairy root was observed after 6 days of infection in case of 48 h co-cultivation only. PCR with rolA and virC gene specific primers confirmed the induced hairy roots were due to Ri T-DNA integration and not due to contaminating A. rhizogenes. The root network as explants showed the maximum transformation efficiency. We tested different media like MS, SHFR (Stage Hog Fern Root) and KNOP's during transformation for hairy root induction. The SHFR based media showed good response in transformation as well as propagation. Further, transformation efficiency was enhanced by addition of TDZ (2 mg/L) and Bevistin (0.1%) in SHFR media. The present work would be helpful in hairy roots-based in vitro production of secondary metabolites and on aspect of functional genomics of S. bryopteris.

Spatio-temporal regulation of the OsHFP gene promoter establishes the involvement of this protein in rice anther development.

Anther development leading to pollen maturation, anther dehiscence and pollen dispersal depends upon the precise timing of programmed cell death (PCD) in specified anther tissues. The PCD necessitates a properly tuned transcriptional regulation of some crucial genes. However, the detailed genetic regulation of this PCD in rice anther is yet to be deciphered. Recently, we have established that the OsHFP, a structurally novel hemopexin fold protein of rice is a flower-specific heme binding protein, and plays a role in chlorophyll degradation. Here, we report the spatio-temporal transcriptional regulation of the OsHFP gene, which is proposed to be involved in anther PCD. The OsHFP was immunodetected in rice anthers, and OsHFP-related proteins were also found to be present in anthers of other monocot (lily) and dicot (tobacco) plant species. Unique cis-acting elements, possibly involved in the activation and anther-specificity of the OsHFP promoter were identified based upon in silico prediction and in planta expression profiling of the reporter gene driven by the OsHFP promoter (2051 bp) and its two deleted versions (1057 bp and 437 bp). The temporal regulation of the OsHFP promoter in different developmental stages of tobacco anther implies the physiological function of this protein in anther PCD.

A structurally novel hemopexin fold protein of rice plays role in chlorophyll degradation.

Proteins containing hemopexin fold domain are suggested to have diverse functions in various living organisms. In order to investigate the structure and function of this type of protein in rice plant (Oryza sativa), the gene encoding a hemopexin fold protein (OsHFP) was cloned, analyzed in silico and characterized. Molecular modeling revealed that the OsHFP is closely related to other hemopexin fold proteins, but is unique with a cylindrical central tunnel as well as extended N- and C-terminal domains. The recombinant OsHFP was found to bind hemin, the oxidized form of heme in vitro. The expression of the single copy OsHFP gene was detected in rice flower buds. Heterologous expression of OsHFP in green leaf tissues resulted in chlorophyll degradation; however, stable expression of OsHFP was observed in transgenic hairy roots, a non-green tissue. The possible role of OsHFP in regulating programmed cell death in anther green tissues of rice is proposed.

Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation.

Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.