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Traditional maize possesses low concentrations of provitamin-A and vitamin-E, leading to various health concerns. Mutant alleles of crtRB1 and vte4 that enhance β-carotene (provitamin-A) and α-tocopherol (vitamin-E), respectively, in maize kernels have been explored in several biofortification programs. For genetic improvement of these target nutrients, uniplex-PCR assays are routinely used in marker-assisted selection. However, due to back-to-back breeding seasons, the time required for genotyping individually for each target gene in large backcross populations becomes a constraint for advancing the generations. Additionally, multiple PCR assays for various genes increase the required costs and resources. Here, we aimed to develop a multiplex-PCR assay to simultaneously identify different allelic forms of crtRB1 and vte4 genes and validate them in a backcross-based segregating population. The PCR assay was carried out using newly developed primers for crtRB1 and a gene-specific primer for vte4. The uniplex-PCR assay was standardized for selected primer pairs in the BC1F1 population segregating for crtRB1 and vte4 genes. Subsequently, a multiplex-PCR assay for crtRB1 and vte4 genes was developed and employed for genotyping in the BC1F1 population. The assay differentiated among four possible genotypic classes, namely crtRB1+crtRB1/vte4+vte4, crtRB1crtRB1/vte4+vte4, crtRB1+crtRB1/vte4+vte4+, and crtRB1crtRB1/vte4+vte4+. This newly developed multiplex-PCR assay saved 41.7% of the cost and 35.6% of the time compared to two individual uniplex-PCR assays. The developed assay could accelerate maize nutritional quality breeding programs through rapid and cost-effective genotyping for the target genes. This is the first report of a multiplex-PCR assay specific to crtRB1 and vte4 genes for its use in genomics-assisted breeding in maize.

Maize is an important food, feed, fodder and industrial crop in addition to being a valuable source of micronutrients. Phytic acid (PA), an anti-nutritional factor in maize, makes crucial minerals inaccessible to monogastric animals. The low phytic acid-1 (lpa1) gene located on chromosome-1S is 7292 bp long with 11 exons, and the recessive lpa1-1 allele reduces the accumulation of PA thereby enhances the bioavailability of essential minerals in maize kernels. Here, we characterized the full-length Lpa1 gene sequence in three mutants (lpa1-1) and seven wild-type (Lpa1) maize inbreds. Sequence analysis revealed 607 polymorphic sites across Lpa1 sequences, indicating wide variability for Lpa1 among the inbreds. Further, SNP from “C” to “T” differentiated wild-type and mutant-type alleles at 1432 amino acid position. Gene-based diversity among 48 diverse maize inbreds using 15 InDel markers revealed the formation of 42 distinct haplotypes; six of which (Hap6, Hap16, Hap17, Hap19 Hap27 and Hap31) were shared by more than one genotype. The number of exons in Lpa1 ranged from 11–19 among maize genotypes and 6–14 among 26 orthologues. Major functional motifs of Lpa1 detected were ATP-binding Cassette (ABC) transporter trans-membrane region and ABC transporter. Phylogenetic tree using nucleotide and protein sequences revealed a closer relationship of maize Lpa1 sequences with Sorghum bicolor, Panicum hallii, Setaria italica and S. viridis. This study offered newer insights into the understanding of the genetic diversity of the Lpa1 gene in maize and related crop-species, and information generated here would further help in exploiting the lpa1 mutant for the enhancement of nutritional value in maize kernels.

Phytic acid (PA) acts as a storehouse for the majority of the mineral phosphorous (P) in maize; ~80% of the total P stored as phytate P is not available to monogastric animals and thereby causes eutrophication. In addition, phytic acid chelates positively charged minerals making them unavailable in the diet. The mutant lpa1-1 allele reduces PA more than the wild-type LPA1 allele. Further, mutant gene opaque2 (o2) enhances lysine and tryptophan and crtRB1 enhances provitamin-A (proA) more than wild-type O2 and CRTRB1 alleles, respectively. So far, the expression pattern of the mutant lpa1-1 allele has not been analysed in maize genotypes rich in lysine, tryptophan and proA. Here, we analysed the expression pattern of wild and mutant alleles of LPA1, O2 and CRTRB1 genes in inbreds with (i) mutant lpa1-1, o2 and crtRB1 alleles, (ii) wild-type LPA1 allele and mutant o2 and crtRB1 alleles and (iii) wild-type LPA1, O2 and CRTRB1 alleles at 15, 30 and 45 days after pollination (DAP). The average reduction of PA/total phosphorous (TP) in lpa1-1 mutant inbreds was 29.30% over wild-type LPA1 allele. The o2 and crtRB1-based inbreds possessed ~two-fold higher amounts of lysine and tryptophan, and four-fold higher amounts of proA compared to wild-type alleles. The transcript levels of lpa1-1, o2 and crtRB1 genes in lpa1-1-based inbreds were significantly lower than their wild-type versions across kernel development. The lpa1-1, o2 and crtRB1 genes reached their highest peak at 15 DAP. The correlation of transcript levels of lpa1-1 was positive for PA/TP (r = 0.980), whereas it was negative with inorganic phosphorous (iP) (r = −0.950). The o2 and crtRB1 transcripts showed negative correlations with lysine (r = −0.887) and tryptophan (r = −0.893), and proA (r = −0.940), respectively. This is the first comprehensive study on lpa1-1 expression in the maize inbreds during different kernel development stages. The information generated here offers great potential for comprehending the dynamics of phytic acid regulation in maize.

To explore the quality rootstocks which impart better quality fruits in mango varieties, we studied the interactive effect of the scion and rootstock using five mango varieties (Mallika, Amrapali, Dashehari, Pusa Arunima, and Pusa Surya) grafted on three rootstocks (Olour, Kurukkan, and K-5). A total of 25 physico-chemical parameters were studied in the five grafted varieties viz., fruit weight, yield efficiency, fruit per plant, pulp percent, total soluble solids (TSS), acidity, physiological loss in weight (PLW), peel thickness, respiration rate, etc., and were found to be altered through scion–rootstock interaction. Among the five mango varieties, Olour rootstock proved best to improve the fruit quality and shelf life using the grafting approach. Physico-chemical-traits-based clustering was unable to precisely group scion varieties according to their grafting rootstock. A total of 35 shelf-life specific markers were designed from ripening genes, such as expansin, polygalactouranase, ethylene insensitive, ethylene sensitive, etc. Of these specific primers, 24 showed polymorphism among the studied genotypes. The gene diversity (GD), allele per locus (An), polymorphism information content (PIC), and major allele frequency (MAF) observed were 0.43, 2.00, 0.34, and 0.63, respectively. Cluster analysis clearly showed that scion grafted on Kurukkan and Olour rootstock, and scion varieties grafted on K-5 rootstock grouped together have more similarity. A total of eight simple sequence repeats loci (SSRs) markers were associated with eight physiological traits. Strong association of SSR loci NMSLC-12 and NMSLC-14 with yield efficiency and fruit weight were observed with a phenotypic variance of 85% and 70%, respectively.

Tocopherol is vital for the nutritional value and stability of Indian mustard (Brassica juncea L. Czern and Coss) oil; nonetheless, the lack of information on genetic control is hampering its improvement. In this study, six populations (P1, P2, F1, F2, BC1P1, and BC1P2) of RLC3 × NPJ203 were evaluated in a family block design to evaluate the inheritance pattern, gene effects, and various other genetic parameters of tocopherol content (α, γ, and total), using generation mean analysis. The comparison of direct and reciprocal crosses indicated that the tocopherol content was not influenced by maternal inheritance. Negative directional heterosis showed that ATC, GTC, and TTC are governed by recessive genes. Potence ratio and degree of dominance highlighted an over-dominance type of gene interaction for GTC and TTC, whereas ATC was governed by epistatic interactions. Furthermore, the six-parameter model revealed a duplicate gene action for α-tocopherol content. Broad and narrow sense heritability coupled with genetic advances were high.