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The recessive opaque2 (o2) gene enhances lysine and tryptophan by ~ twofold in maize kernels. Despite > 6 decades of research, no functional marker for o2 has been reported yet. Here, we sequenced 2848 bp of entire o2 gene in 10 diverse inbreds, and identified 7 exonic SNPs and 3 exonic InDels that distinguished the normal and quality protein maize (QPM) inbreds. Of these, three potential SNPs (SNP601_T/C, SNP2359_C/T and SNP2360_G/T) and three InDels (InDel_1, InDel_8 and InDel_9) were responsible for modification of amino acids. Clustering analysis grouped 24 QPM and 24 normal inbreds into two clusters, and led to the identification of 24 haplotypes of o2. Insertion of alanine was found to regulate the binding of o2 protein with promoter of zein genes. QPM inbreds possessed significantly higher lysine (0.271–0.406%) and tryptophan (0.069–0.101%) compared to normal (lysine: 0.124–0.213%; tryptophan: 0.029–0.047%). A functional marker (MGU-InDel10-o2) was developed, and it clearly differentiated the QPM and normal inbreds. MGU-InDel10-o2 successfully identified desirable plants in BC1F1, BC2F1 and BC2F2 populations. BC2F3 progenies possessed higher lysine (0.379%) and tryptophan (0.084%) over original inbreds (lysine: 0.189% and tryptophan: 0.039%). Among haplotypes, Hap-3 showed the highest average lysine (0.380%) and tryptophan (0.094%), while Hap-14 and Hap-16 had the lowest lysine (0.124%) and tryptophan (0.029%), respectively. This is the first report on understanding allelic variation, haplotypes and functional marker development for o2 gene in maize. [ABSTRACT FROM AUTHOR]

Maize kernel protein is deficient in sulfur-containing essential amino acid such as methionine. The dzs18 gene encodes methionine-rich 18-kDa δ-zein in maize kernels. In this study, we sequenced full-length of dzs18 gene (820 bp) among 10 maize inbreds, revealing 43 SNPs and 22 InDels (average length-7.58 bp). Three InDels (4 bp at 113th, 15 bp at 463rd and 3 bp at 615th position) distinguished the wild-type (functional) from the mutant (non-functional) allele of dzs18. The 4 bp (TTAT) insertion caused a frameshift mutation, resulting in truncated DZS18 protein. The 15 bp insertion (ATG–TCT–TCG–ATG–ATA) added methionine–serine–serine–methionine–isoleucine, while the 3 bp deletion (CAA) led to loss of a glutamine residue in the mutant allele. Three gene-based PCR markers were developed for diversity analysis of dzs18 gene among 48 inbreds, which had an average methionine content of 0.136 %. (range: 0.031–0.340 %). Eight haplotypes were identified with methionine content varying from 0.066 % (Hap7) to 0.262 % (Hap3). Haplotypes with 4 bp deletion accumulated more methionine (0.174 %) than haplotypes with 4 bp insertion (0.082 %). The average methionine in 15 bp deletion and insertion haplotypes was 0.106 % and 0.150 %, respectively. The 3 bp insertion had 0.140 % methionine, while the deletion possessed 0.117 % methionine. Protein–protein association analysis predicted that DZS18 protein interacts with 19-kDa α-zein, 27- and 16-kDa γ-zeins, WAXY and O2 protein. A paralogue of dzs18 gene with 74 % sequence identity was identified. The functional markers reported here could facilitate the development of high methionine maize cultivars, which holds great significance to combat malnutrition, especially in developing countries. [ABSTRACT FROM AUTHOR]

SUMMARY: Enhancing maize kernel oil is vital for improving the bioavailability of fat‐soluble vitamins. Here, we combined favourable alleles of dgat1‐2 and fatb into parental lines of four multi‐nutrient‐rich maize hybrids (APTQH1, APTQH4, APTQH5 and APTQH7) using marker‐assisted selection (MAS). Parental lines possessed favourable alleles of crtRB1, lcyE, vte4 and opaque2 genes. Gene‐specific markers enabled successful foreground selection in BC1F1, BC2F1 and BC2F2, while background selection using genome‐wide microsatellite markers (127–132) achieved 93% recurrent parent genome recovery. Resulting inbreds exhibited significantly higher oil (6.93%) and oleic acid (OA, 40.49%) and lower palmitic acid (PA, 14.23%) compared to original inbreds with elevated provitamin A (11.77 ppm), vitamin E (16.01 ppm), lysine (0.331%) and tryptophan (0.085%). Oil content significantly increased from 4.80% in original hybrids to 6.73% in reconstituted hybrids, making them high‐oil maize hybrids. These hybrids displayed 35.70% increment in oil content and 51.56% increase in OA with 36.32% reduction in PA compared to original hybrids, while maintaining higher provitamin A (two‐fold), vitamin E (nine‐fold), lysine (two‐fold) and tryptophan (two‐fold) compared to normal hybrids. Lipid health indices showed improved atherogenicity, thrombogenicity, cholesterolaemic, oxidability, peroxidizability and nutritive values in MAS‐derived genotypes over original versions. Besides, the MAS‐derived inbreds and hybrids exhibited comparable grain yield and phenotypic characteristics to the original versions. The maize hybrids developed in the study possessed high‐yielding ability with high kernel oil and OA, low PA, better fatty acid health and nutritional properties, higher multi‐vitamins and balanced amino acids, which hold immense significance to address malnutrition and rising demand for oil sustainably in a fast‐track manner. [ABSTRACT FROM AUTHOR]

Bacterial stalk rot (BSR), caused by Dickeya zeae (syn. Erwinia chrysanthemi pv. zeae), has emerged as a significant disease affecting maize crops worldwide. In this study, symptomatic maize plants were collected from diverse agroclimatic zones in India over the period of kharif 2019–2021. Various approaches, including pathogenicity tests, cultural characteristics, biochemical profiling and molecular analysis, were employed to accurately identify the collected bacterial isolates as Dickeya. Pathogenicity assessments were conducted on 40‐day‐old maize plants, following Koch's postulates, as well as through potato maceration assays. Furthermore, cross‐infectivity studies were conducted on rice, potato, tomato and banana plants. Phenotypic and biochemical characterization confirmed that all the isolates belonged to the Dickeya genus. Additionally, PCR amplification of a pel gene fragment, specific to the genus Dickeya, further verified the pathogenic isolates as Dickeya. Molecular characterization studies were performed on four isolates (UKMDZ‐3, PBMDZ‐7, TSMDZ‐11 and HPMDZ‐16), selected to represent distinct maize agroclimatic zones and four states of India, and which caused severe infections on susceptible maize cv. Early Composite. Amplification of six characteristic genome regions (16S rRNA, recN, gyrB, dnaX, recA and dnaJ) from these isolates facilitated individual and concatenated gene phylogenetic analyses, confirming their resemblance to Dickeya zeae. This study represents the first comprehensive molecular analysis of D. zeae isolates from India, providing valuable insights for future crop improvement strategies. The findings contribute to our understanding of the genetic basis of BSR in maize and offer potential avenues for genetic enhancement to mitigate the disease's impact on maize cultivation. [ABSTRACT FROM AUTHOR]

Key message: ‘Sikkim Primitive’ maize landrace, unique for prolificacy (7–9 ears per plant) possesses unique genomic architecture in branching and inflorescence-related gene(s), and locus Zm00001eb365210 encoding glycosyltransferases was identified as the putative candidate gene underlying QTL (qProl-SP-8.05) for prolificacy. The genotype possesses immense usage in breeding high-yielding baby-corn genotypes. ‘Sikkim Primitive’ is a native landrace of North Eastern Himalayas, and is characterized by having 7–9 ears per plant compared to 1–2 ears in normal maize. Though ‘Sikkim Primitive’ was identified in the 1960s, it has not been characterized at a whole-genome scale. Here, we sequenced the entire genome of an inbred (MGUSP101) derived from ‘Sikkim Primitive’ along with three non-prolific (HKI1128, UMI1200, and HKI1105) and three prolific (CM150Q, CM151Q and HKI323) inbreds. A total of 942,417 SNPs, 24,160 insertions, and 27,600 deletions were identified in ‘Sikkim Primitive’. The gene-specific functional mutations in ‘Sikkim Primitive’ were classified as 10,847 missense (54.36%), 402 non-sense (2.015%), and 8,705 silent (43.625%) mutations. The number of transitions and transversions specific to ‘Sikkim Primitive’ were 666,021 and 279,950, respectively. Among all base changes, (G to A) was the most frequent (215,772), while (C to G) was the rarest (22,520). Polygalacturonate 4-α-galacturonosyltransferase enzyme involved in pectin biosynthesis, cell-wall organization, nucleotide sugar, and amino-sugar metabolism was found to have unique alleles in ‘Sikkim Primitive’. The analysis further revealed the Zm00001eb365210 gene encoding glycosyltransferases as the putative candidate underlying QTL (qProl-SP-8.05) for prolificacy in ‘Sikkim Primitive’. High-impact nucleotide variations were found in ramosa3 (Zm00001eb327910) and zeaxanthin epoxidase1 (Zm00001eb081460) genes having a role in branching and inflorescence development in ‘Sikkim Primitive’. The information generated unraveled the genetic architecture and identified key genes/alleles unique to the ‘Sikkim Primitive’ genome. This is the first report of whole-genome characterization of the ‘Sikkim Primitive’ landrace unique for its high prolificacy. [ABSTRACT FROM AUTHOR]

Diverse haploid inducer lines with > 6% of haploid induction rate are now routinely used to develop doubled haploid lines. Though MTL gene regulates haploid induction, its molecular characterization and haplotype analysis in maize and its related species have not been undertaken so far. In the present study, the entire 1812 bp long MTL gene was sequenced among two mutant and eight wild-type inbreds. A 4 bp insertion differentiated the mutant from the wild-type allele. Sequence analysis further revealed 103 polymorphic sites including 38 InDels and 65 SNPs. A total of 15 conserved regions were detected, of which exon-4 was the most conserved. Ten gene-based markers specific to MTL revealed the presence of 40 haplotypes among diverse 48 inbreds of exotic and indigenous origin. It generated 20 alleles with an average of two alleles per locus. The mean polymorphic information content was 0.3247 with mean gene diversity of 0.4135. A total of 15 paralogous sequences of MTL were detected in maize genome with 3–7 exons. Maize MTL proteins of both wild-type and mutant were non-polar in nature, and they possessed four domains. R1-nj-based haploid inducer (HI) lines viz., Pusa-HI-101 and Pusa-HI-102 had an average haploid induction rate of 8.45 ± 0.96% and 10.46 ± 1.15%, respectively. Lines wild-type MTL gene did not generate any haploid. In comparison with 27 orthologues of 21 grass species, maize MTL gene had the closest ancestry with Saccharum spontaneum and Sorghum. The information generated here assumes great significance in understanding the diversity of MTL gene and presence of paralogues and orthologues. This is the first report on haplotype analysis and molecular characterization of MTL gene in maize and related grass species. [ABSTRACT FROM AUTHOR]

Sweet corn is one of the most popular vegetables worldwide. However, traditional shrunken2 (sh2)- based sweet corn varieties are poor in nutritional quality. Here, we analysed the effect of (1) β-carotene hydroxylase1 (crtRB1), (2) opaque2 (o2) and (3) o2 + crtRB1 genes on nutritional quality, germination, seed vigour and physico-biochemical traits in a set of 27 biofortified sh2-based sweet corn inbreds. The biofortified sweet corn inbreds recorded significantly higher concentrations of proA (16.47 μg g-1), lysine (0.36%) and tryptophan (0.09%) over original inbreds proA: 3.14 μg g-1, lysine: 0.18%, tryptophan: 0.04%). The crtRB1-based inbreds had the lowest electrical conductivity (EC), whereas o2-based inbreds possessed the highest EC. The o2 + crtRB1- based inbreds showed similar EC to the original inbreds. Interestingly, o2-based inbreds also had the lowest germination and seed vigour compared to original inbreds, whereas crtRB1 and o2 + crtRB1 introgressed sweet corn inbreds showed similar germination and seed vigour traits to their original versions. This suggested that the negative effect of o2 on germination, seed vigour and EC is nullified by crtRB1 in the double mutant sweet corn. Overall, o2 + crtRB1-based sweet corn inbreds were found the most desirable over crtRB1- and o2-based inbreds alone. [ABSTRACT FROM AUTHOR]

Traditional maize grain is deficient in methionine, an essential amino acid required for proper growth and development in humans and poultry birds. Thus, development of high methionine maize (HMM) assumes great significance in alleviating malnutrition through sustainable and cost-effective approach. Of various genetic loci, aspartate kinase2 (ask2) gene plays a pivotal role in regulating methionine accumulation in maize. Here, we sequenced the entire ask2 gene of 5394 bp with 13 exons in five wild and five mutant maize inbreds to understand variation at nucleotide level. Sequence analysis revealed that an SNP in exon-13 caused thymine to adenine transversion giving rise to a favourable mutant allele associated with leucine to glutamine substitution in mutant ASK2 protein. Gene-based diversity analysis with 11 InDel markers grouped 48 diverse inbreds into three major clusters with an average genetic dissimilarity of 0.570 (range, 0.0–0.9). The average major allele frequency, gene diversity and PIC are 0.693, 0.408 and 0.341, respectively. A total of 45 haplotypes of the ask2 gene were identified among the maize inbreds. Evolutionary relationship analysis performed among 22 orthologues grouped them into five major clusters. The number of exons varied from 7 to 17, with length varying from 12 to 495 bp among orthologues. ASK2 protein with 565 amino acids was predicted to be in homo-dimeric state with lysine and tartaric acid as binding ligands. Amino acid kinase and ACT domains were found to be conserved in maize and orthologues. The study depicted the presence of enough genetic diversity in ask2 gene in maize, and development of HMM can be accelerated through introgression of favourable allele of ask2 into the parental lines of elite hybrids using molecular breeding. [ABSTRACT FROM AUTHOR]

The mutant matrilineal (mtl) gene encoding patatin-like phospholipase activity is involved in in-vivo maternal haploid induction in maize. Doubling of chromosomes in haploids by colchicine treatment leads to complete fixation of inbreds in just one generation compared to 6–7 generations of selfing. Thus, knowledge of patatin-like proteins in other crops assumes great significance for in-vivo haploid induction. So far, no online tool is available that can classify unknown proteins into patatin-like proteins. Here, we aimed to optimize a machine learning-based algorithm to predict the patatin-like phospholipase activity of unknown proteins. Four different kernels [radial basis function (RBF), sigmoid, polynomial, and linear] were used for building support vector machine (SVM) classifiers using six different sequence-based compositional features (AAC, DPC, GDPC, CTDC, CTDT, and GAAC). A total of 1170 protein sequences including both patatin-like (585 sequences) from various monocots, dicots, and microbes; and non-patatin-like proteins (585 sequences) from different subspecies of Zea mays were analyzed. RBF and polynomial kernels were quite promising in the prediction of patatin-like proteins. Among six sequence-based compositional features, di-peptide composition attained > 90% prediction accuracies using RBF and polynomial kernels. Using mutual information, most explaining dipeptides that contributed the highest to the prediction process were identified. The knowledge generated in this study can be utilized in other crops prior to the initiation of any experiment. The developed SVM model opened a new paradigm for scientists working in in-vivo haploid induction in commercial crops. This is the first report of machine learning of the identification of proteins with patatin-like activity. [ABSTRACT FROM AUTHOR]

Background: Deficiency of vitamin E results in several neurological and age-related disorders in humans. Utilization of maize mutants with favourable vte4-allele led to the development of several α-tocopherol (vitamin E) rich (16–19 µg/g) maize hybrids worldwide. However, the degradation of tocopherols during post-harvest storage substantially affects the efficacy of these genotypes. Methods and results: We studied the role of lipoxygenase enzyme and Lipoxygenase 3 (LOX3) gene on the degradation of tocopherols at monthly intervals under traditional storage up to six months in two vte4-based contrasting-tocopherol retention maize inbreds viz. HKI323-PVE and HKI193-1-PVE. The analysis revealed significant degradation of tocopherols across storage intervals in both the inbreds. Lower retention of α-tocopherol was noticed in HKI193-1-PVE. HKI323-PVE with the higher retention of α-tocopherol showed lower lipoxygenase activity throughout the storage intervals. LOX3 gene expression was higher (~ 1.5-fold) in HKI193-1-PVE compared to HKI323-PVE across the storage intervals. Both lipoxygenase activity and LOX3 expression peaked at 120 days after storage (DAS) in both genotypes. Further, a similar trend was observed for LOX3 expression and lipoxygenase activity. The α-tocopherol exhibited a significantly negative correlation with lipoxygenase enzyme and expression of LOX3 across the storage intervals. Conclusions: HKI323-PVE with high tocopherol retention, low -lipoxygenase activity, and -LOX3 gene expression can act as a potential donor in the vitamin E biofortification program. Protein-protein association network analysis also indicated the independent effect of vte4 and LOX genes. This is the first comprehensive report analyzing the expression of the LOX3 gene and deciphering its vital role in the retention of α-tocopherol in biofortified maize varieties under traditional storage. [ABSTRACT FROM AUTHOR]

Traditional maize is poor in vitamin-E [α-tocopherol (α-T): 6–8 ppm], vitamin-A [provitamin-A (proA): 1–2ppm], lysine (0.150–0.2–50%), and tryptophan (0.030–0.040%). Here, we combined favourable alleles of vte4, crtRB1, and opaque2 (o2) genes in the parents of maize hybrids, viz., APQH-10 (PMI-PV-9 × PMI-PV-14) and APQH-11 (PMI-PV-9 × PMI-PV-15) using molecular breeding. Gene-specific markers were successfully used to select vte4, crtRB1, and o2 in BC1F1, BC2F1, and BC2F2 generations. Simple sequence repeats (104–109) were used for background selection, leading to an average recovery of 94% recurrent parent genome. The introgressed inbreds possessed significantly higher α-T: 18.38 ppm, α-/γ-tocopherol (α-/γ-T: 52%), and α-/total tocopherol (α-/TT: 32%) compared to original inbreds (α-T: 8.17 ppm, α-/γ-T: 25%, α-/TT: 18%). These newly derived inbreds also possessed higher β-carotene (BC: 8.91 ppm), β-cryptoxanthin (BCX: 1.27 ppm), proA (9.54 ppm), lysine (0.348%), and tryptophan (0.082%) compared to traditional maize inbreds. The reconstituted hybrids recorded higher α-T (2.1-fold), α-/γ-T (1.9-fold), and α-/TT (1.6-fold) over the original hybrids. These reconstituted hybrids were also rich in BC (5.7-fold), BCX (3.3-fold), proA (5.3-fold), lysine (1.9-fold), and tryptophan (2.0-fold) over the traditional hybrids. The reconstituted hybrids had similar grain yield and phenotypic characteristics to original versions. These multinutrient-rich maize hybrids hold great potential to alleviate malnutrition in sustainable and cost-effective manner. [ABSTRACT FROM AUTHOR]

Waxy corn possessing high amylopectin is widely employed as an industrial product. Traditional corn contains ~ 70–75% amylopectin, whereas waxy corn with the mutant waxy1 (wx1) gene possesses ~ 95–100% amylopectin. Marker-assisted breeding can greatly hasten the transfer of the wx1 allele into normal corn. However, the available gene-based marker(s) for wx1 are not always polymorphic between recipient and donor parents, thereby causing a considerable delay in the molecular breeding program. Here, a 4800 bp sequence of the wx1 gene was analyzed among seven wild-type and seven mutant inbreds employing 16 overlapping primers. Three polymorphisms viz., 4 bp InDel (at position 2406 bp) in intron-7 and two SNPs (C to A at position 3325 bp in exon-10 and G to T at position 4310 bp in exon-13) differentiated the dominant (Wx1) and recessive (wx1) allele. Three breeder-friendly PCR markers (WxDel4, SNP3325_CT1, and SNP4310_GT2) specific to InDel and SNPs were developed. WxDel4 amplified 94 bp among mutant-type inbreds, while 90 bp was amplified among wild-type inbreds. SNP3325_CT1 and SNP4310_GT2 revealed the presence-absence polymorphisms with an amplification of 185 bp and 189 bp of amplicon, respectively. These newly developed markers showed 1:1 segregation in both BC1F1 and BC2F1 generations, while 1:2:1 segregation was observed in BC2F2. The recessive homozygotes (wx1wx1) of BC2F2 identified by the markers possessed significantly higher amylopectin (97.7%) compared to the original inbreds (Wx1Wx1: 72.7% amylopectin). This is the first report of novel wx1 gene-based markers. The information generated here would help in accelerating the development of waxy maize hybrids. [ABSTRACT FROM AUTHOR]

Temperature is one of the critical abiotic factors influencing the range, richness, activity, life history and viability of insects. The persistence trend of thermal stress on biological and reproductive traits were studied for invasive fall armyworm, Spodoptera frugiperda (J. E. Smith) by exposing early fourth instar larvae to thermal stress treatments (42° and 45 °C) for single and multiple times along with control (27 °C) and reared them till F2 generation. Survival of larvae was minimum in 45 °C single (88%) and multiple thermal stress (92.5%). The carry over effect of thermal stress was evident up to F2 generation for developmental attributes with prolonged developmental duration, reduced maturation success and female biased sex ratio. Reproductive parameters such as gonadosomatic index, eupyrene sperm count, fecundity and mating frequency reduced drastically as a result of thermal stress in F0 generation and the effect persisted up to F2 generation on fecundity and gonadosomatic index. The biochemical response due to thermal stress was investigated by analyzing metabolic reserves in the adult stage. The reduction in water content and accumulation of total sugar, glycogen, lipid and protein content was observed in adults of all three generations. A trade-off between reproduction and survival strategies was evident under thermal stress with the allocation of resources between them. Thus, our study provides a basic understanding on persistence of thermal stress across generations both at biological and biochemical level and offers insights into further studies on demography of fall armyworm under current and future climate change scenario. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Malnutrition affects large section of population worldwide. Vitamin A and protein deficiencies have emerged as the major global health-issue. Traditional shrunken2 (sh2)-based sweet corn is deficient in provitamin A (proA), lysine and tryptophan. Natural variant of β-carotene hydroxylase1 (crtRB1) and opaque2 (o2) enhances proA, lysine and tryptophan in maize. So far, no sweet corn hybrid rich in these nutrients has been released elsewhere. Development of biofortified sweet corn hybrids would help in providing the balanced nutrition. Methods and Results: We targeted three sh2-based sweet corn inbreds (SWT-19, SWT-20 and SWT-21) for introgression of mutant crtRB1 and o2 genes using molecular breeding. The gene-based 3′TE-InDel and simple sequence repeat (SSR) (umc1066) markers specific to crtRB1 and o2, respectively were utilized in foreground selection in BC1F1, BC2F1 and BC2F2. Segregation distortion was observed for crtRB1 and o2 genes in majority of populations. Background selection using 91–100 SSRs revealed recovery of recurrent parent genome (RPG) up to 96%. The introgressed progenies possessed significantly higher proA (13.56 µg/g) as compared to the original versions (proA: 2.70 µg/g). Further, the introgressed progenies had accumulated moderately higher level of lysine (0.336%) and tryptophan (0.082%) over original versions (lysine: 0.154% and tryptophan: 0.038%). Kernel sweetness among introgressed progenies (17.3%) was comparable to original sweet corn (17.4%). The introgressed inbreds exhibited higher resemblance with their recurrent parents for yield and morphological characters. Conclusion: These newly developed biofortified sweet corn genotypes hold immense promise to alleviate malnutrition. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: In-vivo maternal haploids serve as the basis of doubled haploid (DH) breeding in maize. R1-navajo (R1-nj) gene governing anthocyanin colouration in the endosperm and embryo is widely used to identify haploid seeds. However, the expression of R1-nj depends on genetic-background of source-germplasm used for deriving DH-lines. Further, presence of C1-Inhibitor (C1-I) gene suppresses the expression of R1-nj, thus makes the selection of haploids difficult. Methods: In the present study, 178 subtropically-adapted maize inbreds were crossed with two R1-nj donors 'that do not have haploid induction genes'. Of these, 76.4% inbreds developed purple colour in endosperm, while 23.6% did not show any colouration. In case of scutellum, 62.9% inbreds possessed colour and 37.1% were colourless. The anthocyanin intensity varied greatly, with 19.66% and 42.98% inbreds displayed the least intensity, while 16.85% and 0.84% inbreds showed the highest intensity in endosperm and scutellum, respectively. Two C1-I specific breeder-friendly markers (MGU-CI-InDel8 and MGU-C1-SNP1) covering (i) 8 bp InDel and (ii) A to G SNP, respectively, were developed. MGU-CI-InDel8 and MGU-C1-SNP1 markers predicted presence of C1-I allele with 92.9% and 84.7% effectiveness, respectively. However, when both markers were considered together, they provided 100% effectiveness. Conclusions: These markers of C1-I gene would help in saving valuable resources and time during haploid induction in maize. The information generated here assume great significance in DH breeding of maize. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sweet corn possessing recessive shrunken2 (sh2) gene is popular worldwide. Traditional sweet corn is poor in vitamin A and vitamin E. Plant breeders during the selection of sweet corn genotypes mainly emphasize on plant architecture and yield. Seed germination and seedling vigour play important role for early establishment in field, thereby increasing yield and income. Here, we analysed a set of 15 sh2-based biofortified sweet corn inbreds with crtRB1 (β-carotene hydroxylase1) and vte4 (γ-tocopherol methyltransferase) genes and three traditional sh2-based sweet corn inbreds for nutritional quality, seed vigour and various physico-biochemical traits. The newly developed inbreds possessed significantly higher provitamin A (proA: 15.60 µg/g) and vitamin E [α-tocopherol (α-T): 20.42 µg/g] than the traditional sweet corn inbreds (proA: 2.51 µg/g, α-T: 11.16 µg/g). The biofortified sweet corn inbreds showed wide variation for germination (80.67–87.33%), vigour index-I (2097.17–2925.28 cm), vigour index-II (134.27–216.19 mg) and electrical conductivity (10.19–13.21 μS cm−1 g−1). Wide variation was also observed for dehydrogenase (1.29–1.59 OD g−1 ml−1), super oxide dismutase (4.01–9.82 g−1), peroxidase (11.66–16.47 μM min−1 g−1), esterase (22.98–34.76 nM min−1 g−1) and α-amylase (5.91–8.15 OD g−1 ml−1). Enrichment of proA and vitamin E in sweet corn did not affect seed vigour and physico-biochemical traits. Correlation analysis revealed that electrical conductivity and α-amylase activity was the reliable indicator for assessing seed germination and vigour. The study identified superior biofortified sweet corn genotypes that would contribute to better vigour and establishment in field. This is the first report of analysis of biofortified sweet corn genotypes for seed vigour and physico-biochemical traits. [ABSTRACT FROM AUTHOR]

Sweet corn has become a popular food worldwide. It possesses six-times more sugar than field corn due to the presence of recessive shrunken2 (sh2) gene. Despite availability of diverse sweet corn germplasm, comprehensive characterization of sh2 has not been undertaken so far. Here, entire Sh2 gene (7320 bp) among five field corn-(Sh2Sh2) and six sweet corn-(sh2sh2) inbreds was sequenced. A total of 686 SNPs and 372 InDels were identified, of which three SNPs differentiated the wild-(Sh2) and mutant-(sh2) allele. Ten InDel markers were developed to assess sh2 gene-based diversity among 23 sweet corn and 25 field corn lines. Twenty-five alleles and 47 haplotypes of sh2 were identified among 48 inbreds. Among markers, MGU-InDel-2, MGU-InDel-3, MGU-InDel-5 and MGU-InDel-8 had PIC>0.5. Major allele frequency varied from 0.458–0.958. The gene sequence of these maize inbreds was compared with 25 orthologues of monocots. Sh2 gene possessed 15–18 exons with 6-225bp among maize, while it was 6–21 exons with 30-441bp among orthologues. While intron length across maize genotypes varied between 67-2069bp, the same among orthologues was 57–2713 bp. Sh2-encoded AGPase domain was more conserved than NTP transferase domain. Nucleotide and protein sequences of sh2 in maize and orthologues revealed that rice orthologue was closer to maize than other monocots. The study also provided details of motifs and domains present in sh2 gene, physicochemical properties and secondary structure of SH2 protein in maize inbreds and orthologues. This study reports detailed characterization and diversity analysis in sh2 gene of maize and related orthologues in various monocots. [ABSTRACT FROM AUTHOR]

Waxy maize is popular for food-, feed- and industrial usage. It possesses a recessive waxy1 (wx1) gene that enhances amylopectin to ~ 95–100%, compared to ~ 70–75% in traditional maize. Marker-assisted selection (MAS) is a preferred approach to converting normal maize into a waxy version. However, it requires specialized expertise, a well-equipped laboratory, and high cost. Here, pollen staining was used as an alternative approach to MAS. BC1F1, BC1F2 and BC2F2 populations in seven genetic backgrounds segregating for the wx1 gene were used. Pollens treated with iodine-potassium iodide showed that wild types (Wx1Wx1) were dark purple, while waxy pollens (wx1wx1) exhibited red colour. Heterozygotes (Wx1wx1) showed a mix of both dark purple and red colour. Staining of endosperm flour also confirmed the same findings. Wx1-based genotyping using phi022 and wx2507F/RG confirmed the same genotypic status. The average amylopectin among genotypes having red coloured pollens was 97.6%, while it was 72.5% among dark purple. Heterozygotes with both dark purple and red pollens had 85.2% amylopectin. Pollen staining showed complete agreement with the genotyping as well as amylopectin contents. Pollen staining saved 81% cost, and 54% time compared to MAS. This is the first report on the utilization of pollen staining for selecting the wx1 allele in segregating populations used for the development of waxy maize hybrids. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Maize (Zea mays L.) is the leading cereal crop and staple food in many parts of the world. This study aims to develop nutrient-rich maize genotypes by incorporating crtRB1 and o2 genes associated with increased β-carotene, lysine, and tryptophan levels. UMI1200 and UMI1230, high quality maize inbreds, are well-adapted to tropical and semi-arid regions in India. However, they are deficient in β-carotene, lysine, and tryptophan. We used the concurrent stepwise transfer of genes by marker-assisted backcross breeding (MABB) scheme to introgress crtRB1 and o2 genes. In each generation (from F1, BC1F1–BC3F1, and ICF1–ICF3), foreground and background selections were carried out using gene-linked (crtRB1 3′TE and umc1066) and genome-wide simple sequence repeats (SSR) markers. Four independent BC3F1 lines of UMI1200 × CE477 (Cross-1), UMI1200 × VQL1 (Cross-2), UMI1230 × CE477 (Cross-3), and UMI1230 × VQL1 (Cross-4) having crtRB1 and o2 genes and 87.45–88.41% of recurrent parent genome recovery (RPGR) were intercrossed to generate the ICF1-ICF3 generations. Further, these gene pyramided lines were examined for agronomic performance and the β-carotene, lysine, and tryptophan contents. Six ICF3 lines (DBT-IC-β1σ4-4-8-8, DBT-IC-β1σ4-9-21-21, DBT-IC-β1σ4-10-1-1, DBT-IC-β2σ5-9-51-51, DBT-IC-β2σ5-9-52-52 and DBT-IC-β2σ5-9-53-53) possessing crtRB1 and o2 genes showed better agronomic performance (77.78–99.31% for DBT-IC-β1σ4 population and 85.71–99.51% for DBT-IC-β2σ5 population) like the recurrent parents and β-carotene (14.21–14.35 μg/g for DBT-IC-β1σ4 and 13.28–13.62 μg/g for DBT-IC-β2σ5), lysine (0.31–0.33% for DBT-IC-β1σ4 and 0.31–0.34% for DBT-IC-β2σ5), and tryptophan (0.079–0.082% for DBT-IC-β1σ4 and 0.078–0.083% for DBT-IC-β2σ5) levels on par with that of the donor parents. In the future, these improved lines could be developed as a cultivar for various agro-climatic zones and also as good genetic materials for maize nutritional breeding programs. [ABSTRACT FROM AUTHOR]

Maize possesses higher concentration of phytic acid (PA) compared with other cereals, reducing bioavailability of iron and zinc. Reduction of PA in maize grains thus assumes significance to alleviate micronutrient malnutrition. Here, a set of 24 inbreds possessing mutant lpa2‐1 gene and seven inbreds with wild‐type LPA2 gene was field evaluated at Delhi and Hyderabad. The performance of lpa2‐1‐based inbreds was at par with wild‐type inbreds for grain yield (P =.09645) and key agronomic traits (P >.54100). Mutant inbreds possessed significantly low mean PA (1.90 mg/g) over wild‐type inbreds (2.56 mg/g) (P <.00001), with average reduction of 26% PA among lpa2‐1 mutants. Mean inorganic phosphorous (iP) was 0.81 mg/g in lpa2‐1 mutants and 0.27 mg/g in wild types, revealing significantly (P <.00001) higher increment (three‐fold) of iP in mutants compared with wild‐type inbreds. Characterization using 61 simple sequence repeats (SSRs) generated 181 alleles with mean polymorphism information content of 0.40 and mean gene diversity of 0.46. Cluster analyses grouped lpa2‐1 inbreds into three‐major clusters. The study identified potential cross combinations for development of low phytate maize with higher bioavailability of iron and zinc. [ABSTRACT FROM AUTHOR]

Maize possesses wide variation in amylose and amylopectin which assumes significance as a part of both food-chain and different industrial applications. Estimation of amylose and amylopectin in maize kernels is important for developing suitable hybrids. The existing protocols for estimation of amylose and amylopectin in maize are elaborate and lengthy, and involve high cost. Here, we developed a rapid and cost-effective method for estimation of amylose and amylopectin in maize kernels. 10% toluene and 80% ethanol were used for removal of proteins (~ 10%) and lipids (~ 4%) from maize flour. The over-estimation of amylose was minimized using NaOH with KI to stop free KI to bind with amylopectin. Standards were improved by mixing amylose and amylopectin in different concentrations (0–100%), rather than using amylose or amylopectin alone. Standard curve generated regression equation of y = 90.436x + 0.8535 with R2 = 0.9989. Two types of samples viz., (1) protein, amylose and amylopectin (2) amylose and amylopectin, showed that starch fractions were highly comparable to expected values with correlation coefficient (r) of 0.9998 and mean standard deviation of 0.54. The protocol successfully estimated wide range of amylose (2.79–50.04%) and amylopectin (59.96–97.21%) among diverse maize inbreds including amylose extender1 (ae1) and waxy1 (wx1) mutants. Present protocol required 75% less time and 92.5% less cost compared to existing protocols. The newly developed method would be highly useful in developing maize hybrids high in amylose or amylopectin. This is the first report of rapid and cost-effective protocol for estimation of starch fractions in maize kernels. [ABSTRACT FROM AUTHOR]

Phytic acid (PA) is a major anti-nutritional factor in maize grains and significantly reduces the bioavailability of minerals such as iron and zinc in humans. Reduction of PA in maize (Zea mays L.) thus assumes great significance. Despite the availability of low phytic-acid (lpa) mutants in maize, their utilisation in breeding has been limited because of non-availability of well adapted lpa-based inbreds. A set of 24 subtropically adapted lpa1-1-based maize inbreds derived through molecular breeding was evaluated along with seven wild-type inbreds at multilocation. The lpa1-1 inbreds possessed 35.8% lower PA (1.68 mg/g) than the wild-type inbreds (2.61 mg/g). Mean proportion of PA was lower in the lpa1-1-based inbreds (62.8%) than in the wild-type inbreds (91.6%). The lpa1-1 inbreds showed similar performance for plant- and ear-height besides flowering behaviour as did wild types. Average grain yield among lpa1-1 inbreds (2735.9 kg/ha) was at par with the original versions (2907.3 kg/ha). Molecular profiling of these lpa1-1 inbreds using 60 genome-wide single-sequence repeats generated 172 alleles, with a mean of 2.87 alleles per locus. Mean polymorphism information content and mean gene diversity were 0.41 and 0.48 respectively. Genetic dissimilarity ranged from 0.23 to 0.81, with an average of 0.64. Cluster analyses grouped 24 lpa1-1 genotypes into three major clusters, and principal-coordinate analysis depicted the diverse nature of genotypes. The study also identified a set of potential hybrid combinations with low PA for their direct utilisation in biofortification program. This is the first study on comprehensive characterisation of lpa1-1-based inbreds adapted to subtropical conditions. [ABSTRACT FROM AUTHOR]

Waxy maize rich in amylopectin has emerged as a preferred food. However, waxy maize is poor in lysine and tryptophan, deficiency of which cause severe health problems. So far, no waxy hybrid with high lysine and tryptophan has been developed and commercialized. Here, we combined recessive waxy1 (wx1) and opaque2 (o2) genes in the parental lines of four popular hybrids (HQPM1, HQPM4, HQPM5, and HQPM7) using genomics-assisted breeding. The gene-based markers, wx-2507F/RG and phi057 specific for wx1 and o2, respectively were successfully used to genotype BC1F1, BC2F1 and BC2F2 populations. Background selection with > 100 SSRs resulted in recovering > 94% of the recurrent parent genome. The reconstituted hybrids showed 1.4-fold increase in amylopectin (mean: 98.84%) compared to the original hybrids (mean: 72.45%). The reconstituted hybrids also showed 14.3% and 14.6% increase in lysine (mean: 0.384%) and tryptophan (mean: 0.102%), respectively over the original hybrids (lysine: 0.336%, tryptophan: 0.089%). Reconstituted hybrids also possessed similar grain yield (mean: 6248 kg/ha) with their original versions (mean: 6111 kg/ha). The waxy hybrids with high lysine and tryptophan assume great significance in alleviating malnutrition through sustainable and cost-effective means. This is the first report of development of lysine and tryptophan rich waxy hybrids using genomics-assisted selection. [ABSTRACT FROM AUTHOR]

Sweet corn has gained worldwide popularity. Traditional sweet corn possesses low concentration of essential nutrients such as lysine (0.15–0.25%), tryptophan (0.03–0.04%) and provitamin-A (proA 3–4 ppm), and deficiency leads to serious health problems in humans. Here, stacking of shrunken2 (sh2), opaque2 (o2), lycopene epsilon cyclase (lcyE) and β‐carotene hydroxylase (crtRB1) genes were undertaken in the parents of four hybrids viz., APQH1, APHQ4, APHQ5 and APHQ7 using marker-assisted backcross breeding (MABB). Gene-linked markers (umc2276 and umc1320) for sh2, while gene-based markers for o2 (umc1066 and phi057), lcyE (5′TE-InDel) and crtRB1 (3′TE-InDel), were used for genotyping in BC1F1, BC2F1 and BC2F2. Selected backcross progenies showed high recovery of recurrent parent genome (92.4–97.7%). The reconstituted sweet corn hybrids possessed significantly high lysine (0.390%), tryptophan (0.082%) and proA (21.14 ppm), coupled with high kernel sweetness (brix 18.96%). The improved sweet corn hybrids had high cob yield (12.22–15.33 t/ha) across three environments. These newly developed biofortified sweet corn hybrids possess great significance in providing balanced nutrition. This is the first report of combining sh2, o2, lcyE and crtRB1 genes for enrichment of sweet corn hybrids with multiple essential nutrients. [ABSTRACT FROM AUTHOR]

Sweet corn is popular worldwide as vegetable. Though large numbers of sugary1 (su1)-based sweet corn germplasm are available, allelic diversity in su1 gene encoding SU1 isoamylase among diverse maize inbreds has not been analyzed. Here, we characterized the su1 gene in maize and compared with allied species. The entire su1 gene (11,720 bp) was sequenced among six mutant (su1) and five wild (Su1) maize inbreds. Fifteen InDels of 2–45 bp were selected to develop markers for studying allelic diversity in su1 gene among 19 mutant- (su1) and 29 wild-type (Su1) inbreds. PIC ranged from 0.15 (SU-InDel7) to 0.37 (SU-InDel13). Major allele frequency varied from 0.52 to 0.90, while gene diversity ranged from 0.16 to 0.49. Phylogenetic tree categorized 48 maize inbreds in two clusters each for wild- type (Su1) and mutant (su1) types. 44 haplotypes of su1 were observed, with three haplotypes (Hap6, Hap22 and Hap29) sharing more than one genotype. Further, comparisons were made with 23 orthologues of su1 from 16 grasses and Arabidopsis. Maize possessed 15–19 exons in su1, while it was 11–24 exons among orthologues. Introns among the orthologues were longer (77–2206 bp) than maize (859–1718 bp). SU1 protein of maize and orthologues had conserved α-amylase and CBM_48 domains. The study also provided physicochemical properties and secondary structure of SU1 protein in maize and its orthologues. Phylogenetic analysis showed closer relationship of maize SU1 protein with P. hallii, S. bicolor and E. tef than Triticum sp. and Oryza sp. The study showed that presence of high allelic diversity in su1 gene which can be utilized in the sweet corn breeding program. This is the first report of comprehensive characterization of su1 gene and its allelic forms in diverse maize and related orthologues. [ABSTRACT FROM AUTHOR]

Malnutrition is a widespread problem that affects human health, society, and the economy. Traditional maize that serves as an important source of human nutrition is deficient in vitamin-E, vitamin-A, lysine, and tryptophan. Here, favorable alleles of vte4 (α -tocopherol methyl transferase), crtRB1 (β -carotene hydroxylase), lcyE (lycopene ε -cyclase), and o2 (opaque2) genes were combined in parental lines of four popular hybrids using marker-assisted selection (MAS). BC1F1, BC2F1, and BC2F2 populations were genotyped using gene-based markers of vte4, crtRB1, lcyE , and o2. Background selection using 81–103 simple sequence repeats (SSRs) markers led to the recovery of recurrent parent genome (RPG) up to 95.45%. Alpha (α)-tocopherol was significantly enhanced among introgressed progenies (16.13 μg/g) as compared to original inbreds (7.90 μg/g). Provitamin-A (proA) (10.42 μg/g), lysine (0.352%), and tryptophan (0.086%) were also high in the introgressed progenies. The reconstituted hybrids showed a 2-fold enhancement in α-tocopherol (16.83 μg/g) over original hybrids (8.06 μg/g). Improved hybrids also possessed high proA (11.48 μg/g), lysine (0.367%), and tryptophan (0.084%) when compared with traditional hybrids. The reconstituted hybrids recorded the mean grain yield of 8,066 kg/ha, which was at par with original hybrids (mean: 7,846 kg/ha). The MAS-derived genotypes resembled their corresponding original hybrids for the majority of agronomic and yield-related traits, besides characteristics related to distinctness, uniformity, and stability (DUS). This is the first report for the development of maize with enhanced vitamin-E, vitamin-A, lysine, and tryptophan. [ABSTRACT FROM AUTHOR]

Traditional sweet corn possesses low levels of provitamin-A (proA), lysine and tryptophan. Mutant version of β-carotene hydroxylase1 (crtRB1) gene affecting the accumulation of β-carotene (BC), β-cryptoxanthin (BCX) and proA, and opaque2 (o2) gene governing the enhancement of lysine and tryptophan were introgressed together into elite sweet corn inbreds through marker-assisted selection. Here, we analyzed the expression pattern of crtRB1 and o2 genes among introgressed and traditional sweet corn inbreds at 20-, 24- and 28-days after pollination (DAP). The introgressed inbreds possessed two- to sevenfolds higher BC, BCX, proA, lysine and tryptophan compared to their original inbreds. However, all the nutrients attained the peak at 20-DAP (BC: 9.95 µg/g, BCX: 8.21 µg/g, proA: 14.05 µg/g, lysine: 0.301%, tryptophan: 0.074%), which gradually reduced through 24-DAP (BC: 8.24 µg/g, BCX: 7.53 µg/g, proA: 12.01 µg/g, lysine: 0.273%, tryptophan: 0.057%) and 28-DAP (BC: 5.84 µg/g, BCX: 5.82 µg/g, proA: 8.75 µg/g, lysine: 0.202%, tryptophan: 0.037%). Biofortified sweet corn inbreds possessed significantly lower expression levels of crtRB1 (4.1-fold) and o2 (2.2-fold) compared to their wild type alleles in traditional sweet corn inbreds across DAPs. The expression of crtRB1 and o2 increased from 20-DAP to attain the highest peak at 24-DAP, and further decreased by 28-DAP. The transcript levels of crtRB1 were negatively correlated with BC (r = − 0.83), BCX (r = − 0.79) and proA (r = − 0.83) across dates of harvest. Lysine (r = − 0.83) and tryptophan (r = − 0.73) were also inversely associated with o2 transcript levels. This is the first report on expression of crtRB1 and o2 genes during kernel development in biofortified sweet corn. This information holds immense promise in understanding the dynamics of gene-regulation during kernel development in sweet corn. [ABSTRACT FROM AUTHOR]

Harnessing the benefits of plant–microbe interactions towards better nutrient mobilization and plant growth is an important challenge for agriculturists globally. In our investigation, the focus was towards analyzing the soil–plant–environment interactions of cyanobacteria-based formulations (Anabaena–Nostoc consortium, BF1–4 and Anabaena–Trichoderma biofilm, An–Tr) as inoculants for ten maize genotypes (V1–V10). Field experimentation using seeds treated with the formulations illustrated a significant increase of 1.3- to 3.8-fold in C–N mobilizing enzyme activities in plants, along with more than five- to six-fold higher values of nitrogen fixation in rhizosphere soil samples. An increase of 22–30% in soil available nitrogen was also observed at flag leaf stage, and 13–16% higher values were also recorded in terms of cob yield of V6 with An-Tr biofilm inoculation. Savings of 30 kg N ha−1 season−1 was indicative of the reduced environmental pollution, due to the use of microbial options. The use of cyanobacterial formulations also enhanced the economic, environmental and energy use efficiency. This was reflected as 37–41% reduced costs lowered GHG emission by 58–68 CO2 equivalents and input energy requirement by 3651–4296 MJ, over the uninoculated control, on hectare basis. This investigation highlights the superior performance of these formulations, not only in terms of efficient C–N mobilization in maize, but also making maize cultivation a more profitable enterprise. Such interactions can be explored as resource-conserving options, for future evaluation across ecologies and locations, particularly in the global climate change scenario. [ABSTRACT FROM AUTHOR]

Summary: Vitamin A‐rich maize hybrids provide sustainable solutions to malnutrition. However, significant loss of carotenoids during storage reduces its efficacy. Grains of nine sub‐tropically adapted crtRB1‐based biofortified hybrids along with six normal hybrids were stored under conventional storage for five months. PVAC (β‐carotene and β‐cryptoxanthin) among crtRB1‐based hybrids degraded from initial level of 18.77 to 3.24 µg g−1, while NPVAC (lutein and zeaxanthin) reduced to 10.79 µg g−1 from 19.00 µg g−1 during storage. Among PVAC, β‐cryptoxanthin (21.8%) possessed more stability than β‐carotene (16.4%). For NPVAC, lutein (61.2%) showed the highest retention than zeaxanthin (50.4%). Majority of the PVAC loss occurred within first three months of storage. Retention for PVAC among crtRB1‐based hybrids varied from 14% to 23% indicating the role of favourable genetic factors. APQH1, APQH7 and APH2 were the promising hybrids with higher retention (>20%) of PVAC. This is the first report on identification of provitamin A‐rich crtRB1‐based biofortified maize hybrids with higher retention during sub‐tropical storage. [ABSTRACT FROM AUTHOR]

Malnutrition affects growth and development in humans and causes socio-economic losses. Normal maize is deficient in essential amino acids, lysine and tryptophan; and vitamin-A. Crop biofortification is a sustainable and economical approach to alleviate micronutrient malnutrition. We combined favorable alleles of crtRB1 and lcyE genes into opaque2 (o2)-based four inbreds viz. QLM11, QLM12, QLM13, and QLM14 using marker-assisted backcross breeding. These are parents of quality protein maize versions of two elite hybrids viz. Buland and PMH1, grown in India. Gene-based SSRs for o2 and InDel markers for crtRB1 and lcyE were successfully employed for foreground selection in BC1F1, BC2F1, and BC2F2 generations. The recurrent parent genome recovery ranged from 88.9 to 96.0% among introgressed progenies. Kernels of pyramided lines possessed a high concentration of proA (7.14–9.63 ppm), compared to 1.05 to 1.41 ppm in the recurrent parents, while lysine and tryptophan ranged from 0.28–0.44% and 0.07–0.09%, respectively. The reconstituted hybrids (RBuland and RPMH1) showed significant enhancement of endosperm proA (6.97–9.82 ppm), tryptophan (0.07–0.09%), and lysine (0.29–0.43%), while grain yield was at par with their original versions. The dissemination of reconstituted hybrids holds significant promise to alleviate vitamin-A deficiency and protein-energy malnutrition in developing countries. [ABSTRACT FROM AUTHOR]

Traditional sweet corn possesses low levels of provitamin-A (proA), lysine and tryptophan. Mutant version of β-carotene hydroxylase1 (crtRB1) gene affecting the accumulation of β-carotene (BC), β-cryptoxanthin (BCX) and proA, and opaque2 (o2) gene governing the enhancement of lysine and tryptophan were introgressed together into elite sweet corn inbreds through marker-assisted selection. Here, we analyzed the expression pattern of crtRB1 and o2 genes among introgressed and traditional sweet corn inbreds at 20-, 24- and 28-days after pollination (DAP). The introgressed inbreds possessed two- to sevenfolds higher BC, BCX, proA, lysine and tryptophan compared to their original inbreds. However, all the nutrients attained the peak at 20-DAP (BC: 9.95 µg/g, BCX: 8.21 µg/g, proA: 14.05 µg/g, lysine: 0.301%, tryptophan: 0.074%), which gradually reduced through 24-DAP (BC: 8.24 µg/g, BCX: 7.53 µg/g, proA: 12.01 µg/g, lysine: 0.273%, tryptophan: 0.057%) and 28-DAP (BC: 5.84 µg/g, BCX: 5.82 µg/g, proA: 8.75 µg/g, lysine: 0.202%, tryptophan: 0.037%). Biofortified sweet corn inbreds possessed significantly lower expression levels of crtRB1 (4.1-fold) and o2 (2.2-fold) compared to their wild type alleles in traditional sweet corn inbreds across DAPs. The expression of crtRB1 and o2 increased from 20-DAP to attain the highest peak at 24-DAP, and further decreased by 28-DAP. The transcript levels of crtRB1 were negatively correlated with BC (r = − 0.83), BCX (r = − 0.79) and proA (r = − 0.83) across dates of harvest. Lysine (r = − 0.83) and tryptophan (r = − 0.73) were also inversely associated with o2 transcript levels. This is the first report on expression of crtRB1 and o2 genes during kernel development in biofortified sweet corn. This information holds immense promise in understanding the dynamics of gene-regulation during kernel development in sweet corn. [ABSTRACT FROM AUTHOR]

'Sikkim Primitive' is a prolific maize landrace with five to nine ears per plant. Though the landrace was discovered in 1960s, loci governing the prolificacy was not identified. Here, a prolific inbred 'MGUSP101' developed from 'Sikkim Primitive' was crossed with two nonprolific inbreds, namely, HKI1128 and UMI1200. Two F2:3 populations (MGUSP101 × HKI1128 and MGUSP101 × UMI1200) were evaluated at three locations. Across locations, number of ears per plant varied from 1.35 to 5.38 in MGUSP101 × HKI1128 population. Bulked‐segregant analysis coupled with targeted QTL mapping identified a major QTL (bin: 8.05) explaining 31.7% phenotypic variation amongst 145 F2:3 individuals. The QTL was further validated in 138 F2:3 individuals of MGUSP101 × UMI1200 (range: 1.10–4.88). The identified QTL explained 29.2% of phenotypic variance at the same interval. We designated this novel QTL as 'qProl‐SP‐8.05' which has not been reported earlier. Six candidate genes responsible for prolificacy were also identified. This novel QTL can be introgressed through marker‐assisted selection for induction of prolificacy in elite maize. This is the first report of identification of locus governing prolificacy in 'Sikkim Primitive'. [ABSTRACT FROM AUTHOR]

Sweet corn has emerged as a popular vegetable worldwide. Commercial shrunken2 (sh2)-based sweet corn lacks lysine, tryptophan and provitamin-A, deficiency of which cause various health problems. To overcome these problems, marker-assisted stacking of sh2, opaque2 (o2), β-carotene hydroxylase (crtRB1) and lycopene epsilon cyclase (lcyE) was attempted to combine nutritional quality traits in maize endosperm. Gene-based simple sequence marker (SSR) specific to o2, InDel-based marker for crtRB1 and lcyE, and SSR linked to sh2 were used in marker-assisted selection. Four backcross populations segregating for the alleles of crtRB1, lcyE, o2 and sh2 were developed. The favourable alleles of sh2 and o2 produced 103 and 150 bp amplicons, while the unfavourable alleles were 96 and 160 bp, respectively. CrtRB1 produced 543 bp favourable and 296 bp unfavourable allele, while the same for lcyE was 650 and 300 bp, respectively. To detect desirable alleles of the target genes simultaneously, multiplex-PCR (Mx-PCR) assay was developed and validated in BC2F2 populations. Mx-PCR assay successfully identified the favourable alleles of four genes simultaneously, and validated the results of individual uniplex-PCR assays. Mx-PCR assay saved 68% of cost and approximately 63% of time. This is the first report of multiplexing sh2, o2, crtRB1 and lcyE genes in maize. The information generated here would be immensely useful in molecular breeding programme. [ABSTRACT FROM AUTHOR]

Traditional maize (Zea mays L.) is deficient in iron (Fe) and zinc (Zn). Metal transporter proteins play pivotal role in uptake, translocation and accumulation of minerals. Here, a set of 70 intergenic SSRs, 20 intragenic SSRs and 20 intragenic InDel markers encompassing 62 metal transporter genes were used for characterization of 24 diverse inbreds varying for Fe (13.95–37.34 µg/g) and Zn (11.83–37.90 µg/g). A total of 356 alleles, with 26 unique (7.03%) and 36 rare (10.11%) alleles were observed. Cluster analysis categorized the inbreds into two main clusters. Five candidate gene-based intragenic SSRs were significantly associated with Fe (r = 0.46–0.52). Two intragenic InDel markers and 11 SSRs were significantly correlated with Zn (r = 0.44–0.76). HUZM-185 and HKI-323 possessed favourable allele of five genes that showed significantly high correlation with Fe. At least six significantly associated genes for Zn were found in BQPML-5207-4-2, CML152, CML169 and HKI193-1. Based on genetic distance, presence of genes and high micronutrients, potential cross combinations were identified for the exploitation of heterosis. This study is the first report on characterizing maize inbreds using markers for metal transporter genes. [ABSTRACT FROM AUTHOR]

Malnutrition has emerged as one of the major health problems worldwide. Traditional yellow maize has low provitamin-A (proA) content and its genetic base in proA biofortification breeding program of subtropics is extremely narrow. To diversify the proA rich germplasm, 10 elite low proA inbreds were crossed with a proA rich donor (HP702-22) having mutant crtRB1 gene. The F2 populations derived from these crosses were genotyped using InDel marker specific to crtRB1. Severe marker segregation distortion was observed. Seventeen crtRB1 inbreds developed through marker-assisted pedigree breeding and seven inbreds generated using marker-assisted backcross breeding were characterized using 77 SSRs. Wide variation in gene diversity (0.08 to 0.79) and dissimilarity coefficient (0.28 to 0.84) was observed. The inbreds were grouped into three major clusters depicting the existing genetic diversity. The crtRB1-based inbreds possessed high β-carotene (BC: 8.72μg/g), β-cryptoxanthin (BCX: 4.58μg/g) and proA (11.01μg/g), while it was 2.35μg/g, 1.24μg/g and 2.97μg/g in checks, respectively. Based on their genetic relationships, 15 newly developed crtRB1-based inbreds were crossed with five testers (having crtRB1 gene) using line × tester mating design. 75 experimental hybrids with crtRB1 gene were evaluated over three locations. These experimental hybrids possessed higher BC (8.02μg/g), BCX (4.69μg/g), proA (10.37μg/g) compared to traditional hybrids used as check (BC: 2.36 μg/g, BCX: 1.53μg/g, proA: 3.13μg/g). Environment and genotypes × environment interaction had minor effects on proA content. Both additive and dominance gene action were significant for proA. The mean proportion of proA to total carotenoids (TC) was 44% among crtRB1-based hybrids, while 11% in traditional hybrids. BC was found to be positively correlated with BCX (r = 0.68) and proA (r = 0.98). However, no correlation was observed between proA and grain yield. Several hybrids with >10.0 t/ha grain yield with proA content >10.0 μg/g were identified. This is the first comprehensive study on development of diverse proA rich maize hybrids through marker-assisted pedigree breeding approach. The findings provides sustainable and cost-effective solution to alleviate vitamin-A deficiency. [ABSTRACT FROM AUTHOR]

Provitamin-A (proA) is essentially required for vision in humans but its deficiency affects children and pregnant women especially in the developing world. Biofortified maize rich in proA provides new opportunity for sustainable and cost-effective solution to alleviate malnutrition, however, significant loss of carotenoids during storage reduces its efficacy. Here, we studied the role of carotenoid cleavage dioxygenase 1 (ccd1) gene on degradation of carotenoids in maize. A set of 24 maize inbreds was analyzed for retention of proA during storage. At harvest, crtRB1-based maize inbreds possessed significantly high proA (β-carotene: 12.30 µg/g, β-cryptoxanthin: 4.36 µg/g) than the traditional inbreds (β-carotene: 1.74 µg/g, β-cryptoxanthin: 1.28 µg/g). However, crtRB1-based inbreds experienced significant degradation of proA carotenoids (β-carotene: 20%, β-cryptoxanthin: 32% retention) following 5 months. Among the crtRB1-based genotypes, V335PV had the lowest retention of proA (β-carotene: 1.63 µg/g, β-cryptoxanthin: 0.82 µg/g), while HKI161PV had the highest retention of proA (β-carotene: 4.17 µg/g, β-cryptoxanthin: 2.32 µg/g). Periodical analysis revealed that ~ 60–70% of proA degraded during the first three months. Expression analysis revealed that high expression of ccd1 led to low retention of proA carotenoids in V335PV, whereas proA retention in HKI161PV was higher due to lower expression. Highest expression of ccd1 was observed during first 3 months of storage. Copy number of ccd1 gene varied among yellow maize (1–6 copies) and white maize (7–35 copies) while wild relatives contained 1–4 copies of ccd1 gene per genome. However, copy number of ccd1 gene did not exhibit any correlation with proA carotenoids. We concluded that lower expression of ccd1 gene increased the retention of proA during storage in maize. Favourable allele of ccd1 can be introgressed into elite maize inbreds for higher retention of proA during storage. [ABSTRACT FROM AUTHOR]

The recessive shrunken2‐Reference (sh2‐Ref) allele is traditionally used in sweet corn cultivar development worldwide. However, non‐availability of suitable gene‐based marker(s) for sh2‐Ref has considerably delayed its utilization in molecular breeding. Here, 7,320 bp sequence of Sh2 gene among five wild‐ and six sh2‐Ref‐based inbreds was analysed by employing 13 overlapping primers. SNP583 (A in wild and G in mutant) and SNP755 (T in wild and C in mutant) in 5'UTR, and SNP5112 (C in wild and T in mutant) in intron‐12 clearly differentiated dominant (Sh2) and recessive (sh2‐Ref) allele. These SNPs were used to develop four gene‐based PCR markers. The newly developed markers were validated in six F2 populations segregating for sh2‐Ref allele, and 230 diverse inbreds of normal and sweet corn types. These markers were further used in genotyping of BC1F1 populations leading to successful selection of sh2‐Ref allele in the marker‐assisted breeding. Globally, this assumes great significance in molecular breeding of sweet corn. [ABSTRACT FROM AUTHOR]

Maize is the predominant food source for the world population, but lack of lysine and tryptophan in maize endosperm cannot fulfill the nutritional requirements of humans. Hence, the improvement of lysine and tryptophan content is the ultimate goal of maize biofortification programs. In the present study, the marker-assisted backcross (MABC) breeding strategy was used to enhance the lysine and tryptophan content of the elite maize inbred line UMI1230 by introgressing opaque 2 (o2) gene from the VQL1. During the transfer of the gene into UMI1230, SSR marker umc1066 tightly associated with o2 used for foreground selection. Background recovery was estimated using 168 SSR markers. Phenotype screening for morphological traits was adopted to choose plants parallel to UMI1230. As a result, four BC2F3 improved lines (DBT5-1-14/25-5/25-8/25-8/25, DBT5-1-14/25-5/25-8/25-7/25, DBT5-1-14/25-5/25-8/25-10/25 and DBT5-1-14/25-5/25-8/25-12/25) with o2 were developed. The improved line's background genome recovery varied between 90.60 and 94.80%. Also, the improved lines had better agronomic performance along with increased lysine (0.311–0.331%) and tryptophan (0.040–0.048%) contents. In summary, the MABC breeding strategy has successfully improved the levels of lysine and tryptophan in UMI1230 without affecting agronomic performance. The improved line's hold great potential as donors in biofortification programs in maize. [ABSTRACT FROM AUTHOR]

Biofilm formation of a nitrogen-fixing cyanobacterium Anabaena torulosa with a beneficial fungus Trichoderma viride (An-Tr) was examined under laboratory conditions. A gradual enhancement in growth over A. torulosa alone was recorded in the biofilm, with 15–20% higher values in nitrogen fixation, IAA and exopolysaccharide production illustrating the synergism among the partners in the biofilm. To investigate the role of such biofilms in priming seed attributes, mesocosm studies using primed seeds of two maize inbred lines (V6, V7) were undertaken. Beneficial effects of biofilm (An-Tr) were recorded, as compared to uninoculated treatment and cyanobacterial consortium (Anabaena–Nostoc; BF 1-4) at both stages (7 and 21 DAS, days after sowing) with a significant increase of more than 20% in seedling attributes, along with 5–15% increment in seed enzyme activities. More than three- to fivefold higher values in nitrogen fixation and C-N mobilizing enzyme activities, and significant increases in leaf chlorophyll, proteins and PEP carboxylase activity were observed with V7-An-Tr biofilm. Cyanobacterial inoculation brought about distinct changes in the soil phospholipid fatty acid profiles (PLFA); particularly, significant changes in those representing eukaryotes and anaerobic bacteria. Principal component analyses illustrated the significant role of dehydrogenase activity and microbial biomass carbon and distinct elicited effects on soil microbial communities, as evidenced by the PLFA. This investigation highlighted the promise of cyanobacteria as valuable priming options to improve mobilization of nutrients at seed stage, modulating the abundance and activities of various soil microbial communities, thereby, enhanced plant growth and vigour of maize plants. [ABSTRACT FROM AUTHOR]

Maize is a major source of food security and economic development in sub-Saharan Africa (SSA), Latin America, and the Caribbean, and is among the top three cereal crops in Asia. Yet, maize is deficient in certain essential amino acids, vitamins, and minerals. Biofortified maize cultivars enriched with essential minerals and vitamins could be particularly impactful in rural areas with limited access to diversified diet, dietary supplements, and fortified foods. Significant progress has been made in developing, testing, and deploying maize cultivars biofortified with quality protein maize (QPM), provitamin A, and kernel zinc. In this review, we outline the status and prospects of developing nutritionally enriched maize by successfully harnessing conventional and molecular marker-assisted breeding, highlighting the need for intensification of efforts to create greater impacts on malnutrition in maize-consuming populations, especially in the low- and middle-income countries. Molecular marker-assisted selection methods are particularly useful for improving nutritional traits since conventional breeding methods are relatively constrained by the cost and throughput of nutritional trait phenotyping. [ABSTRACT FROM AUTHOR]

Based on C (wild) to T (mutant) transition at amino acid position 1432 bp of lpa1-1 gene, two dominant markers each specific to wild type (LPA1) and mutant (lpa1-1) allele were developed and validated across seven F2 populations. Joint segregation of these markers behaved in co-dominant fashion, clearly distinguishing heterozygote from two other homozygote genotypes. Full length sequence alignment between wild type (LPA2) and mutant (lpa2-1) allele revealed one transition mutation (A to G) and a co-dominant CAPS marker was developed which differentiated all three types of segregants across seven F2 populations. Across populations, segregants with lpa1-1/lpa1-1 (1.77 mg/g) and lpa2-1/lpa2-1 (1.85 mg/g) possessed significantly lower phytic acid compared to LPA1/LPA1 (2.58 mg/g) and LPA2/LPA2 (2.53 mg/g). Inorganic phosphorus was however higher in recessive homozygotes (lpa1-1/lpa1-1: 0.77 mg/g, lpa2-1/lpa2-1: 0.53 mg/g) than the dominant homozygotes (LPA1/LPA1: 0.33 mg/g, LPA2/LPA2: 0.19 mg/g). Overall, homozygous segregants of lpa1-1 and lpa2-1 showed 31% and 27% reduction of phytic acid, respectively. Analysis of phytate and inorganic phosphorous in the maize kernel in these segregating populations confirmed co-segregation of trait and markers specific to lpa1-1 and lpa2-1. This is the first report of the development of breeder-friendly gene-based markers for lpa1-1 and lpa2-1; and it holds great significance for maize biofortification. [ABSTRACT FROM AUTHOR]

We sequenced the entire tb1 gene in six maize inbreds and its wild relatives (parviglumis, mexicana, perennis and luxurians) to characterize it at molecular level. Hopscotch and Tourist transposable elements were observed in the upstream of tb1 in all maize inbreds, while they were absent in wild relatives. In maize, tb1 consisted of 431–443 bp 5′UTR, 1101 bp coding sequence and 211–219 bp 3′UTR. In promoter region, mutations in the light response element in mexicana (~ 35 bp and ~ 55 bp upstream of TSS) and perennis (at ~ 35 bp upstream of TSS) were found. A 6 bp insertion at 420 bp downstream of the polyA signal site was present among teosinte accessions, while it was not observed in maize. A codominant marker flanking the 6 bp InDel was developed, and it differentiated the teosintes from maize. In Tb1 protein, alanine (12.7–14.6%) was the most abundant amino acid with tryptophan as the rarest (0.5–0.9%). The molecular weight of Tb1 protein was 38757.15 g/mol except 'Palomero Toluqueno' and HKI1128. R and TCP motifs in Tb1 protein were highly conserved across maize, teosinte and orthologues, while TCP domain differed for tb1 paralogue. Tb1 possessed important role in light-, auxin-, stress-response and meristem identity maintenance. Presence of molecular signal suggested its localization in mitochondria, nucleus and nucleolus. Parviglumis and mexicana shared closer relationship with maize than perennis and luxurians. A highly conserved 59–60 amino acids long bHLH region was observed across genotypes. Information generated here assumes significance in evolution of tb1 gene and breeding for enhancement of prolificacy in maize. [ABSTRACT FROM AUTHOR]