Mutant crtRB1 gene negates the unfavourable effects of opaque2 gene on germination and seed vigour among shrunken2-based biofortified sweet corn genotypes.

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) ß-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 (16.47µg g-1 ), lysine (0.36%) and tryptophan (0.09%) over original inbreds (proA: 3.14µg 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.

Allelic variation in shrunken2 gene affecting kernel sweetness in exotic-and indigenous-maize inbreds.

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.

Development of Maize Hybrids With Enhanced Vitamin-E, Vitamin-A, Lysine, and Tryptophan Through Molecular Breeding.

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 (ß-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.

Expression analysis of ß-carotene hydroxylase1 and opaque2 genes governing accumulation of provitamin-A, lysine and tryptophan during kernel development in biofortified sweet corn.

Traditional sweet corn possesses low levels of provitamin-A (proA), lysine and tryptophan. Mutant version of ß-carotene hydroxylase1 (crtRB1) gene affecting the accumulation of ß-carotene (BC), ß-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.

Development of multinutrient-rich biofortified sweet corn hybrids through genomics-assisted selection of shrunken2, opaque2, lcyE and crtRB1 genes.

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 ß-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.

Development of sub-tropically adapted diverse provitamin-A rich maize inbreds through marker-assisted pedigree selection, their characterization and utilization in hybrid breeding.

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 ß-carotene (BC: 8.72µg/g), ß-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.

Low expression of carotenoids cleavage dioxygenase 1 (ccd1) gene improves the retention of provitamin-A in maize grains during storage.

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 (ß-carotene: 12.30 µg/g, ß-cryptoxanthin: 4.36 µg/g) than the traditional inbreds (ß-carotene: 1.74 µg/g, ß-cryptoxanthin: 1.28 µg/g). However, crtRB1-based inbreds experienced significant degradation of proA carotenoids (ß-carotene: 20%, ß-cryptoxanthin: 32% retention) following 5 months. Among the crtRB1-based genotypes, V335PV had the lowest retention of proA (ß-carotene: 1.63 µg/g, ß-cryptoxanthin: 0.82 µg/g), while HKI161PV had the highest retention of proA (ß-carotene: 4.17 µg/g, ß-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.

Marker-assisted pyramiding of opaque2 and novel opaque16 genes for further enrichment of lysine and tryptophan in sub-tropical maize.

The improvement of protein quality in maize so far has been based on recessive opaque2 (o2) mutant that along with endosperm-modifiers led to development of quality protein maize (QPM). Recent discovery of nutritional benefits of recessive opaque16 (o16) mutant was of immense significance for further improvement of protein quality. In the present study, o16 was introgressed into o2-based parental inbreds (HKI161, HKI193-1, HKI193-2 and HKI163) of four commercial QPM hybrids (HQPM-1, HQPM-4, HQPM-5 and HQPM-7) released in India, using marker-assisted backcross breeding. Background selection led to high recovery of recurrent parent genome (RPG) to maximum of 95%, and introgressed progenies showed considerable phenotypic resemblance for plant-, ear- and grain- characteristics to their respective recurrent parents. Selection of markers for o2 and o16 led to development of pyramided lines (o2o2/o16o16) that possessed as high as 76% and 91% more lysine and tryptophan over the recurrent parents, respectively. Reconstituted hybrids showed an average enhancement of 49% and 60% in lysine and tryptophan over the original hybrids, with highest enhancement amounting 64% and 86%, respectively. This is first report of enhancement of both lysine and tryptophan by o16 in maize genotypes adaptable to sub-tropics. Moderate variation in lysine and tryptophan was also observed in pyramided lines. Multi-location evaluation of reconstituted hybrids revealed similar grain yield and attributing traits to their original versions. This study signified the role of o16 as supplementary to o2 for nutritional quality enhancement in maize, and improved elite inbreds and hybrids developed here hold great significance in maize biofortification programme.

Marker-assisted introgression of opaque2 allele for rapid conversion of elite hybrids into quality protein maize.

Maize is a valuable source of food and feed worldwide. Maize endosperm protein is, however nutritionally poor due to the reduced levels of two essential amino acids, lysine and tryptophan. In this study, recessive opaque2 (o2) allele that confers enhanced endosperm lysine and tryptophan, was introgressed using marker-assisted backcross breeding into three normal inbred lines (HKI323, HKI1105 and HKI1128). These are the parental lines of three popular medium-maturing single cross hybrids (HM4, HM8 and HM9) in India. Gene-based simple sequence repeat (SSR) markers (umc1066 and phi057) were successfully deployed for introgression of o2 allele. Background selection using genome-based SSRs helped in recovering > 96% of recurrent parent genome. The newly developed quality protein maize (QPM) inbreds showed modified kernels (25-50% opaqueness) coupled with high degree of phenotypic resemblance to the respective recipient lines, including grain yield. In addition, endosperm protein quality showed increased lysine and tryptophan in the inbreds to the range of 52-95% and 47-118%, respectively. The reconstituted QPM hybrids recorded significant enhancement of endosperm lysine (48-74%) and tryptophan (55-100%) in the endosperm. The QPM hybrids exhibited high phenotypic similarity with the original hybrids for morphological and yield contributing traits along with responses to some major diseases like turcicum leaf blight and maydis leaf blight. The grain yield of QPM hybrids was at par with their original versions under multilocation testing. These elite, high-yielding QPM hybrids with improved protein quality have been released and notified for commercial cultivation, and hold significant promise for improving nutritional security.

Development of Biofortified Maize Hybrids through Marker-Assisted Stacking of ß-Carotene Hydroxylase, Lycopene-ε-Cyclase and Opaque2 Genes.

Traditional yellow maize though contains high kernel carotenoids, the concentration of provitamin A (proA) is quite low (<2 µg/g), compared to recommended level (15 µg/g). It also possesses poor endosperm protein quality due to low concentration of lysine and tryptophan. Natural variant of crtRB1 (ß-carotene hydroxylase) and lcyE (lycopene-ε-cyclase) cause significant enhancement of proA concentration, while recessive allele, opaque2 (o2) enhances the level of these amino acids. Development of biofortified maize enriched in proA, lysine and tryptophan thus holds significance in alleviation of micronutrient malnutrition. In the present study, marker-assisted stacking of crtRB1, lcyE and o2 was undertaken in the genetic background of four maize hybrids (HQPM1, HQPM4, HQPM5, and HQPM7) popularly grown in India. HP704-22 and HP704-23 were used as donors, while four elite QPM parents viz., HKI161, HKI163, HKI193-1, and HKI193-2 were used as recipients. CrtRB1 showed severe segregation distortion, while lcyE segregated as per the expectation. Recovery of recurrent parent genome (RPG) among selected backcross progenies ranged from 89 to 93%. Introgressed progenies possessed high concentration of proA (7.38-13.59 µg/g), compared to 1.65-2.04 µg/g in the recurrent parents. The reconstituted hybrids showed an average of 4.5-fold increase in proA with a range of 9.25-12.88 µg/g, compared to original hybrids (2.14-2.48 µg/g). Similar plant-, ear-, and grain- characteristics of improved versions of both inbreds and hybrids were observed when evaluated with their respective original versions. Mean lysine (0.334%) and tryptophan (0.080%) of the improved hybrids were at par with the original versions (lysine: 0.340%, tryptophan: 0.083%). Improved hybrids also possessed similar grain yield potential (6,301-8,545 kg/ha) with their original versions (6,135-8,479 kg/ha) evaluated at two locations. This is the first study of staking crtRB1-, lcyE-, and o2-, favorable alleles in single genetic background. The improved inbreds can be effectively used as potential donor for independent and/or simultaneous introgression of crtRB1, lcyE, and o2 in the future breeding programme. These biofortified maize hybrids, rich in proA, lysine and tryptophan will hold great promise for nutritional security.

Molecular characterization of 5' UTR of the lycopene epsilon cyclase (lcyE) gene among exotic and indigenous inbreds for its utilization in maize biofortification.

Maize grains are the important source of food and energy, but possess very low proA (< 2.5 µg/g) compared to target level of 15 µg/g set by HarvestPlus to alleviate VAD. Favorable allele having variation in 5' untranslated region (UTR) of lycopene epsilon cyclase (lcyE) gene enhances concentration of proA in maize. To identify the sequence variation in 5' UTR of lcyE, a set of diverse 13 inbreds of indigenous and exotic origin was characterized for allelic constitution of lcyE. Inbreds possessed wide variation in proA (1.62-23.12 µg/g) with a mean of 9.64 µg/g. The proA in CIMMYT-HarvestPlus genotypes having favorable allele of lcyE was very high (22.28 µg/g), whereas the Indian inbreds with the same allele possessed very low proA (2.48 µg/g). Eight genotypes viz., HKI161, HKI163, HKI161-PV, HKI163-PV, HKI193-1-PV, HKI193-2-PV, HP704-22 and HP704-23 revealed the presence of favorable allele, while VQL1, DMRIL47, MGU-PV-123/C6, HKI193-1 and HKI193-2 showed the presence of unfavorable allele of lcyE gene. Sequence comparison of favorable allele of Indian (HKI161 and HKI163) and exotic genotypes (HP704-22 and HP704-23) revealed seven SNPs having three transitions (SNP1 and SNP3: G to A, SNP2: C to T) and four transversions (SNP4: C to G, SNP5: T to G, SNP6: G to C and SNP7: G to T). Four SNPs (SNP1: position 446, SNP2: position 458, SNP3: position 459 and SNP4: position 483) discriminated the low- and high- proA lines having favorable allele of lcyE 5'TE. These SNPs hold significance in enrichment of proA in maize for marker development and their use in marker-assisted selection.

Opaque16, a high lysine and tryptophan mutant, does not influence the key physico-biochemical characteristics in maize kernel.

The enhancement of lysine and tryptophan in maize is so far basedon opaque2(o2) mutant, that along with the endosperm-modifiersled to development of Quality Protein Maize[QPM]. Though many mutants improving the endospermic protein quality were discovered, they could not be successfully deployed. Recently discovered opaque16 (o16)mutant enhances the lysine and tryptophan content in maize endosperm. In the present study, the influence of o16 on the endosperm modification was analyzed in four F2 populations, two each segregating for o16 allele alone and in combination with o2. The recessive o16o16 seed endosperm was found to be vitreousphenotypically similar to wild-O16O16. The mutant did not influence the degree of kernel opaqueness in o2o2 genetic background as opaqueness in o2o2/O16O16 and o2o2/o16o16 was similar. Grain hardness of o16o16 was comparable with the normal and QPM maize. The pattern of microscopic organization of proteinaceous matrix and starch granules, and zein profiling of the storage protein in o16o16 were found to be similar with normal maize endosperm, but distinct from the o2o2-soft genotype. The pattern in o2o2/o16o16 was unique and different from o2o2 and o16o16 as well. Here we demonstrated the effects of o16 on physico-biochemical characteristics of endosperm and report of o16 possessing negligible influence on kernel modification and hardness, which holds a great significance in maize quality breeding programme.

Microsatellite marker-based characterization of waxy maize inbreds for their utilization in hybrid breeding.

Waxy corn possesses 95-100% amylopectin, compared to 70-75% in normal maize, owing to mutation in Wx gene encoding a granule-bound starch synthase I. Amylopectin is used as an ingredient in textile, adhesive and paper industries. Further, waxy green cob is popular as breakfast item in South Asia and an important constituent of diet in north-eastern states of India as well. We developed a series of waxy inbreds from diverse exotic sources and through introgression breeding. To characterize and unravel the genetic relationships, 24 diverse waxy inbreds were analysed using 77 SSRs distributed throughout the genome. The study generated a total of 203 polymorphic alleles, with a mean of 2.69 alleles per locus. A total of nine unique and 20 rare alleles were detected. The polymorphism information content ranged from 0.08 to 0.68 with an average value of 0.40. Molecular profiling suggested sufficient attainment of homozygosity among the inbreds. Jaccard's dissimilarity coefficient between pairs of genotypes varied from 0.26 to 0.83 which revealed the diverse nature of the inbred lines. Cluster analyses grouped 24 genotypes into three major clusters. Principle coordinate analysis based on SSR also depicted the diverse origin of the genotypes as per the pedigree more reliably than agro-morphological traits. These inbreds were also promising for various cob and grain characteristics including grain yield. The study identified a set of potential cross-combinations that can be planned to develop highly heterotic waxy hybrid combinations. This is the first report of development and characterization of waxy inbreds in India.

Microsatellite-based genetic diversity analyses of sugary1-, shrunken2- and double mutant- sweet corn inbreds for their utilization in breeding programme.

Sweet corn has recently experienced sharp rise in demand worldwide. Recessive sugary1 (su1) and shrunken2 (sh2) that enhances kernel sweetness have been abundantly used in sweet corn breeding. Analyses of genetic diversity among sweet corn inbreds assume great significance for their effective utilization in hybrid breeding. A set of 48 diverse sweet corn genotypes encompassing su1su1, sh2sh2 and su1su1/sh2sh2 types were analyzed using 56 microsatellite markers. A total of 213 alleles with mean of 3.8 alleles per locus were generated. Two unique- and 12 rare- alleles were identified. The average PIC and genetic dissimilarity was 0.50 and 0.73, respectively. Cluster analysis grouped the inbreds into three major clusters, with each of the su1su1-, sh2sh2- and su1su1/sh2sh2-types were broadly clustered together. Principal coordinate analyses also depicted the diverse origin of the genotypes. The study identified inbreds for synthesis of pools and pedigree populations to develop novel inbreds. The study led to the identification of prospective heterotic combinations in various genetic backgrounds (sh2sh2 × sh2sh2, su1su1 × su1su1, su1su1/sh2sh2 × su1su1/sh2sh2, sh2sh2 × su1su1/sh2sh2 and su1su1 × su1su1/sh2sh2).