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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 BC 1 F 1 , BC 2 F 1 and BC 2 F 2 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., (© 2022. The Author(s).)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

Background and Objectives: Maize protein lacks essential amino acids such as methionine, lysine, and tryptophan. Limited information exists on the composition and variation of kernel methionine. This study assessed methionine variability and its relationship with lysine and tryptophan in 48 maize inbreds at three locations. Findings: Analysis of variance revealed significant variations for kernel methionine (0.031%–0.305%), lysine (0.118%–0.389%), and tryptophan (0.014%–0.090%). CML591 (0.305%), CML596 (0.286%), and CML421 (0.284%) showed the highest methionine content. The average methionine contents were comparable in quality protein maize (0.156%) and normal (0.150%) lines. Methionine showed no correlation with lysine (r = 0.001) and tryptophan (r = 0.032), while lysine and tryptophan showed a strong positive correlation (r = 0.740). CML165 (methionine: 0.272%, lysine: 0.320%, and tryptophan: 0.090%), CML186 (methionine: 0.254%, lysine: 0.389%, and tryptophan: 0.083%), and HKI163 (methionine: 0.252%, lysine: 0.331%, and tryptophan: 0.084%) were promising genotypes considering all three amino acids. Conclusions: Multilocation experiment revealed genetic variation in methionine, lysine, and tryptophan among the 48 inbreds. No negative correlation between the amino acids indicates the possibility of developing cultivars high in methionine, lysine, and tryptophan. Significance and Novelty: Identified nutrient‐rich inbreds will serve as valuable germplasm in maize biofortification programs. This is the first report on methionine variability and its relationship with lysine and tryptophan in subtropical maize. [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]

Maize is an important agricultural plant valued for its productivity and nutritive qualities. However, a deficiency of two essential amino acids (lysine and tryptophan) significantly reduces the nutritional quality of maize proteins. The recessive opaque-2 (o2) mutant has a greater content of lysine and tryptophan in their endosperm proteins and their bioavailability is better. Therefore, marker-assisted backcross breeding (MABB) was attempted to incorporate the o2 allele from the donor line VQL 1 into the genetic background of UMI 1200 to develop quality protein maize (QPM) lines. Foreground selection for the gene o2 was effected using tightly linked molecular marker umc1066, in UMI 1200 × VQL 1 backcross series. Further, background selection was done together with stringent phenotypic selection for agronomic traits, to accelerate recurrent parent phenome recovery. As a result, three advanced QPM maize lines (DBT 4-1-1/25-10/25-10/25-16, DBT 4-1-1/25-10/25-17/25-11 and DBT 4-1-1/25-10/25-17/25-13) carrying the opaque-2 allele in a homozygous state, similar to VQL 1 were developed. The lysine and tryptophan content of these lines ranged from 0.35% to 0.40% and 0.03 to 0.05%, respectively, which is on par with the VQL 1. Background analysis using 250 simple sequence repeats (SSRs) revealed up to 97% recurrent parent genome recovery. In conclusion, the newly developed QPM lines can be used in future maize breeding programmes to improve the nutritional traits. [ABSTRACT FROM AUTHOR]

Vitamin A deficiency in humans is a widespread health problem. Quality protein maize (QPM) is a popular food rich in lysine and tryptophan, but poor in provitamin A (proA). Here, we report the improvement of an elite QPM inbred, HKI1128Q for proA using marker‐assisted introgression of crtRB1‐favourable allele. HKI1128 was one of the parental lines of three popular hybrids in India and was converted to QPM in our earlier programme. Severe segregation distortion for crtRB1 was observed in BC1F1, BC2F1 and BC2F2. Background selection by 100 SSRs revealed mean recovery of 91.07% recurrent parent genome varying from 88.78% to 93.88%. Across years, introgressed progenies possessed higher mean β‐carotene (BC: 9.22 µg/g), β‐cryptoxanthin (BCX: 3.05 µg/g) and provitamin A (proA: 10.75 µg/g) compared to HKI1128Q (BC: 2.26 µg/g, BCX: 2.26 µg/g and proA: 3.38 µg/g). High concentration of essential amino acids, viz. lysine (mean: 0.303%) and tryptophan (0.080%) in endosperm, was also retained. Multi‐year evaluation showed that introgressed progenies possessed similar grain yield (1,759–1,879 kg/ha) with HKI1128Q (1,778 kg/ha). Introgressed progenies with higher lysine, tryptophan and proA hold immense potential as donors and parents in developing biofortified hybrids. [ABSTRACT FROM AUTHOR]

Modifier loci in QPM play a vital role in achieving acceptable degree of kernel hardness and accumulation of lysine and tryptophan. This study was undertaken to characterize a set of diverse QPM inbreds using SSRs linked to endosperm and amino acids modifier loci for their effective utilization in the breeding programme. Significant variation was observed for endosperm modification (25-100% opaqueness), tryptophan (0.056-0.111%) and lysine (0.223-0.444%). Generally, inbreds with soft endosperm possessed more tryptophan and lysine than inbreds with higher vitreousness. SSRs generated 341 alleles with two to seven alleles per locus. The frequency of unique and rare alleles was more for amino acid modifications, compared to endosperm modifications. Phylogenetic analyses grouped the inbreds into three major clusters, and the study identified suitable crosses for accumulation of endosperm and amino acids modifiers. QPM inbreds with desirable modifications identified here would serve as suitable donor for both opaque2 and modifier loci in the marker-assisted backcross breeding. Further, contrasting inbreds can be used for generating mapping populations to identify new modifier loci underlying both endosperm and amino acids modifications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Normal maize possesses low lysine (0.150−0.250%) & tryptophan (0.030−0.040%). • Improved inbreds with o2 & o16 possessed 0.520 % lysine and 0.132 % tryptophan. • MAS-hybrids recorded higher lysine (152 %) & tryptophan (332 %) over normal maize. • First report of white hybrids with novel combination of o2 & o16 genes. Traditional maize possesses low lysine (0.150−0.250%) and tryptophan (0.030−0.040%) essential for human growth and development. So far, no maize hybrid with recessive opaque2 (o2) and opaque16 (o16) genes that significantly enhance lysine and tryptophan is released for commercial cultivation. Here, we introgressed both o2 and o16 genes in the parental lines of two popular white hybrids viz., HM5 (HKI1344 × HKI1348−6-2) and HM12 (HKI1344 × HKI1378) through molecular breeding. Gene-specific simple sequence repeat (SSRs) markers (umc1066 and phi057) for o2 and gene-linked SSRs (umc1149 and umc1141) for o16 were utilized for genotyping BC 1 F 1 , BC 2 F 1 and BC 2 F 2 populations. Use of >100 SSRs led to 90–95 % recovery of recurrent parent genome. The mean lysine and tryptophan among introgressed progenies was 0.520 % and 0.132 %, respectively compared to original lines (lysine: 0.212 % and tryptophan: 0.039 %). The reconstituted hybrids recorded 152 % and 332 % higher lysine and tryptophan, respectively over original hybrids. The o2o2/o16o16 -based hybrids also possessed 51 % and 43 % higher lysine and tryptophan, respectively over o2 -based checks. The grain yield of improved versions of HM5 (7203 kg/ha) and HM12 (6247 kg/ha) was at par with original versions (HM5: 7282 kg/ha; HM12: 6482 kg/ha). These newly developed maize hybrids hold immense promise to alleviate malnutrition. [ABSTRACT FROM AUTHOR]

• Biofortified sweet corn hybrids were analysed at different harvest and sowing time. • These hybrids possessed significantly higher provitamin A, lysine and tryptophan. • 20 days after pollination was the ideal harvest time for nutritional traits. • Late sowing during rainy season favoured accumulation of nutrients in sweet corn. • This study holds significance in harnessing the nutritional benefit of sweet corn. Traditional sweet corn is poor in nutritional quality. Here, a set of biofortified sweet corn hybrids were analyzed for nutritional traits at 20-, 24- and 28- days after pollination (DAP) under three sowing dates. The biofortified hybrids possessed significantly higher lysine (0.34 %), tryptophan (0.08 %), β-carotene (BC: 13.77 μg/g), β-cryptoxanthin (BCX: 11.19 μg/g) and provitamin-A (proA: 19.38 μg/g) over traditional yellow sweet corn (0.21 %, 0.03 %, 1.96 μg/g, 2.32 μg/g and 3.12 μg/g). Sowing time, harvest time and their interactions had significant influence on nutritional traits. Decreasing trend of accumulation for lysine (0.367 %, 0.345 % and 0.315 %), tryptophan (0.086 %, 0.078 % and 0.068 %), BC (15.18 μg/g, 14.00 μg/g and 12.12 μg/g), BCX (12.28 μg/g, 11.48 μg/g and 9.82 μg/g) and proA (21.32 μg/g, 19.74 μg/g and 17.07 μg/g) was observed at 20-, 24- and 28-DAP, respectively. However, mean brix attained the peak at 24-DAP (18.53 %), followed by 20-DAP (17.86 %) and 28-DAP (17.60 %). Considering both nutrition and sweetness, 20-DAP was the best suitable time for harvesting of sweet corn ears. Nutritional traits had higher concentration with delay in sowing time. This is the first report on accumulation of lysine, tryptophan and proA at different stages of kernel development in biofortified sweet corn. [ABSTRACT FROM AUTHOR]

Traditional sweet corn is poor in provitamin-A, lysine and tryptophan, deficiency of which causes serious health problems. Here, parental lines of two shrunken2 (sh2) -based sweet corn hybrids viz., ASKH-1 and ASKH-2 were targeted for introgression of crtRB1 and opaque2 (o2) genes through marker-assisted backcross breeding. Gene-based markers; umc1066 (SSR) and 3′TE-InDel were utilized for foreground selection of o2 and crtRB1 , respectively in BC 1 F 1 , BC 2 F 1 and BC 2 F 2 generations. Background selection employing 102–113 polymorphic SSRs led to >90% recovery of recurrent parent genome. Reconstituted hybrids recorded high mean provitamin-A (18.98 μg/g) with a maximum of 7.7-fold increase over original hybrids (3.12 μg/g). High mean lysine (0.39%) and tryptophan (0.10%) with an average enhancement of 1.71- and 1.79-fold, respectively was recorded among reconstituted hybrids over original versions (lysine: 0.23%, tryptophan: 0.06%). Improved hybrids exhibited high phenotypic resemblance with their original hybrids. The average cob yield (11.82 t/ha) and brix (17.66%) of improved hybrids was at par with their original versions (cob yield: 11.27 t/ha, brix: 17.04%). These biofortified sweet corn hybrids rich in provitamin-A, lysine and tryptophan hold immense significance as multinutrient-rich balanced food. This is the first report to stack sh2 , crtRB1 and o2 genes to improve nutritional quality in sweet corn. Image 1 • Traditional sweet corn lacks adequate provitamin-A, lysine and tryptophan. • Two sweet corn hybrids were biofortified by introgression of o2 and crtRB1 genes. • Improved hybrids possessed significantly high provitamin-A, lysine and tryptophan. • Biofortified sweet corn hybrids would provide multinutrient-rich balanced food. • This is the first report of stacking o2 and crtRB1 genes in sweet corn. [ABSTRACT FROM AUTHOR]

Traditional maize is poor in amylopectin (75%), lysine (0.200%), and tryptophan (0.045%). While the recessive waxy1 (wx1) gene enhances amylopectin and the recessive opaque2 (o2) gene increases lysine and tryptophan. Here, we analyzed transcripts of wx1 and o2 genes in (i) three wild-type (Wx1Wx1/O2O2), (ii) six waxy (wx1wx1/O2O2), (iii) four QPM (Wx1Wx1/o2o2), and (iv) eight waxy-QPM (wx1wx1/o2o2) inbreds at 20-, 30- and 40-days after pollination (DAP). The waxy inbreds possessed ∼1.4-fold higher amylopectin, while QPM inbreds showed 1.5- and 1.7-fold more accumulation of lysine and tryptophan over wild types. Interestingly, double mutant (wx1wx1/o2o2) had 2.1% higher amylopectin, and 15.6% and 16.6% more lysine and tryptophan, respectively over single mutants. Amylopectin showed an increase from 20-DAP (94.66%) to 30-DAP (96.56%) with a peak at 40-DAP (97.32%). On contrary, lysine and tryptophan peaked at 20-DAP (lysine: 0.552%, tryptophan: 0.267%), which gradually reduced through 30-DAP (lysine: 0.448%, tryptophan: 0.166%) and 40-DAP (lysine: 0.325%, tryptophan: 0.081%). Waxy and QPM inbreds possessed significantly lower expression of wx1 (∼3.0-fold) and o2 (∼1.1-fold) alleles over wild-type alleles (Wx1 and O2). Expression of wx1 and o2 in double mutant was 57.8% and 80.7% lower over single mutants, respectively. This is the first report on the expression of wx1 and o2 genes during maize kernel development. • wx1 and o2 had higher amylopectin, lysine and tryptophan over Wx1 and O2 type. • wx1wx1/o2o2 had higher amylopectin, lysine and tryptophan over wx1 and o2 alone. • Amylopectin peaked at 40-DAP, while lysine and tryptophan peaked at 20-DAP. • Mutant wx1 and o2 alleles had lower expression over wild Wx1 and O2 alleles. • Expression of wx1 and o2 in double mutant was lower over single mutants. [ABSTRACT FROM AUTHOR]

Traditional maize protein is nutritionally poor due to low levels of lysine (0.150–0.250%) and tryptophan (0.030–0.040%). The recessive opaque2 (o2) gene that enhances lysine (0.300–0.400%) and tryptophan (0.070–0.100%) is used in developing quality protein maize hybrids. Mutant opaque16 (o16) in the presence of o2 further enhances lysine and tryptophan. Here we analyzed a set of seven diverse maize inbreds possessing o2 and o16 for their potential to develop heterotic hybrids. Characterization of inbreds using 75 SSRs revealed 177 alleles with wide variation in dissimilarity coefficient (0.34–0.80). Phylogenetic analysis grouped the o2o2/o16o16 -based inbreds into three clusters. Inbreds were further crossed in a half-diallel mating design, resulting in 21 experimental hybrids were evaluated at two locations. Experimental o2o2/o16o16 -based hybrids possessed significantly higher lysine (0.506%) and tryptophan (0.126%) compared to commercial o2 -based QPM hybrids (lysine: 0.346%, tryptophan: 0.086%). Combining ability for grain yield identified several promising inbreds and hybrids. Grain yield was significantly correlated with genetic distance (r = 0.57**) and SCA effect (r = 0.67**). Selected hybrids with >7500 kg/ha grain yield, >0.500% lysine and >0.120% tryptophan possess great potential in providing both food- and nutritional-security. The improved hybrids with a novel combination of o2 and o16 provide sustainable and cost-effective solution to alleviate protein energy malnutrition. [Display omitted] • Inbreds with o2o2/o16o16 revealed wide genetic diversity. • Phylogenetic analysis grouped the o2o2/o16o16 -based inbreds into three clusters. • o2o2/o16o16 -based hybrids possessed significantly higher lysine and tryptophan. • Combining ability identified promising o2o2/o16o16 -based inbreds and hybrids. • Grain yield was significantly correlated with genetic distance and SCA effect. [ABSTRACT FROM AUTHOR]

Sweet corn is a popular food worldwide. Traditional shrunken2 (sh2)-derived sweet corn possesses low lysine (0.150–0.250%), tryptophan (0.030–0.040%) and provitamin-A (proA: 3–4 μg/g). Here transcript profiles of a newly developed 12 sh2 -based sweet corn inbreds possessing mutant opaque2 (o2) and β-carotene hydroxylase (crtRB1) genes were analyzed during three stages of kernel development viz., 20-, 24- and 28- days after pollination (DAP). Introgressed inbreds had higher lysine (0.309%), tryptophan (0.077%) and proA (17.28 μg/g). Across stages of kernel development, transcript levels of mutant sh2 , o2 and crtRB1 genes were significantly lower than the wild type (sh2 +, o2 + and crtRB1 +) alleles. Among genes, sh2 had the highest level of transcripts accumulation followed by crtRB1 and o2. While o2 and crtRB1 transcripts attained peaks at 24-DAP, the highest peak for sh2 was recorded at 28-DAP. Strong negative correlation (r = −0.99 to −0.71) was observed between the transcripts and corresponding nutrients. The study provided insight of complex regulation of sh2 , o2 and crtRB1 genes during various stages of kernel development. This is a first of its kind study to analyze the transcript level variation of mutant allele of sh2 , o2 and crtRB1 genes among novel biofortified sweet corn genotypes. [Display omitted] • Introgressed inbreds possessed higher lysine, tryptophan & provitamin-A. • Transcript of mutant sh2 , o2 & crtRB1 had lower levels than the wild type allele. • sh2 had the highest level of transcript accumulation followed by crtRB1 and o2. • o2 and crtRB1 transcripts had peak at 24-DAP & peak for sh2 was recorded at 28-DAP. • Negative correlation was observed between the transcripts and respective nutrients. [ABSTRACT FROM AUTHOR]