Vignesh Muthusamy

Development of β-carotene rich maize hybrids through marker-assisted introgression of β-carotene hydroxylase allele.

Development of vitamin A-rich cereals can help in alleviating the widespread problem of vitamin A deficiency. We report here significant enhancement of kernel β-carotene in elite maize genotypes through accelerated marker-assisted backcross breeding. A favourable allele (543 bp) of the β-carotene hydroxylase (crtRB1) gene was introgressed in the seven elite inbred parents, which were low (1.4 µg/g) in kernel β-carotene, by using a crtRB1-specific DNA marker for foreground selection. About 90% of the recurrent parent genome was recovered in the selected progenies within two backcross generations. Concentration of β-carotene among the crtRB1-introgressed inbreds varied from 8.6 to 17.5 µg/g - a maximum increase up to 12.6-fold over recurrent parent. The reconstituted hybrids developed from improved parental inbreds also showed enhanced kernel β-carotene as high as 21.7 µg/g, compared to 2.6 µg/g in the original hybrid. The reconstituted hybrids evaluated at two locations possessed similar grain yield to that of original hybrids. These β-carotene enriched high yielding hybrids can be effectively utilized in the maize biofortification programs across the globe.

Molecular Characterization of Exotic and Indigenous Maize Inbreds for Biofortification with Kernel Carotenoids

Maize, consumed as food and feed, possesses a number of carotenoids for which ample genetic variability exists. Two of them, lutein and zeaxanthin, function as antioxidants while β-cryptoxanthin and β-carotene serve as precursors to vitamin A in human body. We report here genetic diversity in 48 diverse maize inbreds for their utilization in the biofortification program. While there was wide genetic variation for lutein (0.36–15.75 μg/g), zeaxanthin (0.25–22.76 μg/g), and β-carotene (0.07–17.41 μg/g), low variation was observed for β-cryptoxanthin (0.06–4.37 μg/g). Fifty-eight microsatellite markers having genome-wide distribution generated a total of 225 polymorphic alleles among the inbreds. Two to seven alleles with a mean of 3.88 alleles per marker locus were detected. A total of 35 rare alleles and 11 unique alleles were obtained. The polymorphism information content ranged from 0.14 to 0.75 with an average of 0.52. Mean pair-wise genetic dissimilarity of 0.74 revealed the diverse nature of the inbreds. Neighbor-joining based clustering pattern grouped the inbreds into six major clusters. Principal coordinate analysis also depicted the diverse origin of the genotypes consistent with their pedigree. The CIMMYT-HarvestPlus bred inbreds with high kernel β-carotene concentration were genetically distant from the inbreds of Indian origin. Based on genetic distance and carotenoid concentration, potential heterotic combinations have been identified for biofortification of lutein and zeaxanthin. The CIMMYT-HarvestPlus inbreds with high β-carotene can serve as potential donors of β-carotene hydroxylase (crtRB1) favorable allele for introgression into Indian maize inbreds through marker-assisted selection.

Exploration of novel opaque16 mutation as a source for high lysine and tryptophan in maize endosperm

In the present study, two F2 populations derived by crossing wild type-(CML533 and CML537) and o16-donor line (QCL3024) were raised and genotyped. The recessive o16o16 possessed nearly two-fold more lysine (0.247%) and tryptophan (0.072%) in mutants, than wild type (0.125% lysine and 0.035% tryptophan) across populations. However, o16o16 showed wide variation for both lysine (0.111–0.376%) and tryptophan (0.027–0.117%) across populations, suggesting the role of modifier loci. The study did not show any significant correlation (r=0.14) between lysine and tryptophan, however some segregants with highlysine (>0.300%) and-tryptophan (>0.090%) were identified. Some of the segregants with o16o16 possessed comparable lysine and tryptophan with o2o2 genotypes. These o16o16 segregants may serve as novel genetic resource in the QPM breeding programme, and some of them can be used for enhancement of both lysine and tryptophan in place of o2 mutant. This is the first ever report of influence of o16 on accumulation of tryptophan in maize.

Allelic variations for lycopene-ε-cyclase and β-carotene hydroxylase genes in maize inbreds and their utilization in β-carotene enrichment programme

Abstract Vitamin A deficiency is a global health problem and can be effectively alleviated through crop biofortification. Quantification of carotenoids using high-performance liquid chromatography is expensive and time-consuming, thereby posing a challenge in the selection of genotypes with high provitamin A. Favourable alleles possessing rare genetic variation in lycopene-ε-cyclase (lcyE) and β-carotene hydroxylase (crtRB1) genes are associated with higher accumulation of provitamin A, especially β-carotene; and selection of these alleles holds immense promise in reducing large-scale phenotypic assays. Screening of a diverse set of 385 maize inbred lines of indigenous and exotic origin detected the presence of two alleles (amplicon size: 250 and 650 bp) of lcyE and three alleles (amplicon size: 296, 543 and 875 bp) of crtRB1 in the inbred panel. Favourable alleles of both the genes were rare among the traditional maize germplasm; 3.38% of the inbreds possessed the favourable allele (650 bp) of lcyE, while 3.90% inbreds had the favourable allele (543 bp) of crtRB1. Five inbreds (1.3%) with favourable alleles of both the genes were found. Inbreds with favourable alleles of crtRB1 and lcyE serve as rich genetic resources for effective utilization in the maize biofortification programme.

Characterization of β-carotene rich MAS-derived maize inbreds possessing rare genetic variation in β-carotene hydroxylase gene

Traditional yellow maize kernels though contain enough carotenoids, the concentration of -carotene is quite low. A rare natural variant of crtRB1 gene enhances -carotene in the kernel by blocking its conversion to further components. β-carotene rich versions of inbreds, VQL1, VQL2, V335, V345, HKI161, HKI323 and HKI1105 developed through marker-assisted backcross breeding strategy were characterized for selected DUS-characters, yield and yield, attributing traits. The crtRB1-introgressed inbreds displayed high degree of phenotypic resemblance with their respective original parents for majority of the characters. V335 based introgressed inbreds displayed 100% phenotypic similarity to original parent, followed by VQL1 (88.9–100%), HKI161 (88.9–100%), VQL2 (94.4%),V345 (88.9%), HKI323 (83.3–94.4%) and HKI1105 (77.8–83.3%) based introgressed inbreds. Introgressed inbreds having contrast for pigmentation in glume base and silk (with respective recurrent parents) possess great utility for registration and unambiguous identification in the field. These newly developed β-carotene rich maize inbreds hold enormous potential in maize biofortification programme.

Evaluation of specialty corn inbreds for responses to stored grain weevil (Sitophilus oryzae L.) infestation

Evaluation of a diverse set of 68 specialty corn inbreds (pop corn, sweet corn and QPM) against stored grain weevil (Sitophilus oryzae L.) revealed existence of wide genetic variations for grain weight loss (3.40–41.21%), number of insect progeny emerged (5.00–76.33), germination of seeds after infestation (0.00–81.33%), pericarp thickness (36.18–178.13 m) and grain hardness (62.33–600.33 Newton). Pop corn inbreds recorded lowest mean grain weight loss (8.19%) and number of insect progeny emerged (9.23) with highest mean germination (78.28%). Grain weight loss and number of insect progeny emerged were positively correlated, while both showed negative correlation with germination percentage. Pericarp thickness and grain hardness could not contribute to confer resistance. Based on cumulative resistance index (CRI), pop corn inbreds were in general more resistant (mean CRI=1.84) to weevil infestation as compared to shrunken sweet corn (mean CRI=1.51), sugary sweet corn (mean CRI=1.26) and QPM (mean CRI=0.76) inbreds. However, resistant inbreds were also identified in sweet corn and QPM types that can be utilized effectively as donors in the resistance breeding programme. The contrasting inbreds could serve as rich genetic resources for unravelling the basis of resistance.

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.

Popping quality attributes of popcorn hybrids in relation to weevil (Sitophilus oryzae) infestation

A set of 21 popcorn hybrids were evaluated at multilocations to study the effects of weevil (Sitophilus oryzae) infestation on popping quality traits. Wide genetic variation for percent popping expansion (PPE) and grain popping percentage (GPP) after weevil infestation were observed. PPE and GPP varied from 6.09–85.21% and 4.33–66.67%, across locations, respectively. The mean PPE was 52.90%, while same for GPP was 23.46%. Significant effects of environment and genotype × environment interactions were observed for both PPE and GPP. Strong positive correlation (r=0.89) across locations was recorded between PPE and GPP. Despite weevil infestation, popcorn hybrids viz., PH114 (PPE: 83.18%, GPP: 54.11%), PH110 (PPE: 69.27%, GPP: 33.11%), PH103 (PPE: 65.84%, GPP: 26.78%), PH112 (PPE: 64.91%, GPP: 29.22%) and PH109 (PPE: 64.48%, GPP: 29.00%) were identified as promising with desirable popping quality traits. Many of the popcorn hybrids possessed undesirable popping characteristics (12 hybrids with <60% PPE; 18 hybrids with <30% GPP) upon infestation. Despite having a common notion that popcorn genotypes are relatively resistant than other types of maize grain, many of the popcorn hybrids were highly susceptible to weevils leading to undesirable popping quality attributes. The study emphasizes the need for breeding weevil resistant popcorn genotypes possessing desirable popping quality attributes.

Genetics of resistance to stored grain weevil (Sitophilus oryzae L.) in maize

Abstract Stored grain weevil (Sitophilus oryzae) has emerged as important storage grain pest of maize, causing substantial economic losses. Owing to high costs and environmental hazards of pesticides, host plant resistance holds promise for effective control of weevils. In the present study, a set of experimental maize hybrids generated using line × tester mating design were evaluated against S. oryzae. Significant variation for grain weight loss (GWL) (6.0–49.1%), number of insect progeny emerged (NIP) (17.8–203.3), grain hardness (GH) (263.1–495.4 N), and pericarp thickness (PT) (60.3–161.0 μm) was observed. Strong positive association was observed between GWL and NIP. GH and PT did not show any correlation with GWL and NIP. Additive and non-additive gene actions were important for both GWL and NIP. Promising inbreds and experimental crosses identified can be effectively utilized in the resistance breeding programme. In majority of promising crosses having desirable SCA effects, one of the parents had desirable GCA effects, indicating that selection of inbred parents based on per se performance for generating resistant crosses may be possible. The commercial hybrid checks were highly susceptible compared to experimental hybrids. The inbreds and experimental hybrids identified hold promise in developing weevil resistant maize cultivars offering sustainable solution to management of weevils in maize.

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