Maize
ICAR-IARI, ICAR-IIMR, ICAR-VPKAS, CCS-HAU (Uchani), TNAU, and ICAR-DPR (6)
Objectives:
- Heterotic grouping and development of biofortified maize genotypes (Provitamin A, QPM, Fe, Zn and low phytate) with high grain potentia
- Mapping of QTLs for high -Fe, -Zn, and storability of provitamin A in maize kernels (IIMR: Fe and Zn and IARI: provitamin A).
- Designing and validation of gene-based marker(s) for lpa1 and lpa2 in maize (TNAU: lpa1 and VPKAS: lpa2).
- Studying the effects of QPM and/or provitamin A rich maize grains on nutritional status (lysine, tryptophan, provitamin-A) of eggs and meat (DPR).
Target traits:
Provita
| Maize Target content | ||
|---|---|---|
| Iron | >30 ppm | 45 ppm |
| Zinc | >25 ppm | 38 ppm |
| Provitamin A | 2 ppm | 15 ppm |
| Protein-Lysine | 2% | 4% |
Coordinator: Dr. Sujay Rakshit, ICAR-IIMR, Ludhiana
Sub-leader: Dr. Firoz Hossain, ICAR-IARI, New Delhi
Principal Investigators: (i) Dr. Firoz Hossain, ICAR-IARI, New Delhi, (ii) Dr. Bhupender Kumar, ICAR-IIMR, Ludhiana, (iii) Dr. Rajesh Khulbe, ICAR-VPKAS, Almora, (iv) Dr. Mehar Chand Kamboj, CCS-HAU, Uchani, (iv) Dr. John Joel, TNAU, Tamil Nadu, and (vi) Dr. Bhukya Praksh, ICAR-DPR, Hyderabad.
Maize: under evaluation
| Hybrid/Variety | Status | CRP centers | |
| 1 | IIMR-QPM-1530 (QPM) | AVT-II | IIMR |
| 2 | IARI-APH-27 (proA) | AVT-II | IARI |
| 3 | IARI- APH-1 (ProA) | AVT-I | IARI |
| 4 | IARI-APH-2 (ProA) | NIVT | IARI |
Objective-1: Heterotic grouping and development of biofortified maize genotypes (Provitamin A, QPM, Fe, Zn and low phytate) with high grain potential
Provitamin-A:
Traditional maize possesses 1-2 ppm of provitamin-A (proA), constitutes around 3-10% of the total carotenoids which is dominated by non-proA carotenoids such as lutein and zeaxanthin. Mutant allele of crtRB1 that blocks the hydroxylation of beta-carotene enhances proA by 2-10 folds. So far targeted breeding efforts in India have led to the development of crtRB1-based proA rich inbreds that contain 10-15 ppm of proA. However, their numbers and genetic background they are present is very limited (10-12). In order to develop diverse proA rich inbreds and hybrids, 10 elite normal maize inbreds (1-2 ppm of proA and good combiners for grain yield) were crossed with HarvestPlus donor inbred, HP704-22 (15-20 ppm of proA but poor adaptation in Indian conditions). The elite inbreds include viz., UMI1200, UMI1230, BML6Q, BML7Q, LM11Q, LM12Q, LM13Q, LM14Q, AH7000F and AH7000M. The F2 populations of the 10 crosses were genotyped using crtRB1-specific InDel marker. Segregants homozygous for crtRB1 mutant allele were selected (Fig. 1). The F3 families were evaluated during kharif 2017 for their plant-, ear-,
grain- characteristics (Fig. 2) and quality traits.
A set of 75 progenies were selected and selfed for estimation of kernel quality traits. Due to the presence of crtRB1 mutant allele, 82% of the non-proA carotenoids in the elite inbreds have been reduced to 27% in the newly derived proA rich lines (Fig. 3). In donor inbred, proA carotenoids constituted 75% of the total carotenoids, while it was only 8% in the elite inbreds. Among newly developed inbreds, 70% of the the total carotenoids were proA fractions. The proA among the elite inbreds varied from 1.2-2.2 ppm, while the same was 20.6 ppm in the donor inbred. The F2-derived progenies possessed high mean proA (15.9 ppm) with a range of 9.4-21.5 ppm. The comparison of donor, recipient and newly derived 75 crtRB1-based progenies are presented in Fig. 4.
Figure 1: Segregation of crtRB1 alleles (543 bp and 296 bp) in F2 populations. 543 bp allele is favourable, star indicates homozygous individuals with 543 bp allele.
Figure 2: Ear- and grain- characteristics of selected newly derived proA rich line
Figure 3: Mean proportion of different carotenoid fractions among donor, elite inbreds and newly developed crtRB1-based inbreds. (LUT: lutein, ZEA: zeaxanthin, BC: beta-carotene, BCX: beta-cryptoxanthin)
Based on the maturity of the newly developed inbreds, a set of 68 newly developed proA rich inbreds were crossed with five proA rich testers (HKI1105-ProA, HKI161-ProA, HKI163-proA, HKI193-1-ProA and CE-16) during rabi 2017-18. The test cross hybrids would be evaluated at multi-locations for grain yield potential and proA. The data would be used for heterotic grouping of the newly derived proA rich inbreds.
Figure 4: ProA levels among donor, elite inbreds and newly developed crtRB1-based inbreds.
Quality protein maize:
A set of 197 new QPM lines have been developed through introgressing opaque2 alleles in diverse normal genetic backgrounds. The normal inbred lines used in diversification programme were the parental lines of already released productive public sector hybrids. The detail of inbred lines used as donor for opaque2 alleles are given in table 1. During generation advancement, the segregating lines have been selected based on their opaqueness (45-75%) and useful breeding parameters (Fig. 5). At F4 stage they have been planted in high plant density and simultaneously evaluated for tryptophan and protein content. The tryptophan and protein content was ranging from 0.33 to 0.87 and 7.06 to 12.87% respectively. The desirable QPM segregants were selected and advanced to F5 during kharif 2017. The 46 lines were identified with high lysine (>0.65-0.87) and good in per se performance. The newly developed set has been put in crossing programme during spring 2018 using two diverse tester viz., HKI161 and 193-1. The test crosses will be developed and evaluated during kharif 2018. The DQL 266, DQL 99, DQL 278, DQL 122, DQL 97, DQL 267 and DQL 240 were the promising lines with high tryptophan (> 0.75%) content. Further, one QPM hybrid viz., IMHQPM 1530 has completed two years of testing in AICRP maize programme and based on its significant superiority over the best check the same has been promoted for final year of testing in NHZ (Zone-I). The seed of IMHQPM 1530 has been multiplied through hand pollination for further testing in final year AICRP-QPM trials.
Table 1. Details of QPM lines used as donors in diversification programme.
| Inbred | Grain | Grain | Source | Adaptation | Maturity |
| CML161 | Yellow | Flint | G25Q | Lowland | Late |
| CML170 | Yellow | Dent | G26Q | Lowland | Late |
| CML176 | White | Flint | P63 | Subtropical | Late |
| CML181 | White | Dent | UWO41 | Subtropical | Late |
Figure 5. Genetic diversity for opaqueness and cobs traits in newly developed QPM lines
Low phytate:
- F3 progenies of five crosses (high tryptophan lines VQL1, VQL2, VQL17, VQL373 and SA12-1 with low phytate donor lpa2) were raised at ICAR-VPKAS Almora during kharif 2017.
- Selection for agronomic traits was carried out and selected individuals were selfed.
- F4 progenies of the crosses were raised at IIMR-WNC, Hyderabad during rabi 2017-18 for generation advancement.
Lane 2 – VQL1, Lane 3 – lpa2, F4 individuals in rest of the
Figure 6: Screening of VQL 1 × lpa2 progenies with CAPS marker (ZMGSPR1580)
Lane 2 – VQL1, Lane 3 – Lpa2, F4 individuals in rest of
Figure 7: Screening of VQL 1 × lpa2 progenies with umc1066
- F5 progenies homozygous for lpa2 will be evaluated during kharif 2018 at ICAR-VPKAS Almora for agronomic performance
- Biochemical evaluation of self-seed of the selected progenies will be carried out for tryptophan and phytate content
- Test cross combinations among selected progenies of different crosses will be generated during rabi 2018-19 at IIMR-WNC, Hyderabad
Further, three targeted F2 populations were generated and raised. F3 seeds were analyzed for low phytate content, and segregants with <9.0 mg/g of the phytate were selected (Table 2).
Table 2: Segreants with low phytate content
| S.NO | F2 segregants | Phytate (mg/g) |
| 1 | UMI1200 × UMI1099-F2- 47 | 5.61 |
| 2 | UMI1200 × UMI1099-F2-177 | 8.80 |
| 3 | UMI1200 × UMI1099-F2-175 | 8.93 |
| 4 | UMI1201 × UMI1099-F2-38 | 5.80 |
| 5 | UMI1201 × UMI1099-F2-5 | 6.85 |
| 6 | UMI1201 × UMI1099-F2-39 | 8.33 |
| 7 | UMI1201 × UMI1099-F2-23 | 8.36 |
| 8 | UMI1210 × UMI1099-F2- 247 | 5.73 |
| 9 | UMI1210 × UMI1099-F2- 249 | 8.17 |
| 10 | UMI1210 × UMI1099-F2- 2 | 8.64 |
| 11 | UMI1210 × UMI1099-F2- 241 | 8.78 |
| 12 | UMI1210 × UMI1099-F2- 254 | 8.87 |
| 13 | UMI1210 × UMI1099-F2-248 | 8.96 |
These low phytate progenies along with the already available low phytate inbreds would be crossed with the heterotic testers.
Objective 2: Mapping of QTLs for high -Fe, -Zn, and storability of provitamin-A in maize kernels
Provitamin-A: Seven inbreds (VQL1-PV, V335-PV, HKI161-PV, HKI193-1-PV, CE-13, CE-25 and CE-477). possessing high proA (10.0-17.6 ppm) were stored for five months. Loss of proA during the storage was calculated which varied from 44-88%. Contrasting inbreds were crossed during rabi 2017-18 for generating F2:3 mapping populations. One desirable population would be selected for mapping of QTLs for retention of proA during storage.
Quality protein maize:
A set of 350 diverse inbred lines panel have been constituted (Fig. 8). The seed of same were multiplied through hand pollination and has been evaluated for Fe and Zn at multiple location (total six).The sampling has been completed for Fe and Zn analysis for all environments. The analysis of samples for Fe & Zn is in progress, and so for completed for three environments. Based on available results, the sufficient genetic variation has been reported for Fe (8.1-63.5 ppm) and Zn (5.7-57.5 ppm) (Fig. 9). Depending upon the availability of fund during 2017-18, the DNA of 96 samples of panel was isolated, purified and quantified. The genotyping by sequencing (GBS) data has been generated for these samples. A preliminary study of population structure of association panel using SSR markers has reported six-sub-populations in the panel. The complete analysis of structure, kinship and association studies will be done once the Phenotyping and GBS for 350 samples will get completed
Figure 8. Diversity in association panel
Figure 9. Phenotypic variation for Fe and Zn in association mapping panel
Objective 3: Designing and validation of gene-based marker(s) for lpa-1 and lpa-2 in maize:
Selected maize inbreds contrasting for phytate content were subjected to molecular analysis with the primers designed from lpa-1 gene sequence obtained from NCBI. The Gene specific primers were designed from the primer blast and the following are the details of the lpa-1 gene specific primers.
Lpa-2 gene (GRMZM2G456626) sequence was retrieved from MaizeGDB. Primers were designed from the reference sequence. Complete gene was amplified from available lpa-2, VQL1, V407 and VQL373. Overlapping primers were used to sequence complete gene from all these four genotypes. Only one (G/A) SNP was found in the coding region. As non-low phytate genotype VQL1 and low phytate genotype lpa-2 carry same allele of that SNP (G), it does not appear to be a functional polymorphism.
Objective 4: Studying the effects of QPM and/or provitamin A rich maize grains on nutritional status (lysine, tryptophan, provitamin-A) of eggs and meat
The study was conducted to determine the effect feeding QPM based diets in Vanaraja birds. For the purpose, 175 numbers (day old chicks) were randomly divided into 5 dietary groups each having 7 replicates with 5 birds each. Five diets were formulated to contain normal yellow maize (NMY; Diet-1), Vivek Hyb 9 (Diet-2), QPM + pro A (Diet-3), QPM (Diet-4) and White Maize (Diet-5) (Table 4). The care was taken that all the diets were contained equal amount of essential amino acids, protein and energy. It has been observed that the improved body weight gain and feed efficiency was recorded among the groups fed diet with QPM + pro A compared to other diets during the 6 weeks of age (Table 5 and Fig 10). Though, there was numerical improvement in body weight gain and feed efficiency during the 9 week, it did not result in statistically significant difference between the dietary groups (Fig 11). At the end of the experiment, 7 birds from each treatment (each from a replicate) were sacrificed to study the slaughter variables. It has been recorded that the slaughter parameters did not vary among the various dietary groups. However, there was significant decrease in abdominal fat and increased breast muscle among the birds fed diet contained QPM (Diet-4) and QPM+proA (Diet-3) compared to other dietary groups.
Table 4. Ingredient and chemical composition of basal diet
| Ingredients composition | g/100 g | Ingredients composition | g/100 g |
|---|---|---|---|
| Maize | 61.769 | L-Threonine | 0.024 |
| Soya DOC 45% | 30.834 | L-Tryptophan | 0.000 |
| Deoiled Rice Bran | 3.081 | Trace Mineral Mixture | 0.100 |
| Dicalcium Phosphate | 1.618 | AB2D3K | 0.015 |
| LSP-Powder | 1.720 | B Complex | 0.010 |
| Salt | 0.384 | Choline Cl | 0.060 |
| DL-Methionine | 0.194 | Toxin Binder | 0.100 |
| L-Lysine HCL | 0.111 |
| Nutrients composition | Value |
|---|---|
| M.E (Kcal/kg) | 2800 |
| Protein (%) | 20.0 |
| Lysine (%) | 1.15 |
| Methionine (%) | 0.50 |
| Calcium (%) | 0.94 |
| Available Phosphorus (%) | 0.40 |
| Threonine (%) | 0.80 |
Table 5. Effect of feeding different source of maize on performance in Vanaraja birds during 3, 6 and 9 weeks of age
| Diet (maize genotype) | 3 week | 6 week | 9 week | |||||
|---|---|---|---|---|---|---|---|---|
| BWG | FCR | BWG | FCR | BWG | FCR | |||
| Diet-1 DPR Maize | 205.3 | 2.023 | 529.7 | 2.181ab | 909.2 | 2.361 | ||
| Diet-2 Vivek Hyb 9 | 213.0 | 1.979 | 586.9 | 2.202a | 956.1 | 2.404 | ||
| Diet-3 APQH9 | 210.6 | 1.980 | 583.4 | 2.034c | 972.4 | 2.339 | ||
| Diet-4 Vivek QPM 9 | 186.6 | 2.182 | 584.8 | 2.131bc | 960.7 | 2.357 | ||
| Diet-5 White Maize | 191.4 | 2.096 | 563.6 | 2.177ab | 962.0 | 2.252 | ||
| N | 7 | 7 | 7 | 7 | 7 | 7 | ||
| SEM | 4.98 | 0.02 | 9.93 | 0.02 | 11.7 | 0.02 | ||
| P value | 0.36 | 0.08 | 0.32 | 0.01 | 0.48 | 0.08 | ||
Figure 10: Effect of feeding different source of maize on Vanaraja birds during 6 weeks of age. FCR: feed conversion ratio, BWG: body weight gain, FI: feed intake
Figure 11: Effect of feeding different source of maize on Vanaraja birds during 9 weeks of age. FCR: feed conversion ratio, BWG: body weight gain, FI: feed intake
Table 6. Effect of feeding different source of maize on slaughter variables in Vanaraja birds (g/kg live weight)
Figure 12: Effect of feeding different source of maize on breast and abdominal fat in Vanaraja birds. Ab: Abdominal
Basing on the results obtained, it is concluded that the birds fed QPM and QPM+ProA showed a better body weight gain and improved feed efficiency. Further, QPM and QPM+ProA feeding considerably reduced the abdominal fat and increased breast muscle, which is highly desirable attribute of chicken meat.
Publications:
Dube N, PC Mashurabad, F Hossain, R Pullakhandam, L Thingnganing, DK Bharatraj. 2018. β-Carotene bioaccessibility from biofortified maize (Zea mays) is related to its density and is negatively influenced by lutein and zeaxanthin. Food Funct. 2018 Jan 24;9(1):379-388. doi: 10.1039/c7fo01034f.