Validation of foreground marker for lpa2 allele
The PCR of SSR marker umc2230, tightly linked to gene determining low phytate trait resulted in the amplicon size of 150 bp and 155 bp, in recurrent parents (BML 6 and BML 45) and donor parent, respectively (Fig. 1). The umc2230 marker is located in the maize genome at 0.4 cM away from the lpa2 gene on the short arm of chromosome 1. For foreground selection, umc2230 which showed polymorphism between the donor (LPA 2), and recipient parents (BML 6 and BML 45) was used as a foreground marker at every step in MABB to identify plants carrying the lpa2 allele and also differentiate the zygosity condition.
Marker-assisted introgression of lpa2 gene
The gene determining low phytate trait, lpa2 was introgressed from the donor parent to the genetic background of the recurrent parents BML 45 and BML 6, the female and male parental inbred lines, respectively of commercially released single cross hybrid maize, DHM 121 through MABB to develop low phytate maize.
Confirmation of F1s
The SSR marker umc2230 validated for tight linkage with the gene of interest, lpa2 for polymorphism between recurrent parents and donor parent was used to identify and also confirm plants carrying lpa2 gene through PCR amplifications in different generations during MABB. The F1 plants of crosses namely BML 45/LPA 2 and BML 6/LPA 2 were screened using umc2230. The PCR amplification showed that the F1 plants of the above crosses were heterozygous with a banding pattern of 155/150 bp (Supplementary Fig. 1). The confirmed F1 plants were also screened using unlinked SSR markers to doubly confirm the heterozygous at selected SSR marker loci. The confirmed F1 hybrid plants of each cross were used as female parents to backcross with their respective recurrent parents as males to develop the BC1F1 population.
Foreground and background selection
The true F1 plants namely #1 (1, BML 6/LPA 2) and #4 (4, BML 45/LPA 2) were selected based on conformity of heterozygosity at lpa2 locus and backcrossed with their respective recurrent parents namely BML 6 and BML 45, respectively to generate BC1F1. The number of BC1F1 plants from F1 plants was 168 (1, BML 6/LPA 2) and 324 (4, BML 45/LPA 2). The numbers of plants heterozygous at lpa2 locus were 76 and 169 in BML 6/LPA 2 and BML 45/LPA 2 crosses, respectively (Fig. 2A, 3A). The remaining BC1F1 progenies showed recurrent parent allele (150/150), homozygous for wild type allele, LPA2. Based on the relative resemblance with recurrent parents in morphological traits, ten heterozygous BC1F1 plants were used for background selection to identify plants with higher RPG % recovery. The number of polymorphic SSR markers selected for background selections was 100 and 99 between BML 6 and LPA 2 and BML 45 and LPA 2, respectively. The number of polymorphic markers on each chromosome ranged from 6 (Chromosome 9) to 16 (Chromosome 1) between BML 6 and LPA 2 and 4 (Chromosome 9 and 10) to 19 (Chromosome 1) between BML 45 and LPA 2. The highest number of polymorphic markers was located on Chromosome 1 as the target gene, lpa2 is located on this chromosome. The graphical representation of the polymorphic markers used in backcross generation was given in Supplementary Fig. 2. The PCR amplified products of each polymorphic SSR marker in each plant were scored as AA (amplicon size corresponds to recurrent parent allele) and AB (amplicon size corresponds to both recurrent as well as donor allele) (Fig. 4A & 4B). The RPG % in each plant was estimated and it was ranged from 74.06-79.72% in BC1F1s derived from BML 6/LPA 2 cross whereas 74.53-80.19% in BML 45/LPA 2 derived BC1F1s. The pictorial representation of the RPG recovery of chromosome number 1 of BML 6/LPA 2 and BML 45/LPA 2 crosses in BC1F1 generation is shown in Fig. 5A & 5B, respectively. Based on the RPG recovery across all 10 chromosomes, the BC1F1 plants, #117 (79.72% RPG) of BML 6/LPA 2 cross and #353 (80.19% RPG) of BML 45/LPA 2 crosses with the highest RPG were selected and backcrossed with their respective recurrent parents to develop BC2F1 population.
The number of BC2F1 plants raised was 218 (BML 6/LPA 2) and 165 (BML 45/LPA 2). The foreground selection performed in the BC2F1 generation is similar to that of BC1F1. The number of plants heterozygous at lpa2 locus was 72 and 55 in BML 6/LPA 2 and BML 45/LPA 2 derived BC2F1 population, respectively (Fig. 2B & 3B). Whereas the size of the amplicons in the remaining plants was similar to that of wild type allele LPA2. Similar to BC1F1 generation, ten BC2F1 plants, heterozygous at lpa2 locus were selected, based on relative phenotypic resemblance with their respective recurrent parents for background selection. The background selection in BC2F1 generation was carried out using 99 polymorphic SSR markers covering all 10 chromosomes. The number of polymorphic markers on each chromosome varies from 5 (Chromosome 9) to 15 (Chromosome 1) between BML 6 and LPA 2; 3 (Chromosome 10) to 19 (Chromosome 1) between BML 45 and LPA 2. The PCR amplicons of each polymorphic SSR molecular markers in each individual were scored as AA (recurrent parent allele) and AB (heterozygous containing both recurrent as well as donor allele) (Fig. 4C & 4D). The per cent recovery of RPG in BC2F1 generation ranged from 86.32-89.62% and 84.91-89.15% in BML 6/LPA 2 and BML 45/LPA 2 derived backcrosses respectively. The BC2F1 plants viz., #2734 [2734, {117, BML 6*/(1, BML 6/LPA 2)}/*BML 6], #2741 [2741, {117, BML 6*/(1, BML 6/LPA 2)}/*BML 6], #1996 [1996, {353, BML 45*/(4, BML 45/LPA 2)}/*BML 45], and #2006 [2006, {353, BML 45*/(4, BML 45/LPA 2)}/*BML 45] with highest RPG were selected and advanced through self-pollination to develop BC2F2 population. The RPG % in selected BC2F1 plants was 89.62 (# 2734) 89.15 (# 2741), 88.68 (# 1996), and 89.15 (# 2006). The pictorial representation of RPG recovery of chromosome number 1 of BML 6 and BML 45 crosses in BC2F1 generation is shown in Fig. 5A & 5B, respectively.
The number of plants raised in BC2F2 generation, derived from BML 6/LPA 2 and BML 45/LPA 2 crosses were 241 and 192, respectively. The number of BC2F2 progenies that were homozygous for the lpa2 allele in the genetic background of BML 6 and BML 45 was 46 and 33, respectively. The homozygous plants showed a similar banding pattern (155/155) as that of the donor line (LPA 2) for the low phytate trait. Whereas the number of plants that were homozygous for wild-type allele, LPA2 (150/150) was 51 and 42 in the genetic background of BML 6 and BML 45, respectively; the banding pattern was similar to that of recurrent parents (Fig. 2C & 3C). The PCR amplicons banding pattern in the remaining plants was heterozygous (155/150). Based on relative morphological resemblance with their respective recurrent parents, ten homozygous BC2F2 plants were selected for background selection to identify BC2F2 plants with the highest RPG content. The number of polymorphic SSR markers chosen for background selection in BC2F2 generation derived in the genetic background of BML 6 and BML 45 were 101 and 100, respectively. The polymorphic markers covered all 10 chromosomes and the number of polymorphic markers on each chromosome varies from 5 (Chromosome 9) to 15 (Chromosome 1) between BML 6 and LPA 2; 4 (Chromosome 9 and 10) to 18 (Chromosome 1) between BML 45 and LPA 2. The PCR amplicons of each of the SSR markers in each BC2F2 individual plant were scored as AA (recurrent parent allele), and AB (contain both recurrent as well as donor allele), and BB (donor parent allele) (Fig. 4E & 4F). The RPG % in the selected BC2F2 plants ranged from 88.68-91.04 and 90.09-91.51 in the genetic background of BML 6 and BML 45, respectively. Based on the background selection, the best five BC2F2 plants viz., #3190, #3283, #3230, #3263, and #3292 in the genetic background of BML 6 and #3720, #3776, #3717, #3828, and #3832 in the genetic background of BML 45 with highest RPG were selected and advanced or maintained through self-pollination to develop BC2F3 stage NILs (Fig. 6). The pictorial representation of recurrent parent genome recovery of chromosome number 1 of BML 6 and BML 45 crosses in BC2F2 generation is shown in Fig. 5A & 5B, respectively. The pedigree details of the selected BC2F3 stage NILs are given in Table 2. The stabilized NILs carrying lpa2 gene, determining the low phytate trait would be further used in the hybridization program to reconstitute the original hybrid DHM 121 hybrid with low phytate trait.
Estimation of PA and P i in BC2F3 stage NILs carrying lpa2 allele
The qualitative and quantitative estimation of PA and Pi was done for parents (LPA 2, BML 6, and BML 45) and selected BC2F3 progenies which are homozygous for the lpa2 allele (Table 2). The PA and Pi were estimated as mentioned in materials and methods. The PA and Pi estimation was done to confirm the expression of introgressed lpa2 allele with low phytate phenotypes in the genetic background of recurrent parents.
The PA and Pi levels in recurrent parents were relatively high as compared to donor parents. This was confirmed using the high inorganic phosphorous (HIP) assay which is a quick, easy and inexpensive method to differentiate high, low, or intermediate phytate lines. In the HIP assay, kernels having high phytate content produced light blue colour, and kernels with low phytate content produce a dark blue colour, whereas intermediate phytate content kernels produce a medium blue colour. This colour differentiation helps to differentiate phytic acid levels among various lines. In marker-assisted backcross breeding, the HIP assay was used to confirm lpa2 allele introgression in BC2F3 seeds (Fig. 7). This figure shows that some BC2F2 lines with lpa2 allele introgression produce dark blue colour which is comparable to that of our donor mutant line and these lines were selected as low phytate near-isogenic lines.
Quantitative estimation of phytic acid and inorganic phosphate was analyzed by taking the readings at OD490 and OD820 nm, respectively. The estimation was performed in the lpa2 donor line, recurrent parents and newly developed low phytate BC2F3 lines. The analysis revealed that PA and Pi levels varied among the selected lines (Table 3). The donor line which is homozygous for mutant lpa2 allele having a minimum level of PA (1.72±0.118 mg/g) and maximum level of Pi (1.22±0.49 mg/g), whereas recurrent parents which are homozygous for the wild type LPA2 allele having maximum PA (BML 6: 3.59±0.12 mg/g, BML 45: 3.16±0.1 mg/g) and a minimum Pi (BML 6: 0.65±0.49 mg/g, BML 45: 0.51±0.47 mg/g). The newly developed low phytate BC2F3 lines which are homozygous for lpa2 alleles contains lower levels of PA (ranges from 1.67±0.118 to 2.84±0.12 mg/g in NILs of BML 6 and 1.8±0.1 to 3.01±0.1 mg/g in NILs of BML 45) and higher levels of Pi (ranges from 0.15±0.49 to 1.01±0.49 mg/g in NILs of BML 6 and 0.3±0.47 to 1.48±0.47 mg/g in NILs of BML 45) when compared to their respective recurrent parents. Among the NILs developed, #3190 (1.67±0.118 mg/g) of BML 6 and #3720 (1.8±0.1 mg/g) of BML 45 were statistically on par with that of donor parents and #3230 (2.02±0.118 mg/g), #3283 (2.07±0.118 mg/g) of BML 6 and #3828 (2.13±0.1 mg/g), #3776 (2.17±0.1 mg/g) of BML 45 were significantly low levels of PA when compared to recurrent parents. The NILs viz., #3190 (1.01±0.49 mg/g) of BML 6 and #3720 (1.38±0.47 mg/g) of BML 45 have the highest levels of Pi when compared to recurrent parents and are comparable with that of the donor parent. The results indicate that MAB breeding has successfully introgressed the low phytate allele from donor parent to recurrent parents.
Table 3
Phytic acid (PA) and inorganic phosphorus (Pi) (mg/g) content of newly developed near-isogenic lines (NILs) along with their parents (BML 6, BML 45, LPA 2).
S. No. | Plant No | PA | Pi | Plant No | PA | Pi |
1 | LPABML 6-1 | 2.02d | 0.46de | LPABML 45-1 | 2.17e | 0.80d |
2 | LPABML 6-2 | 2.07d | 0.31f | LPABML 45-2 | 1.80f | 1.38a |
3 | LPABML 6-3 | 1.67e | 1.01b | LPABML 45-3 | 2.18e | 0.65ef |
4 | LPABML 6-4 | 2.22d | 0.32f | LPABML 45-4 | 2.13e | 0.74de |
5 | LPABML 6-5 | 2.22d | 0.38ef | LPABML 45-5 | 2.24de | 0.72de |
6 | LPABML 6-6 | 2.51c | 0.31f | LPABML 45-6 | 2.44cd | 0.30i |
7 | LPABML 6-7 | 2.48c | 0.15g | LPABML 45-7 | 2.46c | 0.58fg |
8 | LPABML 6-8 | 2.84b | 0.20g | LPABML 45-8 | 2.68b | 0.90c |
9 | LPABML 6-9 | 2.54c | 0.48d | LPABML 45-9 | 2.76b | 0.80d |
10 | LPABML 6-10 | 2.52c | 0.45de | LPABML 45-10 | 3.01a | 0.46h |
11 | BML 6 | 3.59a | 0.65c | BML 45 | 3.16a | 0.51gh |
12 | LPA 2 | 1.72e | 1.22a | LPA 2 | 1.72f | 1.22b |
| General Mean | 2.37 | 0.49 | General Mean | 2.40 | 0.76 |
| Mean SS | 0.81** | 0.31** | Mean SS | 0.59** | 0.32** |
| p-Value | <.00 | <.00 | p-Value | <.00 | <.00 |
| CV (%) | 6.1 | 12.0 | CV (%) | 5.1 | 7.6 |
| SE(d) | 0.12 | 0.049 | SE(d) | 0.099 | 0.04 |
| LSD at 5% | 0.25 | 0.10 | LSD at 5% | 0.20 | 0.09 |
Means with at least one letter common are not statistically significant using TUKEY's Honest Significant Difference |
Agronomic Evaluation Of Nils With Low Phytate Trait
The main aim of the agronomic evaluation was to evaluate near-isogenic lines (NILs) developed in the genetic background of BML 6 and BML 45 with low phytate trait along with their respective recurrent parents namely BML 6 and BML 45, respectively, and to identify NILs as similar as that of recurrent parents in most of the traits, if not all traits. The observations were recorded on 18 agronomically important traits and all the traits are quantitative. The descriptive statistics for agronomic traits in this study are shown in Tables 4 and 5.
Table 4
The descriptive statistics of near-isogenic lines derived in the genetic background of BML 6 with low phytate traitfor agronomic traits.
SNo | Genotype | GP (%) | DA (days) | DS (days) | ASI (days) | LW (cm) | PH (cm) | EH (cm) | TL (cm) | EL (cm) | ED (cm) | EC (cm) | KR (no.) | KpR (no.) | TKW (g) | SP (%) | EtoPR (no.) | Barr (no.) | GY (kg/ha) |
1 | BML 6 | 73ab | 61bc | 66ab | 6 | 6.6a | 111abc | 64ab | 23abc | 12ab | 4 | 12 | 14 | 23abc | 170 | 65 | 0.8 | 0.23 | 768f |
2 | LPABML 6-1 | 66ab | 58bc | 65ab | 7 | 7.5a | 129a | 74a | 25abc | 14a | 4 | 13 | 14 | 25ab | 225 | 69 | 0.5 | 0.50 | 1070de |
3 | LPABML 6-2 | 75a | 59bc | 64ab | 5 | 6.4a | 113abc | 59abcd | 24abc | 14a | 4 | 12 | 14 | 25ab | 197 | 61 | 0.6 | 0.37 | 1050de |
4 | LPABML 6-3 | 57ab | 56c | 60b | 4 | 7.8a | 126ab | 65ab | 30abc | 14a | 4 | 13 | 14 | 29a | 242 | 59 | 0.6 | 0.43 | 1183d |
5 | LPABML 6-4 | 78a | 62ab | 64ab | 2 | 7.8a | 108bc | 65ab | 31a | 12ab | 4 | 13 | 15 | 23abc | 245 | 79 | 0.8 | 0.30 | 1291d |
6 | LPABML 6-5 | 60ab | 66a | 69a | 3 | 7.0a | 112abc | 47cd | 24abc | 12ab | 3 | 11 | 13 | 16abc | 175 | 74 | 0.9 | 0.10 | 1050de |
7 | LPABML 6-6 | 66ab | 61ab | 70a | 9 | 6.1a | 107bc | 51bcd | 22c | 11ab | 3 | 10 | 10 | 18abc | 122 | 66 | 0.5 | 0.70 | 661f |
8 | LPABML 6-7 | 80a | 61ab | 66ab | 5 | 6.2a | 115abc | 52bcd | 31a | 12ab | 3 | 10 | 11 | 16abc | 115 | 78 | 0.5 | 0.50 | 2778a |
9 | LPABML 6-8 | 80a | 62ab | 67ab | 5 | 6.8a | 115abc | 58abcd | 30ab | 10ab | 4 | 12 | 15 | 17abc | 200 | 77 | 0.5 | 0.60 | 631f |
10 | LPABML 6-9 | 77a | 57bc | 63ab | 5 | 6.3a | 101c | 44d | 23bc | 12ab | 3 | 8 | 10 | 12bc | 155 | 62 | 0.5 | 0.53 | 1606c |
11 | LPABML 6-10 | 67ab | 62ab | 68ab | 6 | 6.1a | 116abc | 61abc | 24bc | 14a | 3 | 10 | 11 | 15abc | 192 | 64 | 0.7 | 0.70 | 974e |
12 | General Mean | 71 | 60 | 66 | 5 | 6.8 | 114 | 58 | 26 | 12.4 | 4 | 11 | 13 | 20 | 185 | 69 | 0.6 | 0.45 | 1278 |
13 | Mean SS | 791** | 24** | 26** | 10 | 1.2** | 197** | 238** | 40** | 31** | 1.5 | 13 | 17 | 116** | 8272 | 1005 | 0.1 | 0.1 | 1407614** |
13 | p-Value | 0.00 | <.00 | 0.00 | 0.06 | 0.00 | 0.00 | <.00 | <.00 | 0.00 | 0.09 | 0.18 | 0.24 | 0.00 | 0.06 | 0.12 | 0.06 | 0.08 | <.00 |
14 | CV(%) | 21 | 2.4 | 3.7 | 40 | 7.3 | 4.8 | 7.7 | 9 | 22 | 26 | 26 | 27 | 23 | 33 | 39 | 42 | 50 | 3.8 |
15 | SE(d) | 10.8 | 1.2 | 2.0 | 1.7 | 0.4 | 4.5 | 3.7 | 1.9 | 1.9 | 0.7 | 2.3 | 2.8 | 3.54 | 49 | 19 | 0.2 | 0.19 | 39 |
Means with at least one letter common are not statistically significant using TUKEY's Honest Significant Difference |
Germination percentage (GP),days to 50% anthesis (DA), days to 50% silking (DS), anthesis-silking interval (ASI), leaf width (LW), ear height/placement (EH), plant height (PH), tassel length(TL), ear length without husk (EL), ear diameter without husk (ED), ear circumference (EC), number of kernel rows (KR), number of kernels per rows (KpR), 1000 kernel weight (TKW), shelling percentage (SP), ear to plant ratio (EtoPR), barrenness (Barr) andgrain yield (GY) |
Table 5
The descriptive statistics of near-isogenic lines derived in the genetic background of BML 45 with low phytate traitfor agronomic traits.
SNo | Genotype | GP (%) | DA (days) | DS (days) | ASI (days) | LW (cm) | PH (cm) | EH (cm) | TL (cm) | EL (cm) | ED (cm) | EC (cm) | KR (no.) | KpR (no.) | TKW (g) | SP (%) | EtoPR (no.) | Barr (no.) | GY (kg/ha) |
1 | BML 6 | 60a | 62ab | 65a | 3 | 5.8ab | 90ab | 38 | 24a | 10ab | 3b | 10ab | 13ab | 17a | 230ab | 80 | 0.8ab | 0.2ab | 1400bc |
2 | LPABML 45-1 | 78a | 57b | 60a | 3 | 6.0ab | 100ab | 42 | 24a | 11a | 4a | 11a | 12ab | 17a | 225ab | 62 | 0.7ab | 0.3ab | 787cd |
3 | LPABML 45-2 | 93a | 61ab | 63a | 2 | 5.0ab | 84ab | 34 | 24a | 9ab | 3ab | 10ab | 11ab | 15ab | 245ab | 73 | 1.0a | 0.0b | 1564ab |
4 | LPABML 45-3 | 84a | 59ab | 62a | 3 | 4.6b | 95ab | 39 | 22a | 10ab | 3ab | 11a | 12ab | 17a | 185ab | 85 | 0.8ab | 0.3ab | 1050bcd |
5 | LPABML 45-4 | 68a | 61ab | 64a | 3 | 5.9ab | 92ab | 42 | 24a | 7ab | 2ab | 7ab | 8ab | 12ab | 250ab | 72 | 0.6ab | 0.5ab | 1188bcd |
6 | LPABML 45-5 | 64a | 58ab | 61a | 3 | 5.3ab | 101ab | 43 | 23a | 11a | 4a | 11a | 14a | 20a | 258a | 66 | 1.0a | 0.0b | 2041a |
7 | LPABML 45-6 | 69a | 66a | 71a | 5 | 6.0a | 108a | 34 | 28a | 10ab | 4b | 12a | 13ab | 16a | 262a | 53 | 0.07b | 0.9a | 1382bc |
8 | LPABML 45-7 | 71a | 61ab | 65a | 4 | 5.5ab | 77b | 39 | 19a | 10ab | 3ab | 10ab | 11ab | 17a | 233ab | 55 | 0.8ab | 0.2ab | 1040bcd |
9 | LPABML 45-8 | 64a | 64ab | 67a | 3 | 6.4a | 101ab | 44 | 32a | 9ab | 3ab | 10ab | 12ab | 17a | 243ab | 63 | 0.5ab | 0.5ab | 732d |
10 | LPABML 45-9 | 62a | 61ab | 62a | 1 | 5.4ab | 103ab | 41 | 23a | 11a | 4a | 12a | 12ab | 22a | 247ab | 67 | 0.6ab | 0.4ab | 1485ab |
11 | LPABML 45-10 | 87a | 61ab | 69a | 8 | 5.8ab | 97ab | 32 | 24a | 8ab | 3ab | 9ab | 9ab | 12ab | 235ab | 53 | 0.5ab | 0.5ab | 767d |
12 | General Mean | 73 | 61 | 64 | 3 | 5.6 | 95 | 39 | 24.2 | 10 | 3.1 | 10.3 | 12 | 16.5 | 238 | 66.2 | 0.68 | 0.33 | 1221.7 |
13 | MeanSS | 523* | 20** | 37* | 11 | 0.8** | 387* | 51 | 34* | 21** | 2.4** | 20** | 25** | 75** | 11746** | 1531 | 0.2** | 0.2** | 872633** |
13 | p-Value | 0.01 | 0.00 | 0.02 | 0.08 | 0.00 | 0.01 | 0.17 | 0.04 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.1 | 0.00 | 0.00 | <.00 |
14 | CV(%) | 18 | 3.93 | 5.6 | 67 | 7.06 | 11.53 | 14.4 | 15.4 | 25.4 | 22.7 | 24 | 25.5 | 24.1 | 27 | 50 | 30.8 | 63.2 | 15.80 |
15 | SE(d) | 10 | 1.9 | 2.9 | 1.9 | 0.3 | 8.9 | 4.6 | 3.1 | 1.9 | 0.5 | 1.9 | 2.2 | 3.01 | 47 | 23 | 0.17 | 0.16 | 141 |
Means with at least one letter common are not statistically significant using TUKEY's Honest Significant Difference |
Germination percentage (GP),days to 50% anthesis (DA), days to 50% silking (DS), anthesis-silking interval (ASI), leaf width (LW), ear height/placement (EH), plant height (PH), tassel length(TL), ear length without husk (EL), ear diameter without husk (ED), ear circumference (EC), number of kernel rows (KR), number of kernels per rows (KpR), 1000 kernel weight (TKW), shelling percentage (SP), ear to plant ratio (EtoPR), barrenness (Barr) andgrain yield (GY) |
The results of the agronomic evaluation indicated that the NILs developed in the genetic background of BML 6 showed no significant differences in eight (ASI, EC, ED, EtoPR, KR, SP, TKW, and Barrenness) out of 18 traits studied. All the eight traits with no significant differences contribute directly or indirectly to final grain yield. Out of 10 NILs, two NILs viz., LPABML 6-5 for two traits (DA and EH); LPABML 6-9 for EH differed significantly with that of recurrent parent BML 6. Whereas the rest of the eight NILs did not differ significantly from that of the recurrent parent in any of the traits. If LPABML 6-5 excluded then the DA of NILs were ranged from 56 (LPABML 6-3) to 62 (LPABML 6-4, LPABML 6-8, LPABML 6-10) which did not differ significantly with the recurrent parent BML 6 (61). Similarly, the DS among the NILs derived in the genetic background of BML 6 varied between 60 (LPABML 6-3) to 70 (LPABML 6-6), with no significant difference with recurrent parent BML 6 (66).
Similarly, NILs developed in the genetic background of BML 45 were compared with the recurrent parent BML 45. The results indicated that out of 18 traits, three traits namely ASI, EH, and SP did not show any significant differences. However, when compared with the recurrent parent BML 45, none of the traits showed any significant difference between NILs and recurrent parent BML 45. Thus all the traits are comparable between NILs and recurrent parent BML 45. Out of 10 NILs one NIL, LPABML 45-6 has differed significantly with recurrent parent BML 45 for one trait kernel per row (KpR). The present study could able to identify eight and ten NILs that are comparable with the recurrent parents BML 6 and BML 45, respectively in 17 of the 18 traits studied.
Grain yield being a very complex trait, almost all the NILs derived in the genetic background of BML 6 and BML 45 were comparable with their recurrent parent BML 6 and BML 45, respectively. Even though almost all NILs except two (LPABML 6-6 and LPABML 6-7) derived in the genetic background of BML 6 differed significantly from that of the recurrent parent but they were numerically superior over the recurrent parent BML 6 which is desirable. Whereas in the case of NILs developed in the genetic background of BML 45, out of 10 NILs selected based on the foreground and background selection three NILs namely LPABML 45-6, LPABML 45-8, and LPABML 45-10 were significantly inferior over the recurrent parent BML 45.
Percentage of germination is another important trait that requires special attention, especially in low phytate maize. The results obtained in the present study have shown that none of the NILs developed in the genetic background of BML 6 and BML 45 differed significantly for germination which is very much required.
In summary, through MABB it was possible to transfer low phytate traits successfully from donor to recipient parents. The biochemical and agronomic performance for most of the agronomic traits were comparable with that of recurrent parents. Thus the newly developed NILs are not only similar to that of the recurrent parents but also having low phytate content which is very much useful to reconstitute DHM 121 hybrid with low phytate content.