Combining higher accumulation of amylopectin, lysine and tryptophan in maize hybrids through genomics-assisted stacking of waxy1 and opaque2 genes

Marker polymorphism among parents

Gene-based InDel marker, wx-2507F/RG was polymorphic between recurrent (HKI161, HKI163, HKI193-1 and HKI193-2) and donor (MGU-102-wx1) parents. wx-2507F/RG amplified 280 bp fragment in all the four recurrent inbreds, while it amplified 260 bp fragment in waxy donor line (Fig. 2A). Gene-based SSR, phi057 produced 165 bp allele in all four recurrent parents, while the donor generated 153 bp allele (Fig. 2B). A range of 102–273 plants across BC₁F₁, BC₂F₁ and BC₂F₂ were subjected to foreground selection using wx1 and o2 gene (Table 2, Fig. 2). A total of 112, 107, 102 and 105 polymorphic SSRs with polymorphism of 35.00%, 33.44%, 31.86% and 32.81% were observed in HKII61 × MGU-102-wx1, HKI163 × MGU-102-wx1, HKI193-1 × MGU-102-wx1 and HKI193-2 × MGU-102-wx1, respectively (Table 1). The number of polymorphic markers per chromosome ranged from 8 to 16 across crosses.

Genomics-assisted selection

F

₁

generation

The corresponding polymorphic markers for wx1 and o2 showed hybridity among all the F₁s. as all plants revealed 280/260 bp (Wx1wx1) and 165/153 bp (O2o2) amplicons.

BC₁F₁ generation

Foreground selection using wx1 gene in BC₁F₁ identified 51, 46, 47 and 62 heterozygous plants (Wx1wx1) in HKII61 × MGU-102-wx1, HKI163 × MGU-102-wx1, HKI193-1 × MGU-102-wx1 and HKI193-2 × MGU-102-wx1 populations, respectively (Table 2). The chi-square test revealed Mendelian segregation ratio of 1:1 for the wx1 gene in all four crosses (Table 2). These identified heterozygous plants (Wx1wx1) were further subjected to foreground selection using o2 gene. The PCR assay identified 33 homozygous plants (o2o2) in HKII61 × MGU-102-wx1, while it was 26, 20 and 37 plants in HKI163 × MGU-102-wx1, HKI193-1 × MGU-102-wx1 and HKI193-2 × MGU-102-wx1 populations, respectively. Significant segregation distortion of o2 gene was observed in two crosses (HKII61 × MGU-102-wx1 and HKI193-2 × MGU-102-wx1), while rest two crosses (HKI163 × MGU-102-wx1and HKI193-1 × MGU-102-wx1) showed 1:1 ratio (Table 2). Consequently, foreground positive plants with 25–50% opaqueness were studied for background selection using polymorphic markers. Foreground positive plants with 75–100% opaqueness were rejected. Two plants each in HKI161- (82.1% and 83.5% RPG), HKI163- (81.3% and 80.4% RPG), and HKI193-2- (82.4% and 83.3% RPG), while three plants in HKI193-1- (82.4%, 82.8% and 81.4% RPG) based populations were selected for further advancement (Table 3). In BC1F1 generation, the recovery of RPG among the selected individuals varied from 81.3 to 83.5% with an average of 82.2%.

BC₂F₁ generation

A total of 59 heterozygous plants (Wx1wx1) was identified in HKI161 × MGU-102-wx1, while the same was 48 in HKI163 × MGU-102-wx1, 54 in HKI193-1 × MGU-102-wx1 and 46 in HKI193-2 × MGU-102-wx1 (Table 2). In all four crosses, Mendelian inheritance ratio of 1:1 was observed for the wx1 gene. All the heterozygous plants across population also showed homozygosity for o2 gene. Background selection among Wx1wx1/o2o2 plants (with 25–50% opaqueness) using polymorphic SSRs led to the recovery of 89.3–93.3% RPG in HKI161 × MGU-102-wx1, 87.9–91.6% in HKI163 × MGU-102-wx1, 88.7–92.2% in HKI193-1 × MGU-102-wx1 and 89.5–92.4% in HKI193-2 × MGU-102-wx1. Two plants in each of HKI161- (92.9% and 93.3% RPG), HKI193-1- (92.2% and 91.7% RPG), and HKI193-2- (91.9% and 92.4% RPG), while three plants in HKI163- (91.6%, 90.7% and 91.1% RPG) based populations were advanced (Table 3). Across BC₂F₁ generations, the average recovery of RPG was 92.0% with a range from 90.7 to 93.3%.

BC₂F₂ generation

Foreground selection identified 50 homozygous plants (wx1wx1) in HKI161 × MGU-102-wx1, while it was 60, 58 and 51 in HKI163 × MGU-102-wx, HKI193-1 × MGU-102-wx1 and HKI193-2 × MGU-102-wx1, respectively (Table 2). With regard to the wx1 gene, all four crosses followed the Mendelian segregation pattern of 1:2:1. (Table 2). All the homozygous plants (wx1wx1) also revealed the presence of o2 gene in homozygous condition. Screening of double-homozygous plants (wx1wx1/o2o2) having 25–50% opaqueness with background markers led to high recovery of RPG in HKI161 × MGU-102-wx1 (91.6–96.4%), HKI163 × MGU-102-wx1 (90.7–94.4%), HKI193-1 × MGU-102-wx1 (91.7–95.1%) and HKI193-2 × MGU-102-wx1 (91.9–95.2%) (Table 3). Three plants each in HKI161- (95.1%, 95.5% and 96.4%), HKI163- (94.4%, 93.5% and 93.9% RPG), HKI193-1- (94.1%, 95.1% and 94.6% RPG) and HKI193-2- (94.3%, 94.8% and 95.2% RPG) based populations were selected for further advancement (Table 3, Fig. 3). Recovery among the selected progenies ranged from 93.5 to 96.4%, with an average of 95.2%.

Selection of BC₂F₃ progenies for kernel colour

BC₂F₃ seeds borne on BC₂F₂ plants with wx1wx1/o2o2 genotype were selected for white colour in kernels. Seeds with yellow kernel colour were not considered for the present study. The white seeds homozygous for both wx1 and o2 genes were planted in order to generate BC₂F₃ progenies. Three progenies each in HKI161-, HKI1163-, HKI193-1- and HKI193-2- based populations were finally selected for evaluation and reconstitution of hybrids (Table 4).

Evaluation of introgressed inbreds for amylopectin

Amylopectin among MABB-derived progenies of HKI161, HKI163, HKI193-1 and HKI193-2 showed substantial increase (mean: 98.70%, range: 97.68–99.31%) over their respective recurrent parents (mean: 73.76%, range: 72.00–75.17%) (Table 4, Fig. 4). All the introgressed inbreds were statistically superior to their respective recurrent parents for amylopectin content. HKI161 had 73.76% amylopectin, while its waxy versions possessed 98.94% (HKI161-99-30-43-290), 99.00% (HKI161-107-42-1-291) and 98.57% (HKI161-107-42-10-293) amylopectin. HKI163 possessed 72.00% amylopectin, and its MABB versions had 98.24% (HKI163-9-2-13-302), 97.68% (HKI163-9-35-88-303) and 98.46% (HKI163-19-3-107-304) amylopectin. Waxy versions of HKI193-1 had 99.31% [HKI193-1-6-55-9-317)], 98.70% [HKI193-1-6-55-116-319], and 99.06% [HKI193-1-14-1-57-320] amylopectin, compared to 74.10% in HKI193-1 (Table 4, Fig. 3S). HKI193-2 had 75.17% amylopectin, and its MABB versions possessed 99.23% [HKI193-2-4-39-45-321], 98.72% [HKI193-2-4-20-56-322] and 98.52% [HKI193-2-4-20-111-325] amylopectin. Overall, an average of ~ 1.4-fold increase in amylopectin was recorded among introgressed progenies. However, starch content among introgressed- (mean: 69.42%) and original- inbreds (mean: 68.05%) were statistically at par (Table S5).

Evaluation of introgressed inbreds for lysine and tryptophan

MABB-derived progenies of HKI161, HKI163, HKI193-1 and HKI193-2 showed higher lysine (mean: 0.367%) and tryptophan (mean; 0.091%) over their respective recurrent parents (lysine: 0.318%, tryptophan: 0.079%) (Table 4, Figs. 4S, 5S). Each of the introgressed progenies had significantly higher lysine and tryptophan over their respective recurrent parents except HKI163-9-35-88-303 and HKI163-19-3-107-304 which had statistically similar lysine with HKI163. HKI161 had 0.315% lysine and 0.079% tryptophan, while its waxy versions viz., HKI161-99-30-43-290 (lysine: 0.364%, tryptophan: 0.093%), HKI161-107-42-1-291 (lysine: 0.367%, tryptophan: 0.089%) and HKI161-107-42-10-293 (lysine: 0.351%, tryptophan: 0.091%) had higher accumulation. Waxy versions viz., HKI163-9-2-13-302 (lysine: 0.378%, tryptophan: 0.094%), HKI163-9-35-88-303 (lysine: 0.365%, tryptophan: 0.093%) and HKI163-19-3-107-304 (lysine: 0.370%, tryptophan: 0.091%) possessed better nutritional quality compared to HKI163 (lysine: 0.338%, tryptophan: 0.084%). In case of HKI193-1, lysine and tryptophan was 0.320% and 0.078%, respectively, while waxy versions viz., HKI193-1-6-55-9-317 (lysine: 0.391%, and tryptophan: 0.092%), HKI193-1-6-55-116-319 (lysine: 0.372%, tryptophan: 0.092%), and HKI193-1-14-1-57-320 (lysine: 0.381%, tryptophan: 0.095%) possessed higher accumulation. MABB-versions viz., HKI193-2-4-39-45-321 (lysine: 0.351%, tryptophan: 0.087%), HKI193-2-4-20-56-322 (lysine: 0.345%, tryptophan: 0.09%) and HKI193-2-4-20-111-325 (lysine: 0.364%, tryptophan: 0.088%) also possessed superior nutritional quality over original inbred, HKI193-2 (lysine: 0.298%, tryptophan: 0.074%) (Table 4). Overall, introgressed progenies possessed 1.2-fold more lysine and tryptophan over the original inbreds.

Evaluation of introgressed inbreds for yield and morphological characters

In general, introgressed progenies and their respective recurrent parents showed statistically similar levels of grain yield, days to anthesis, days to silking, plant height and ear height (Table 5). The grain yield of HKI161 was 1876 kg/ha, while the same in waxy versions ranged from 1742 to 2142 kg/ha (mean: 1991 kg/ha). HKI163 had grain yield of 2080 kg/ha, while its wx1 introgressed lines produced 1951–2107 kg/ha (mean: 2016 kg/ha) of grain yield. HKI193-1 and HKI193-2 produced grain yield of 1698 kg/ha and 1876 kg/ha, while their introgressed lines had grain yield of 1653–1920 kg/ha (mean: 1754 kg/ha) and 1653–2053 kg/ha (mean: 1949 kg/ha), respectively. However, significant difference was observed in few cases viz., (1) grain yield (HKI161 and HKI161-107–42-1–291, HKI193-1 and HKI193-2-4-20-111-325, and (2) days to anthesis and silking (HKI193-1 and HKI193-1-6-55-9-317, HKI193-1-6-55-116-319 and HKI193-1-14-1-57-320) (Table 5). The waxy inbreds also showed a high degree of phenotypic similarity for DUS characters with their recurrent parents (Table S7). However, the marker-assisted selection (MAS)-derived inbreds differed from their original inbreds for few DUS characteristics as well. For example, anthocyanin colouration of brace root was present in HKI161, while it was absent in HKI161-99-30-43-290, HKI161-107-42-1-291 and HKI161-107-42-10-293 (Table S7a). Similarly, anthocyanin colouration of brace root was absent in HKI193-1, while it was found present in all the three versions (Table S7c).

Evaluation of MAS-derived hybrids for amylopectin

The amylopectin of the reconstituted hybrids increased significantly from 72.45% in the original hybrids to 98.84% in the MABB-derived hybrids across three locations (Table S6, Fig. 6S). The newly derived waxy hybrids possessed amylopectin ranging from 98.07 to 99.37% compared to 70.43–74.36% among the original hybrids (Fig. 5). All the reconstituted hybrids showed statistically higher amount of amylopectin from their original hybrids. The original HQPM1 possessed 71.60% amylopectin, whereas its reconstituted hybrids had 98.07% (HQPM1-A), 98.48% (HQPM1-B), and 98.78% (HQPM1-C) amylopectin. HQPM4 had 70.43% amylopectin, while waxy versions of the hybrids possessed 99.04% (HQPM4-A), 98.42% (HQPM4-A) and 98.81 (HQPM4-A) amylopectin. On the other hand, reconstituted hybrids had 99.14% (HQPM5-A), 98.85% (HQPM5-B), and 99.00% (HQPM5-C) amylopectin, compared to 73.44% in HQPM5. Similarly, amylopectin of HQPM7 was 74.36%, and its waxy versions had 98.95% (HQPM7-A), 99.37% (HQPM7-B), and 99.11% (HQPM7-C) amylopectin. Amylopectin levels in the reconstituted waxy hybrids was increased by 1.4-fold over original versions across locations. However, starch content of the original (mean: 70.20%) and reconstituted (mean: 71.66%) versions of the hybrids were statistically at par (Table S5).

Evaluation of MAS-derived hybrids for lysine and tryptophan

The newly derived waxy hybrids had significantly higher lysine (mean: 0.384%, range: 0.347–0.417%) and tryptophan (mean: 0.102%, range: 0.096–0.107%) compared to lysine (mean: 0.336%, range: 0.314–0.361%) and tryptophan (mean: 0.089%, range: 0.083–0.093%) in the original hybrids (Figs. 6, 7). All the reconstituted hybrids possessed statistically higher amount of lysine and tryptophan over the original versions (Table S6). The lysine and tryptophan in HQPM1 were 0.314% and 0.091%, while waxy HQPM1 version of the reconstituted hybrids viz., HQPM1-A (lysine: 0.353%, tryptophan: 0.103%), HQPM1-B (lysine: 0.354%, tryptophan: 0.102%) and HQPM1-C (lysine: 0.347%, tryptophan: 0.105%) were superior in nutritional quality. HQPM4 had 0.334% lysine and 0.083% tryptophan, while the reconstituted hybrids viz., HQPM4-A (lysine: 0.388%, tryptophan: 0.097%), HQPM4-B (lysine: 0.400%, tryptophan: 0.098%) and HQPM4-C (lysine: 0.389%, tryptophan: 0.096%) possessed higher concentration of amino acids. The reconstituted hybrids of HQPM5 viz., HQPM5-A (lysine: 0.382%, tryptophan: 0.101%); HQPM5-B (lysine: 0.372%, tryptophan: 0.103%) and HQPM5-C (lysine: 0.383%, tryptophan: 0.104%) possessed higher nutritional value over the original hybrid, HQPM5 (lysine: 0.337%, tryptophan: 0.090%). Similarly, lysine and tryptophan concentration of HQPM7 was 0.361% and 0.093%, respectively while waxy version of the hybrids viz., HQPM7-A (lysine: 0.417%, tryptophan: 0.107%), HQPM7-B (lysine: 0.416%, tryptophan: 0.107%) and HQPM7-C (lysine: 0.412%, tryptophan: 0.107%) had higher accumulation (Figs. 6, 7). Across locations, reconstituted hybrids had 1.1-fold and 1.2-fold more lysine and tryptophan, respectively over the original hybrids.

Evaluation of MAS-derived hybrids for yield and morphological characters

In general, reconstituted hybrids showed statistically similar grain yield, days to anthesis, days to silking, plant height and ear height with their original version (Table 6). The grain yield of reconstituted waxy QPM hybrids was 6248 kg/ha (range: 5724–7067 kg/ha), whereas in original QPM hybrids it was 6111 kg/ha (range: 5906–6648 kg/ha) across locations (Table 6). HQPM1 had grain yield of 5953 kg/ha, while its waxy hybrids had 6174 kg/ha (HQPM1-A), 6185 kg/ha (HQPM1-B), and 6056 kg/ha (HQPM1-C). Grain yield of the reconstituted hybrids was 5724 kg/ha (HQPM4-A), 5750 kg/ha (HQPM4-B), and 5971 kg/ha (HQPM4-C), compared to 5935 kg/ha in HQPM4. In case of HQPM5, grain yield was 5906 kg/ha, and its waxy reconstituted hybrids produced grain yield of 5968 kg/ha (HQPM5-A), 6102 kg/ha (HQPM5-B), and 5865 kg/ha (HQPM5-C). Similarly, HQPM7 had grain yield of 6648 kg/ha, whereas the waxy versions had grain yield of 7044 kg/ha (HQPM7-A), 7065 kg/ha (HQPM7-B), and 7067 kg/ha (HQPM7-C) (Table 6, Table S8). The flowering behaviour and plant characteristics of the reconstituted waxy hybrids were quite similar to the original hybrids as well (Table S8). However, in case of plant height and ear height, significant difference was observed between (1) HQPM1 and their reconstitute hybrids viz., HQPM1-C, and (2) HQPM4 and their reconstitute hybrids viz., HQPM1-B and HQPM1-C (Table 6). The improved waxy hybrids were very similar to their respective original hybrids for DUS characters except few traits (Table S9). For examples: anthocyanin colouration of brace root was present in HQPM1, while it was absent in HQPM1-A, HQPM1-B and HQPM1-C (Table S9a). Similarly, anthocyanin colouration of brace root was present in HQPM7, while it was found absent in HQPM7-A and HQPM7-C (Table S9d).