Unique genetic architecture of prolificacy in 'Sikkim Primitive' maize unraveled through whole-genome resequencing-based DNA polymorphism.

KEY MESSAGE: 'Sikkim Primitive' maize landrace, unique for prolificacy (7-9 ears per plant) possesses unique genomic architecture in branching and inflorescence-related gene(s), and locus Zm00001eb365210 encoding glycosyltransferases was identified as the putative candidate gene underlying QTL (qProl-SP-8.05) for prolificacy. The genotype possesses immense usage in breeding high-yielding baby-corn genotypes. 'Sikkim Primitive' is a native landrace of North Eastern Himalayas, and is characterized by having 7-9 ears per plant compared to 1-2 ears in normal maize. Though 'Sikkim Primitive' was identified in the 1960s, it has not been characterized at a whole-genome scale. Here, we sequenced the entire genome of an inbred (MGUSP101) derived from 'Sikkim Primitive' along with three non-prolific (HKI1128, UMI1200, and HKI1105) and three prolific (CM150Q, CM151Q and HKI323) inbreds. A total of 942,417 SNPs, 24,160 insertions, and 27,600 deletions were identified in 'Sikkim Primitive'. The gene-specific functional mutations in 'Sikkim Primitive' were classified as 10,847 missense (54.36%), 402 non-sense (2.015%), and 8,705 silent (43.625%) mutations. The number of transitions and transversions specific to 'Sikkim Primitive' were 666,021 and 279,950, respectively. Among all base changes, (G to A) was the most frequent (215,772), while (C to G) was the rarest (22,520). Polygalacturonate 4-α-galacturonosyltransferase enzyme involved in pectin biosynthesis, cell-wall organization, nucleotide sugar, and amino-sugar metabolism was found to have unique alleles in 'Sikkim Primitive'. The analysis further revealed the Zm00001eb365210 gene encoding glycosyltransferases as the putative candidate underlying QTL (qProl-SP-8.05) for prolificacy in 'Sikkim Primitive'. High-impact nucleotide variations were found in ramosa3 (Zm00001eb327910) and zeaxanthin epoxidase1 (Zm00001eb081460) genes having a role in branching and inflorescence development in 'Sikkim Primitive'. The information generated unraveled the genetic architecture and identified key genes/alleles unique to the 'Sikkim Primitive' genome. This is the first report of whole-genome characterization of the 'Sikkim Primitive' landrace unique for its high prolificacy.

Genetic basis of maize maternal haploid induction beyond MATRILINEAL and ZmDMP.

In maize, doubled haploid (DH) lines are created in vivo through crosses with maternal haploid inducers. Their induction ability, usually expressed as haploid induction rate (HIR), is known to be under polygenic control. Although two major genes (MTL and ZmDMP) affecting this trait were recently described, many others remain unknown. To identify them, we designed and performed a SNP based (~9007) genome-wide association study using a large and diverse panel of 159 maternal haploid inducers. Our analyses identified a major gene near MTL, which is present in all inducers and necessary to disrupt haploid induction. We also found a significant quantitative trait loci (QTL) on chromosome 10 using a case-control mapping approach, in which 793 noninducers were used as controls. This QTL harbors a kokopelli ortholog, whose role in maternal haploid induction was recently described in Arabidopsis. QTL with smaller effects were identified on six of the ten maize chromosomes, confirming the polygenic nature of this trait. These QTL could be incorporated into inducer breeding programs through marker-assisted selection approaches. Further improving HIR is important to reduce the cost of DH line production.

Low expression of lipoxygenase 3 (LOX3) enhances the retention of kernel tocopherols in maize during storage.

BACKGROUND: Deficiency of vitamin E results in several neurological and age-related disorders in humans. Utilization of maize mutants with favourable vte4-allele led to the development of several α-tocopherol (vitamin E) rich (16-19 µg/g) maize hybrids worldwide. However, the degradation of tocopherols during post-harvest storage substantially affects the efficacy of these genotypes. METHODS AND RESULTS: We studied the role of lipoxygenase enzyme and Lipoxygenase 3 (LOX3) gene on the degradation of tocopherols at monthly intervals under traditional storage up to six months in two vte4-based contrasting-tocopherol retention maize inbreds viz. HKI323-PVE and HKI193-1-PVE. The analysis revealed significant degradation of tocopherols across storage intervals in both the inbreds. Lower retention of α-tocopherol was noticed in HKI193-1-PVE. HKI323-PVE with the higher retention of α-tocopherol showed lower lipoxygenase activity throughout the storage intervals. LOX3 gene expression was higher (~ 1.5-fold) in HKI193-1-PVE compared to HKI323-PVE across the storage intervals. Both lipoxygenase activity and LOX3 expression peaked at 120 days after storage (DAS) in both genotypes. Further, a similar trend was observed for LOX3 expression and lipoxygenase activity. The α-tocopherol exhibited a significantly negative correlation with lipoxygenase enzyme and expression of LOX3 across the storage intervals. CONCLUSIONS: HKI323-PVE with high tocopherol retention, low -lipoxygenase activity, and -LOX3 gene expression can act as a potential donor in the vitamin E biofortification program. Protein-protein association network analysis also indicated the independent effect of vte4 and LOX genes. This is the first comprehensive report analyzing the expression of the LOX3 gene and deciphering its vital role in the retention of α-tocopherol in biofortified maize varieties under traditional storage.

Development and validation of rapid and cost-effective protocol for estimation of amylose and amylopectin in maize kernels.

Maize possesses wide variation in amylose and amylopectin which assumes significance as a part of both food-chain and different industrial applications. Estimation of amylose and amylopectin in maize kernels is important for developing suitable hybrids. The existing protocols for estimation of amylose and amylopectin in maize are elaborate and lengthy, and involve high cost. Here, we developed a rapid and cost-effective method for estimation of amylose and amylopectin in maize kernels. 10% toluene and 80% ethanol were used for removal of proteins (~ 10%) and lipids (~ 4%) from maize flour. The over-estimation of amylose was minimized using NaOH with KI to stop free KI to bind with amylopectin. Standards were improved by mixing amylose and amylopectin in different concentrations (0-100%), rather than using amylose or amylopectin alone. Standard curve generated regression equation of y = 90.436x + 0.8535 with R 2 = 0.9989. Two types of samples viz., (1) protein, amylose and amylopectin (2) amylose and amylopectin, showed that starch fractions were highly comparable to expected values with correlation coefficient (r) of 0.9998 and mean standard deviation of 0.54. The protocol successfully estimated wide range of amylose (2.79-50.04%) and amylopectin (59.96-97.21%) among diverse maize inbreds including amylose extender1 (ae1) and waxy1 (wx1) mutants. Present protocol required 75% less time and 92.5% less cost compared to existing protocols. The newly developed method would be highly useful in developing maize hybrids high in amylose or amylopectin. This is the first report of rapid and cost-effective protocol for estimation of starch fractions in maize kernels.

Expression analysis of ß-carotene hydroxylase1 and opaque2 genes governing accumulation of provitamin-A, lysine and tryptophan during kernel development in biofortified sweet corn.

Traditional sweet corn possesses low levels of provitamin-A (proA), lysine and tryptophan. Mutant version of ß-carotene hydroxylase1 (crtRB1) gene affecting the accumulation of ß-carotene (BC), ß-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.

Development of multinutrient-rich biofortified sweet corn hybrids through genomics-assisted selection of shrunken2, opaque2, lcyE and crtRB1 genes.

Sweet corn has gained worldwide popularity. Traditional sweet corn possesses low concentration of essential nutrients such as lysine (0.15-0.25%), tryptophan (0.03-0.04%) and provitamin-A (proA 3-4 ppm), and deficiency leads to serious health problems in humans. Here, stacking of shrunken2 (sh2), opaque2 (o2), lycopene epsilon cyclase (lcyE) and ß-carotene hydroxylase (crtRB1) genes  were undertaken in the parents of four hybrids viz., APQH1, APHQ4, APHQ5 and APHQ7 using marker-assisted backcross breeding (MABB). Gene-linked markers (umc2276 and umc1320) for sh2, while gene-based markers for o2 (umc1066 and phi057), lcyE (5'TE-InDel) and crtRB1 (3'TE-InDel), were used for genotyping in BC1F1, BC2F1 and BC2F2. Selected backcross progenies showed high recovery of recurrent parent genome (92.4-97.7%). The reconstituted sweet corn hybrids possessed significantly high lysine (0.390%), tryptophan (0.082%) and proA (21.14 ppm), coupled with high kernel sweetness (brix 18.96%). The improved sweet corn hybrids had high cob yield (12.22-15.33 t/ha) across three environments. These newly developed biofortified sweet corn hybrids possess great significance in providing balanced nutrition. This is the first report of combining sh2, o2, lcyE and crtRB1 genes for enrichment of sweet corn hybrids with multiple essential nutrients.

Molecular characterization of teosinte branched1 gene governing branching architecture in cultivated maize and wild relatives.

We sequenced the entire tb1 gene in six maize inbreds and its wild relatives (parviglumis, mexicana, perennis and luxurians) to characterize it at molecular level. Hopscotch and Tourist transposable elements were observed in the upstream of tb1 in all maize inbreds, while they were absent in wild relatives. In maize, tb1 consisted of 431-443 bp 5'UTR, 1101 bp coding sequence and 211-219 bp 3'UTR. In promoter region, mutations in the light response element in mexicana (~ 35 bp and ~ 55 bp upstream of TSS) and perennis (at ~ 35 bp upstream of TSS) were found. A 6 bp insertion at 420 bp downstream of the polyA signal site was present among teosinte accessions, while it was not observed in maize. A codominant marker flanking the 6 bp InDel was developed, and it differentiated the teosintes from maize. In Tb1 protein, alanine (12.7-14.6%) was the most abundant amino acid with tryptophan as the rarest (0.5-0.9%). The molecular weight of Tb1 protein was 38757.15 g/mol except 'Palomero Toluqueno' and HKI1128. R and TCP motifs in Tb1 protein were highly conserved across maize, teosinte and orthologues, while TCP domain differed for tb1 paralogue. Tb1 possessed important role in light-, auxin-, stress-response and meristem identity maintenance. Presence of molecular signal suggested its localization in mitochondria, nucleus and nucleolus. Parviglumis and mexicana shared closer relationship with maize than perennis and luxurians. A highly conserved 59-60 amino acids long bHLH region was observed across genotypes. Information generated here assumes significance in evolution of tb1 gene and breeding for enhancement of prolificacy in maize.

Molecular characterization of 5' UTR of the lycopene epsilon cyclase (lcyE) gene among exotic and indigenous inbreds for its utilization in maize biofortification.

Maize grains are the important source of food and energy, but possess very low proA (< 2.5 µg/g) compared to target level of 15 µg/g set by HarvestPlus to alleviate VAD. Favorable allele having variation in 5' untranslated region (UTR) of lycopene epsilon cyclase (lcyE) gene enhances concentration of proA in maize. To identify the sequence variation in 5' UTR of lcyE, a set of diverse 13 inbreds of indigenous and exotic origin was characterized for allelic constitution of lcyE. Inbreds possessed wide variation in proA (1.62-23.12 µg/g) with a mean of 9.64 µg/g. The proA in CIMMYT-HarvestPlus genotypes having favorable allele of lcyE was very high (22.28 µg/g), whereas the Indian inbreds with the same allele possessed very low proA (2.48 µg/g). Eight genotypes viz., HKI161, HKI163, HKI161-PV, HKI163-PV, HKI193-1-PV, HKI193-2-PV, HP704-22 and HP704-23 revealed the presence of favorable allele, while VQL1, DMRIL47, MGU-PV-123/C6, HKI193-1 and HKI193-2 showed the presence of unfavorable allele of lcyE gene. Sequence comparison of favorable allele of Indian (HKI161 and HKI163) and exotic genotypes (HP704-22 and HP704-23) revealed seven SNPs having three transitions (SNP1 and SNP3: G to A, SNP2: C to T) and four transversions (SNP4: C to G, SNP5: T to G, SNP6: G to C and SNP7: G to T). Four SNPs (SNP1: position 446, SNP2: position 458, SNP3: position 459 and SNP4: position 483) discriminated the low- and high- proA lines having favorable allele of lcyE 5'TE. These SNPs hold significance in enrichment of proA in maize for marker development and their use in marker-assisted selection.

Microsatellite marker-based characterization of waxy maize inbreds for their utilization in hybrid breeding.

Waxy corn possesses 95-100% amylopectin, compared to 70-75% in normal maize, owing to mutation in Wx gene encoding a granule-bound starch synthase I. Amylopectin is used as an ingredient in textile, adhesive and paper industries. Further, waxy green cob is popular as breakfast item in South Asia and an important constituent of diet in north-eastern states of India as well. We developed a series of waxy inbreds from diverse exotic sources and through introgression breeding. To characterize and unravel the genetic relationships, 24 diverse waxy inbreds were analysed using 77 SSRs distributed throughout the genome. The study generated a total of 203 polymorphic alleles, with a mean of 2.69 alleles per locus. A total of nine unique and 20 rare alleles were detected. The polymorphism information content ranged from 0.08 to 0.68 with an average value of 0.40. Molecular profiling suggested sufficient attainment of homozygosity among the inbreds. Jaccard's dissimilarity coefficient between pairs of genotypes varied from 0.26 to 0.83 which revealed the diverse nature of the inbred lines. Cluster analyses grouped 24 genotypes into three major clusters. Principle coordinate analysis based on SSR also depicted the diverse origin of the genotypes as per the pedigree more reliably than agro-morphological traits. These inbreds were also promising for various cob and grain characteristics including grain yield. The study identified a set of potential cross-combinations that can be planned to develop highly heterotic waxy hybrid combinations. This is the first report of development and characterization of waxy inbreds in India.