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.

Understanding complex genetic architecture of rice grain weight through QTL-meta analysis and candidate gene identification.

Quantitative trait loci (QTL) for rice grain weight identified using bi-parental populations in various environments were found inconsistent and have a modest role in marker assisted breeding and map-based cloning programs. Thus, the identification of a consistent consensus QTL region across populations is critical to deploy in marker aided breeding programs. Using the QTL meta-analysis technique, we collated rice grain weight QTL information from numerous studies done across populations and in diverse environments to find constitutive QTL for grain weight. Using information from 114 original QTL in meta-analysis, we discovered three significant Meta-QTL (MQTL) for grain weight on chromosome 3. According to gene ontology, these three MQTL have 179 genes, 25 of which have roles in developmental functions. Amino acid sequence BLAST of these genes indicated their orthologue conservation among core cereals with similar functions. MQTL3.1 includes the OsAPX1, PDIL, SAUR, and OsASN1 genes, which are involved in grain development and have been discovered to play a key role in asparagine biosynthesis and metabolism, which is crucial for source-sink regulation. Five potential candidate genes were identified and their expression analysis indicated a significant role in early grain development. The gene sequence information retrieved from the 3 K rice genome project revealed the deletion of six bases coding for serine and alanine in the last exon of OsASN1 led to an interruption in the synthesis of α-helix of the protein, which negatively affected the asparagine biosynthesis pathway in the low grain weight genotypes. Further, the MQTL3.1 was validated using linked marker RM7197 on a set of genotypes with extreme phenotypes. MQTL that have been identified and validated in our study have significant scope in MAS breeding and map-based cloning programs for improving rice grain weight.

Salinity stress tolerance and omics approaches: revisiting the progress and achievements in major cereal crops.

Salinity stress adversely affects plant growth and causes considerable losses in cereal crops. Salinity stress tolerance is a complex phenomenon, imparted by the interaction of compounds involved in various biochemical and physiological processes. Conventional breeding for salt stress tolerance has had limited success. However, the availability of molecular marker-based high-density linkage maps in the last two decades boosted genomics-based quantitative trait loci (QTL) mapping and QTL-seq approaches for fine mapping important major QTL for salinity stress tolerance in rice, wheat, and maize. For example, in rice, 'Saltol' QTL was successfully introgressed for tolerance to salt stress, particularly at the seedling stage. Transcriptomics, proteomics and metabolomics also offer opportunities to decipher and understand the molecular basis of stress tolerance. The use of proteomics and metabolomics-based metabolite markers can serve as an efficient selection tool as a substitute for phenotype-based selection. This review covers the molecular mechanisms for salinity stress tolerance, recent progress in mapping and introgressing major gene/QTL (genomics), transcriptomics, proteomics, and metabolomics in major cereals, viz., rice, wheat and maize.

Meta-analysis and validation of genomic loci governing seedling and reproductive stage salinity tolerance in rice.

Identification of concurrent genomic regions contributing tolerance to salinity at the seedling and reproductive stages were done using 45 quantitative trait loci (QTL) mapping studies reporting 915 individual QTLs. The QTL-data were used to perform a meta-analysis to predict, validate and analyze the Meta-QTLs governing component traits contributing to salinity tolerance. We predicted a total of 65 and 49 Meta-QTLs distributed across the genome governing seedling and reproductive stage salinity tolerance, respectively. Salinity stress (EC ~10.0 dSm-1 ) was evaluated in a set of 32 genotypes grown hydroponically, from these eight extreme (highly tolerant and highly susceptible) genotypes were selected for validation of significant Meta-QTLs. Another set of eight previously known and reported (highly tolerant and highly susceptible) genotypes were evaluated under saline micro plot conditions (EC ~8.0 dSm-1 ) and used for validation of significant Meta-QTLs for reproductive stage salinity tolerance. The microsatellite marker "RM5635" linked to MSQTL4.2 (~295.43 kb) was able to clearly differentiate contrasting genotypes for seedling stage salinity tolerance, whereas at the reproductive stage, none of the markers were able to validate the predicted Meta-QTL for salinity tolerance. Earlier reported, gene expression studies were used for candidate gene analysis of validated MSQTL4.2, which indicated the down regulation of Os04g0423100, a gene encoding Mono-oxygenase-FAD binding domain containing protein. The traits associated with this Meta-QTL were root and shoot sodium and potassium concentration and leaf chlorophyll content. The identified and validated genomic region assumes a great significant role in seedling stage salinity tolerance in rice, and it can be used for marker-assisted backcross breeding programs.

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.

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.