Development of Novel KASP Markers for Improved Germination in Deep-Sown Direct Seeded Rice.

BACKGROUND: The lack of stable-high yielding and direct-seeded adapted varieties with better germination ability from deeper soil depth and availability of molecular markers are major limitation in achieving the maximum yield potential of rice under water and resource limited conditions. Development of high-throughput and trait-linked markers are of great interest in genomics-assisted breeding. The aim of present study was to develop and validate novel KASP (Kompetitive Allele-Specific PCR) markers associated with traits improving germination and seedling vigor of deep sown direct seeded rice (DSR). RESULTS: Out of 58 designed KASP assays, four KASP assays did not show any polymorphism in any of the eleven genetic backgrounds considered in the present study. The 54 polymorphic KASP assays were then validated for their robustness and reliability on the F1s plants developed from eight different crosses considered in the present study. The third next validation was carried out on 256 F3:F4 and 713 BC3F2:3 progenies. Finally, the reliability of the KASP assays was accessed on a set of random 50 samples from F3:F4 and 80-100 samples from BC3F2:3 progenies using the 10 random markers. From the 54 polymorphic KASP, based on the false positive rate, false negative rate, KASP utility in different genetic backgrounds and significant differences in the phenotypic values of the positive (desirable) and negative (undesirable) traits, a total of 12 KASP assays have been selected. These 12 KASP include 5 KASP on chromosome 3, 1 on chromosome 4, 3 on chromosome 7 and 3 on chromosome 8. The two SNPs lying in the exon regions of LOC_Os04g34290 and LOC_Os08g32100 led to non-synonymous mutations indicating a possible deleterious effect of the SNP variants on the protein structure. CONCLUSION: The present research work will provide trait-linked KASP assays, improved breeding material possessing favourable alleles and breeding material in form of expected pre-direct-seeded adapted rice varieties. The marker can be utilized in introgression program during pyramiding of valuable QTLs/genes providing adaptation to rice under DSR. The functional studies of the genes LOC_Os04g34290 and LOC_Os08g32100 possessing two validated SNPs may provide valuable information about these genes.

Corrigendum: Unravelling the genetic framework associated with grain quality and yield-related traits in maize (Zea mays L.).

[This corrects the article DOI: 10.3389/fgene.2023.1248697.].

Integrating Association Mapping, Linkage Mapping, Fine Mapping with RNA Seq Conferring Seedling Vigor Improvement for Successful Crop Establishment in Deep Sown Direct-Seeded Rice.

BACKGROUND: Ongoing large-scale shift towards direct seeded rice (DSR) necessitates a convergence of breeding and genetic approaches for its sustenance and harnessing natural resources and environmental benefits. Improving seedling vigour remains key objective for breeders working with DSR. The present study aims to understand the genetic control of seedling vigour in deep sown DSR. Combined genome-wide association mapping, linkage mapping, fine mapping, RNA-sequencing to identify candidate genes and validation of putative candidate genes were performed in the present study. RESULTS: Significant phenotypic variations were observed among genotypes in both F3:4:5 and BC2F2:3 populations. The mesocotyl length showed significant positive correlation with %germination, root and shoot length. The 881 kb region on chromosome 7 reported to be associated with mesocotyl elongation. RNA-seq data and RT-PCR results identified and validated seven potential candidate genes. The four promising introgression lines free from linkage drag and with longer mesocotyl length, longer root length, semi-dwarf plant height have been identified. CONCLUSION: The study will provide rice breeders (1) the pre breeding material in the form of anticipated DSR adapted introgression lines possessing useful traits and alleles improving germination under deep sown DSR field conditions (2) the base for the studies involving functional characterization of candidate genes. The development and utilization of improved introgression lines and molecular markers may play an important role in genomics-assisted breeding (GAB) during the pyramiding of valuable genes providing adaptation to rice under DSR. Our results offer a robust and reliable package that can contribute towards enhancing genetic gains in direct seeded rice breeding programs.

Unravelling the genetic framework associated with grain quality and yield-related traits in maize (Zea mays L.).

Maize serves as a crucial nutrient reservoir for a significant portion of the global population. However, to effectively address the growing world population's hidden hunger, it is essential to focus on two key aspects: biofortification of maize and improving its yield potential through advanced breeding techniques. Moreover, the coordination of multiple targets within a single breeding program poses a complex challenge. This study compiled mapping studies conducted over the past decade, identifying quantitative trait loci associated with grain quality and yield related traits in maize. Meta-QTL analysis of 2,974 QTLs for 169 component traits (associated with quality and yield related traits) revealed 68 MQTLs across different genetic backgrounds and environments. Most of these MQTLs were further validated using the data from genome-wide association studies (GWAS). Further, ten MQTLs, referred to as breeding-friendly MQTLs (BF-MQTLs), with a significant phenotypic variation explained over 10% and confidence interval less than 2 Mb, were shortlisted. BF-MQTLs were further used to identify potential candidate genes, including 59 genes encoding important proteins/products involved in essential metabolic pathways. Five BF-MQTLs associated with both quality and yield traits were also recommended to be utilized in future breeding programs. Synteny analysis with wheat and rice genomes revealed conserved regions across the genomes, indicating these hotspot regions as validated targets for developing biofortified, high-yielding maize varieties in future breeding programs. After validation, the identified candidate genes can also be utilized to effectively model the plant architecture and enhance desirable quality traits through various approaches such as marker-assisted breeding, genetic engineering, and genome editing.

Genome-wide identification and in silico analysis of NPF, NRT2, CLC and SLAC1/SLAH nitrate transporters in hexaploid wheat (Triticum aestivum).

Nitrogen transport is one of the most important processes in plants mediated by specialized transmembrane proteins. Plants have two main systems for nitrogen uptake from soil and its transport within the system-a low-affinity transport system and a high-affinity transport system. Nitrate transporters are of special interest in cereal crops because large amount of money is spent on N fertilizers every year to enhance the crop productivity. Till date four gene families of nitrate transporter proteins; NPF (nitrate transporter 1/peptide transporter family), NRT2 (nitrate transporter 2 family), the CLC (chloride channel family), and the SLAC/SLAH (slow anion channel-associated homologues) have been reported in plants. In our study, in silico mining of nitrate transporter genes along with their detailed structure, phylogenetic and expression analysis was carried out. A total of 412 nitrate transporter genes were identified in hexaploid wheat genome using HMMER based homology searches in IWGSC Refseq v2.0. Out of those twenty genes were root specific, 11 leaf/shoot specific and 17 genes were grain/spike specific. The identification of nitrate transporter genes in the close proximity to the previously identified 67 marker-traits associations associated with the nitrogen use efficiency related traits in nested synthetic hexaploid wheat introgression library indicated the robustness of the reported transporter genes. The detailed crosstalk between the genome and proteome and the validation of identified putative candidate genes through expression and gene editing studies may lay down the foundation to improve nitrogen use efficiency of cereal crops.

Development and validation of a novel core set of KASP markers for the traits improving grain yield and adaptability of rice under direct-seeded cultivation conditions.

The development and utilization of molecular-markers play an important role in genomics-assisted breeding during pyramiding of valuable genes. The aim of present study was to develop and validate a novel core-set of KASP (Kompetitive Allele-Specific PCR) markers associated with traits improving rice grain yield and adaptability under direct-seeded cultivation conditions. The 110 phenotypically validated KASP assays out of 171 designed KASP, include assays for biotic-resistance genes, anaerobic germination, root-traits, grain yield, lodging resistance and early-uniform emergence. The KASP assays were validated for their robustness and reliability at five different levels using diverse germplasm, segregating and advanced population, comparison with SSR markers and on F1s. The present research work will provide (i) breeding material in form of anticipated pre-direct-seeded adapted rice varieties (ii) single improved breeding line with many useful genes and (iii) KASP assay information for the useful QTL/genes providing grain yield and adaptability to rice under direct-seeded cultivation conditions.

Meta-QTL Analysis in Rice and Cross-Genome Talk of the Genomic Regions Controlling Nitrogen Use Efficiency in Cereal Crops Revealing Phylogenetic Relationship.

The phenomenal increase in the use of nitrogenous fertilizers coupled with poor nitrogen use efficiency is among the most important threats to the environment, economic, and social health. During the last 2 decades, a number of genomic regions associated with nitrogen use efficiency (NUE) and related traits have been reported by different research groups, but none of the stable and major effect QTL have been utilized in the marker-assisted introgression/pyramiding program. Compiling the data available in the literature could be very useful in identifying stable and major effect genomic regions associated with the root and NUE-related trait improving the rice grain yield. In the present study, we performed meta-QTL analysis on 1,330 QTL from 29 studies published in the past 2 decades. A total of 76 MQTL with a stable effect over different genetic backgrounds and environments were identified. The significant reduction in the confidence interval of the MQTL compared to the initial QTL resulted in the identification of annotated and putative candidate genes related to the traits considered in the present study. A hot spot region associated with correlated traits on chr 1, 4, and 8 and candidate genes associated with nitrate transporters, nitrogen content, and ammonium uptake on chromosomes 2, 4, 6, and 8 have been identified. The identified MQTL, putative candidate genes, and their orthologues were validated on our previous studies conducted on rice and wheat. The research-based interventions such as improving nitrogen use efficiency via identification of major genomic regions and candidate genes can be a plausible, simple, and low-cost solution to address the challenges of the crop improvement program.

Genome-wide identification and analysis of GRAS transcription factors in the bottle gourd genome.

GRAS genes belong to the plant-specific transcription factors (TF's) family that are known to be involved in plant growth and development. In this study, we have identified 37 genes from the bottle gourd genome that encodes for GRAS TF's. Except for the SCLA, we were able to identify at least one gene from each of the 17 subfamilies. Gene structure and chromosomal analysis showed that maximum seven genes are present on Chr7 followed by six genes on Chr1. The subcellular location analysis revealed that most of the genes were localized in the nucleus, except for a few in chloroplast and mitochondria. Additionally, we have identified one tandem gene duplication event on Chr7 and three major motifs that were present in all the GRAS genes. Furthermore, the protein-protein interaction prediction and gene expression analysis showed five candidate hub-genes interact with various other genes and thus probably control the expression of interacting partners in different plant tissues. Overall, this study provides a comprehensive analysis of GRAS transcription factors in bottle gourd genome which could be further extended to other vegetable crops.