High-throughput diagnostic markers for foliar fungal disease resistance and high oleic acid content in groundnut.

BACKGROUND: Foliar diseases namely late leaf spot (LLS) and leaf rust (LR) reduce yield and deteriorate fodder quality in groundnut. Also the high oleic acid content has emerged as one of the most important traits for industries and consumers due to its increased shelf life and health benefits. RESULTS: Genetic mapping combined with pooled sequencing approaches identified candidate resistance genes (LLSR1 and LLSR2 for LLS and LR1 for LR) for both foliar fungal diseases. The LLS-A02 locus housed LLSR1 gene for LLS resistance, while, LLS-A03 housed LLSR2 and LR1 genes for LLS and LR resistance, respectively. A total of 49 KASPs markers were developed from the genomic regions of important disease resistance genes, such as NBS-LRR, purple acid phosphatase, pentatricopeptide repeat-containing protein, and serine/threonine-protein phosphatase. Among the 49 KASP markers, 41 KASPs were validated successfully on a validation panel of contrasting germplasm and breeding lines. Of the 41 validated KASPs, 39 KASPs were designed for rust and LLS resistance, while two KASPs were developed using fatty acid desaturase (FAD) genes to control high oleic acid levels. These validated KASP markers have been extensively used by various groundnut breeding programs across the world which led to development of thousands of advanced breeding lines and few of them also released for commercial cultivation. CONCLUSION: In this study, high-throughput and cost-effective KASP assays were developed, validated and successfully deployed to improve the resistance against foliar fungal diseases and oleic acid in groundnut. So far deployment of allele-specific and KASP diagnostic markers facilitated development and release of two rust- and LLS-resistant varieties and five high-oleic acid groundnut varieties in India. These validated markers provide opportunities for routine deployment in groundnut breeding programs.

HOMEOBOX2, the paralog of SIX-ROWED SPIKE1/HOMEOBOX1, is dispensable for barley spikelet development.

The HD-ZIP class I transcription factor Homeobox 1 (HvHOX1), also known as Vulgare Row-type Spike 1 (VRS1) or Six-rowed Spike 1, regulates lateral spikelet fertility in barley (Hordeum vulgare L.). It was shown that HvHOX1 has a high expression only in lateral spikelets, while its paralog HvHOX2 was found to be expressed in different plant organs. Yet, the mechanistic functions of HvHOX1 and HvHOX2 during spikelet development are still fragmentary. Here, we show that compared with HvHOX1, HvHOX2 is more highly conserved across different barley genotypes and Hordeum species, hinting at a possibly vital but still unclarified biological role. Using bimolecular fluorescence complementation, DNA-binding, and transactivation assays, we validate that HvHOX1 and HvHOX2 are bona fide transcriptional activators that may potentially heterodimerize. Accordingly, both genes exhibit similar spatiotemporal expression patterns during spike development and growth, albeit their mRNA levels differ quantitatively. We show that HvHOX1 delays the lateral spikelet meristem differentiation and affects fertility by aborting the reproductive organs. Interestingly, the ancestral relationship of the two genes inferred from their co-expressed gene networks suggested that HvHOX1 and HvHOX2 might play a similar role during barley spikelet development. However, CRISPR-derived mutants of HvHOX1 and HvHOX2 demonstrated the suppressive role of HvHOX1 on lateral spikelets, while the loss of HvHOX2 does not influence spikelet development. Collectively, our study shows that through the suppression of reproductive organs, lateral spikelet fertility is regulated by HvHOX1, whereas HvHOX2 is dispensable for spikelet development in barley.

Integrated multi-omics analysis reveals drought stress response mechanism in chickpea (Cicer arietinum L.).

Drought is one of the major constraints limiting chickpea productivity. To unravel complex mechanisms regulating drought response in chickpea, we generated transcriptomics, proteomics, and metabolomics datasets from root tissues of four contrasting drought-responsive chickpea genotypes: ICC 4958, JG 11, and JG 11+ (drought-tolerant), and ICC 1882 (drought-sensitive) under control and drought stress conditions. Integration of transcriptomics and proteomics data identified enriched hub proteins encoding isoflavone 4'-O-methyltransferase, UDP-d-glucose/UDP-d-galactose 4-epimerase, and delta-1-pyrroline-5-carboxylate synthetase. These proteins highlighted the involvement of pathways such as antibiotic biosynthesis, galactose metabolism, and isoflavonoid biosynthesis in activating drought stress response mechanisms. Subsequently, the integration of metabolomics data identified six metabolites (fructose, galactose, glucose, myoinositol, galactinol, and raffinose) that showed a significant correlation with galactose metabolism. Integration of root-omics data also revealed some key candidate genes underlying the drought-responsive "QTL-hotspot" region. These results provided key insights into complex molecular mechanisms underlying drought stress response in chickpea.

Multilayered regulation of developmentally programmed pre-anthesis tip degeneration of the barley inflorescence.

Leaf and floral tissue degeneration is a common feature in plants. In cereal crops such as barley (Hordeum vulgare L.), pre-anthesis tip degeneration (PTD) starts with growth arrest of the inflorescence meristem dome, which is followed basipetally by the degeneration of floral primordia and the central axis. Due to its quantitative nature and environmental sensitivity, inflorescence PTD constitutes a complex, multilayered trait affecting final grain number. This trait appears to be highly predictable and heritable under standardized growth conditions, consistent with a developmentally programmed mechanism. To elucidate the molecular underpinnings of inflorescence PTD, we combined metabolomic, transcriptomic, and genetic approaches to show that barley inflorescence PTD is accompanied by sugar depletion, amino acid degradation, and abscisic acid responses involving transcriptional regulators of senescence, defense, and light signaling. Based on transcriptome analyses, we identified GRASSY TILLERS1 (HvGT1), encoding an HD-ZIP transcription factor, as an important modulator of inflorescence PTD. A gene-edited knockout mutant of HvGT1 delayed PTD and increased differentiated apical spikelets and final spikelet number, suggesting a possible strategy to increase grain number in cereals. We propose a molecular framework that leads to barley PTD, the manipulation of which may increase yield potential in barley and other related cereals.

Selective and Generic Photocatalytic Oxidation of Alcohol with Pd-TiO2 Thin Films: Butanols to Butanal/Butanone with Different Morphologies of Pd and 0.5θPt -Pd Counterparts.

The present study reports on the photocatalytic oxidation of butanols to butanal/butanone using thin film form of facet-dependent nano-Pd supported on commercial TiO2 under one-sun condition and demonstrates the generic nature. Pd-nanocube (PdNC (100)), Pd-truncated octahedron (PdTO (100) and (111)), polycrystalline (PdPC ), and their counterparts with half-a-monolayer Pt-coated on Pd (0.5θPt -Pd)) have been used as co-catalyst. A potentially scalable thin film form of Pd/TiO2 photocatalyst, prepared by drop-casting method, has been employed to study oxidation of n-butanol, 2-butanol, and iso-butanol to corresponding aldehyde/ketone. 100% selectivity is demonstrated to respective aldehyde/ketone with any catalyst used in the present study with varying degree of butanols conversion by NMR. 0.5θPt -PdTO /TiO2 shows the highest conversion of 2-butanol to butanone (13.6% in 4 h). Continuous 10 h of reaction with the most active 0.5θPt -PdTO /P25 catalyst demonstrates 31% conversion of 2-butanol to butanone, and catalyst recyclability has been demonstrated. The present protocol can be scalable to large scales to maximize the conversion in direct sunlight. Due to its generic nature, the current method can also be applied to many other alcohols and substrate molecules.

Chromosome-length genome assemblies of six legume species provide insights into genome organization, evolution, and agronomic traits for crop improvement.

INTRODUCTION: Legume crops are an important source of protein and oil for human health and in fixing atmospheric N2 for soil enrichment. With an objective to accelerate much-needed genetic analyses and breeding applications, draft genome assemblies were generated in several legume crops; many of them are not high quality because they are mainly based on short reads. However, the superior quality of genome assembly is crucial for a detailed understanding of genomic architecture, genome evolution, and crop improvement. OBJECTIVES: Present study was undertaken with an objective of developing improved chromosome-length genome assemblies in six different legumes followed by their systematic investigation to unravel different aspects of genome organization and legume evolution. METHODS: We employed in situ Hi-C data to improve the existing draft genomes and performed different evolutionary and comparative analyses using improved genome assemblies. RESULTS: We have developed chromosome-length genome assemblies in chickpea, pigeonpea, soybean, subterranean clover, and two wild progenitor species of cultivated groundnut (A. duranensis and A. ipaensis). A comprehensive comparative analysis of these genome assemblies offered improved insights into various evolutionary events that shaped the present-day legume species. We highlighted the expansion of gene families contributing to unique traits such as nodulation in legumes, gravitropism in groundnut, and oil biosynthesis in oilseed legume crops such as groundnut and soybean. As examples, we have demonstrated the utility of improved genome assemblies for enhancing the resolution of "QTL-hotspot" identification for drought tolerance in chickpea and marker-trait associations for agronomic traits in pigeonpea through genome-wide association study. Genomic resources developed in this study are publicly available through an online repository, 'Legumepedia'. CONCLUSION: This study reports chromosome-length genome assemblies of six legume species and demonstrates the utility of these assemblies in crop improvement. The genomic resources developed here will have significant role in accelerating genetic improvement applications of legume crops.

Large-scale genotyping and phenotyping of a worldwide winter wheat genebank for its use in pre-breeding.

Plant genetic resources (PGR) stored at genebanks are humanity's crop diversity savings for the future. Information on PGR contrasted with modern cultivars is key to select PGR parents for pre-breeding. Genotyping-by-sequencing was performed for 7,745 winter wheat PGR samples from the German Federal ex situ genebank at IPK Gatersleben and for 325 modern cultivars. Whole-genome shotgun sequencing was carried out for 446 diverse PGR samples and 322 modern cultivars and lines. In 19 field trials, 7,683 PGR and 232 elite cultivars were characterized for resistance to yellow rust - one of the major threats to wheat worldwide. Yield breeding values of 707 PGR were estimated using hybrid crosses with 36 cultivars - an approach that reduces the lack of agronomic adaptation of PGR and provides better estimates of their contribution to yield breeding. Cross-validations support the interoperability between genomic and phenotypic data. The here presented data are a stepping stone to unlock the functional variation of PGR for European pre-breeding and are the basis for future breeding and research activities.

Genomics-informed prebreeding unlocks the diversity in genebanks for wheat improvement.

The great efforts spent in the maintenance of past diversity in genebanks are rationalized by the potential role of plant genetic resources (PGR) in future crop improvement-a concept whose practical implementation has fallen short of expectations. Here, we implement a genomics-informed prebreeding strategy for wheat improvement that does not discriminate against nonadapted germplasm. We collect and analyze dense genetic profiles for a large winter wheat collection and evaluate grain yield and resistance to yellow rust (YR) in bespoke core sets. Breeders already profit from wild introgressions but PGR still offer useful, yet unused, diversity. Potential donors of resistance sources not yet deployed in breeding were detected, while the prebreeding contribution of PGR to yield was estimated through 'Elite × PGR' F1 crosses. Genomic prediction within and across genebanks identified the best parents to be used in crosses with elite cultivars whose advanced progenies can outyield current wheat varieties in multiple field trials.

FIND-IT: Accelerated trait development for a green evolution.

Improved agricultural and industrial production organisms are required to meet the future global food demands and minimize the effects of climate change. A new resource for crop and microbe improvement, designated FIND-IT (Fast Identification of Nucleotide variants by droplet DigITal PCR), provides ultrafast identification and isolation of predetermined, targeted genetic variants in a screening cycle of less than 10 days. Using large-scale sample pooling in combination with droplet digital PCR (ddPCR) greatly increases the size of low-mutation density and screenable variant libraries and the probability of identifying the variant of interest. The method is validated by screening variant libraries totaling 500,000 barley (Hordeum vulgare) individuals and isolating more than 125 targeted barley gene knockout lines and miRNA or promoter variants enabling functional gene analysis. FIND-IT variants are directly applicable to elite breeding pipelines and minimize time-consuming technical steps to accelerate the evolution of germplasm.

A catalogue of resistance gene homologs and a chromosome-scale reference sequence support resistance gene mapping in winter wheat.

A resistance gene atlas is an integral component of the breeder's arsenal in the fight against evolving pathogens. Thanks to high-throughput sequencing, catalogues of resistance genes can be assembled even in crop species with large and polyploid genomes. Here, we report on capture sequencing and assembly of resistance gene homologs in a diversity panel of 907 winter wheat genotypes comprising ex situ genebank accessions and current elite cultivars. In addition, we use accurate long-read sequencing and chromosome conformation capture sequencing to construct a chromosome-scale genome sequence assembly of cv. Attraktion, an elite variety representative of European winter wheat. We illustrate the value of our resource for breeders and geneticists by (i) comparing the resistance gene complements in plant genetic resources and elite varieties and (ii) conducting genome-wide associations scans (GWAS) for the fungal diseases yellow rust and leaf rust using reference-based and reference-free GWAS approaches. The gene content under GWAS peaks was scrutinized in the assembly of cv. Attraktion.

Genetic variation in CaTIFY4b contributes to drought adaptation in chickpea.

Chickpea production is vulnerable to drought stress. Identifying the genetic components underlying drought adaptation is crucial for enhancing chickpea productivity. Here, we present the fine mapping and characterization of 'QTL-hotspot', a genomic region controlling chickpea growth with positive consequences on crop production under drought. We report that a non-synonymous substitution in the transcription factor CaTIFY4b regulates seed weight and organ size in chickpea. Ectopic expression of CaTIFY4b in Medicago truncatula enhances root growth under water deficit. Our results suggest that allelic variation in 'QTL-hotspot' improves pre-anthesis water use, transpiration efficiency, root architecture and canopy development, enabling high-yield performance under terminal drought conditions. Gene expression analysis indicated that CaTIFY4b may regulate organ size under water deficit by modulating the expression of GRF-INTERACTING FACTOR1 (GIF1), a transcriptional co-activator of Growth-Regulating Factors. Taken together, our study offers new insights into the role of CaTIFY4b and on diverse physiological and molecular mechanisms underpinning chickpea growth and production under specific drought scenarios.

Mining for New Sources of Resistance to Powdery Mildew in Genetic Resources of Winter Wheat.

Genetic pathogen control is an economical and sustainable alternative to the use of chemicals. In order to breed resistant varieties, information about potentially unused genetic resistance mechanisms is of high value. We phenotyped 8,316 genotypes of the winter wheat collection of the German Federal ex situ gene bank for Agricultural and Horticultural Crops, Germany, for resistance to powdery mildew (PM), Blumeria graminis f. sp. tritici, one of the most important biotrophic pathogens in wheat. To achieve this, we used a semi-automatic phenotyping facility to perform high-throughput detached leaf assays. This data set, combined with genotyping-by-sequencing (GBS) marker data, was used to perform a genome-wide association study (GWAS). Alleles of significantly associated markers were compared with SNP profiles of 171 widely grown wheat varieties in Germany to identify currently unexploited resistance conferring genes. We also used the Chinese Spring reference genome annotation and various domain prediction algorithms to perform a domain enrichment analysis and produced a list of candidate genes for further investigation. We identified 51 significantly associated regions. In most of these, the susceptible allele was fixed in the tested commonly grown wheat varieties. Eleven of these were located on chromosomes for which no resistance conferring genes have been previously reported. In addition to enrichment of leucine-rich repeats (LRR), we saw enrichment of several domain types so far not reported as relevant to PM resistance, thus, indicating potentially novel candidate genes for the disease resistance research and prebreeding in wheat.

Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement.

Aegilops tauschii, the diploid wild progenitor of the D subgenome of bread wheat, is a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. Here we sequenced 242 Ae. tauschii accessions and compared them to the wheat D subgenome to characterize genomic diversity. We found that a rare lineage of Ae. tauschii geographically restricted to present-day Georgia contributed to the wheat D subgenome in the independent hybridizations that gave rise to modern bread wheat. Through k-mer-based association mapping, we identified discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of hexaploids incorporating diverse Ae. tauschii genomes. Exploiting the genomic diversity of the Ae. tauschii ancestral diploid genome permits rapid trait discovery and functional genetic validation in a hexaploid background amenable to breeding.

Understanding Heterosis, Genetic Effects, and Genome Wide Associations for Forage Quantity and Quality Traits in Multi-Cut Pearl Millet.

Pearl millet is an important food and fodder crop cultivated in the arid and semi-arid regions of Africa and Asia, and is now expanding to other regions for forage purpose. This study was conducted to better understand the forage quantity and quality traits to enhance the feed value of this crop. Two sets of pearl millet hybrids (80 single cross hybrids in Set-I and 50 top cross hybrids in Set-II) along with their parents evaluated multi-locationally for the forage-linked traits under multi-cut (two cuts) system revealed significant variability for the forage traits in the hybrids and parents. The mean better parent heterosis (BPH) for total dry forage yield (TDFY) was 136% across all the single cross hybrids and 57% across all the top cross hybrids. The mean BPH for in vitro organic matter digestibility (IVOMD) varied from -11 to 7% in the single cross hybrids and -13 to 11% in the top cross hybrids across cuts. The findings of TDFY and IVOMD heterosis in these sets indicated the potential of improvement of the hybrid cultivars for forage quantity and quality in forage pearl millet. The parental lines single cross parent (SCP)-L02, SCP-L06, and top cross parent (TCP)-T08 found superior in the forage quantity and quality traits can be utilized in the future breeding programs. Most of the forage traits were found to be controlled by using the non-additive gene action. A diverse panel of 105 forage-type hybrid parents (Set-III) genotyped following genotyping by sequencing (GBS) and phenotyped for crude protein (CP) and IVOMD under multi-cuts for 2 years identified one stable significant single nucleotide polymorphism (SNP) on LG4 for CP, and nine SNPs for IVOMD distributed across all the linkage groups except on LG2. The identified loci, once validated, then could be used for the forage quality traits improvement in pearl millet through marker-assisted selection.

Global range expansion history of pepper (Capsicum spp.) revealed by over 10,000 genebank accessions.

Genebanks collect and preserve vast collections of plants and detailed passport information, with the aim of preserving genetic diversity for conservation and breeding. Genetic characterization of such collections has the potential to elucidate the genetic histories of important crops, use marker-trait associations to identify loci controlling traits of interest, search for loci undergoing selection, and contribute to genebank management by identifying taxonomic misassignments and duplicates. We conducted a genomic scan with genotyping by sequencing (GBS) derived single nucleotide polymorphisms (SNPs) of 10,038 pepper (Capsicum spp.) accessions from worldwide genebanks and investigated the recent history of this iconic staple. Genomic data detected up to 1,618 duplicate accessions within and between genebanks and showed that taxonomic ambiguity and misclassification often involve interspecific hybrids that are difficult to classify morphologically. We deeply interrogated the genetic diversity of the commonly consumed Capsicum annuum to investigate its history, finding that the kinds of peppers collected in broad regions across the globe overlap considerably. The method ReMIXTURE-using genetic data to quantify the similarity between the complement of peppers from a focal region and those from other regions-was developed to supplement traditional population genetic analyses. The results reflect a vision of pepper as a highly desirable and tradable cultural commodity, spreading rapidly throughout the globe along major maritime and terrestrial trade routes. Marker associations and possible selective sweeps affecting traits such as pungency were observed, and these traits were shown to be distributed nonuniformly across the globe, suggesting that human preferences exerted a primary influence over domesticated pepper genetic structure.

Publisher Correction: A recombination bin-map identified a major QTL for resistance to Tomato Spotted Wilt Virus in peanut (Arachis hypogaea).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Seed protein content and its relationships with agronomic traits in pigeonpea is controlled by both main and epistatic effects QTLs.

The genetic architecture of seed protein content (SPC) and its relationships to agronomic traits in pigeonpea is poorly understood. Accordingly, five F2 populations segregating for SPC and four agronomic traits (seed weight (SW), seed yield (SY), growth habit (GH) and days to first flowering (DFF)) were phenotyped and genotyped using genotyping-by-sequencing approach. Five high-density population-specific genetic maps were constructed with an average inter-marker distance of 1.6 to 3.5 cM, and subsequently, integrated into a consensus map with average marker spacing of 1.6 cM. Based on analysis of phenotyping data and genotyping data, 192 main effect QTLs (M-QTLs) with phenotypic variation explained (PVE) of 0.7 to 91.3% were detected for the five traits across the five populations. Major effect (PVE ≥ 10%) M-QTLs included 14 M-QTLs for SPC, 16 M-QTLs for SW, 17 M-QTLs for SY, 19 M-QTLs for GH and 24 M-QTLs for DFF. Also, 573 epistatic QTLs (E-QTLs) were detected with PVE ranging from 6.3 to 99.4% across traits and populations. Colocalization of M-QTLs and E-QTLs explained the genetic basis of the significant (P < 0.05) correlations of SPC with SW, SY, DFF and GH. The nature of genetic architecture of SPC and its relationship with agronomic traits suggest that genomics-assisted breeding targeting genome-wide variations would be effective for the simultaneous improvement of SPC and other important traits.

Genome-wide analysis of epigenetic and transcriptional changes associated with heterosis in pigeonpea.

Hybrids are extensively used in agriculture to deliver an increase in yield, yet the molecular basis of heterosis is not well understood. Global DNA methylation analysis, transcriptome analysis and small RNA profiling were aimed to understand the epigenetic effect of the changes in gene expression level in the two hybrids and their parental lines. Increased DNA methylation was observed in both the hybrids as compared to their parents. This increased DNA methylation in hybrids showed that majority of the 24-nt siRNA clusters had higher expression in hybrids than the parents. Transcriptome analysis revealed that various phytohormones (auxin and salicylic acid) responsive hybrid-MPV DEGs were significantly altered in both the hybrids in comparison to MPV. DEGs associated with plant immunity and growth were overexpressed whereas DEGs associated with basal defence level were repressed. This antagonistic patterns of gene expression might contribute to the greater growth of the hybrids. It was also noticed that some common as well as unique changes in the regulatory pathways were associated with heterotic growth in both the hybrids. Approximately 70% and 67% of down-regulated hybrid-MPV DEGs were found to be differentially methylated in ICPH 2671 and ICPH 2740 hybrid, respectively. This reflected the association of epigenetic regulation in altered gene expressions. Our findings also revealed that miRNAs might play important roles in hybrid vigour in both the hybrids by regulating their target genes, especially in controlling plant growth and development, defence and stress response pathways. The above finding provides an insight into the molecular mechanism of pigeonpea heterosis.

Genotyping-by-sequencing and multilocation evaluation of two interspecific backcross populations identify QTLs for yield-related traits in pigeonpea.

This study has identified single-nucleotide polymorphism (SNP) markers associated with nine yield-related traits in pigeonpea by using two backcross populations (BP) developed through interspecific crosses and evaluating them at two locations and 3 years. In both the populations, markers have shown strong segregation distortion; therefore, a quantitative trait locus (QTL) mapping mixed model was used. A total of 86 QTLs explaining 12-21% phenotypic variation were detected in BP-1. On the other hand, 107 QTLs explaining 11-29% phenotypic variation were detected in BP-2. Although most QTLs were environment and trait specific, few stable and consistent QTLs were also detected. Interestingly, 11 QTLs in BP-2 were associated with more than one trait. Among these QTLs, eight QTLs associated with days to 50% flowering and days to 75% maturity were located on CcLG07. One SNP "S7_14185076" marker in BP-2 population has been found associated with four traits, namely days to 50% flowering, days to 75% maturity, primary branches per plant and secondary branches per plant with positive additive effect. Hence, the present study has not only identified QTLs for yield-related traits, but also discovered novel alleles from wild species, which can be used for improvement of traits through genomics-assisted breeding.