Candidate effectors for leaf rust resistance gene Lr28 identified through transcriptome and in-silico analysis.

Puccinia spp. causing rust diseases in wheat and other cereals secrete several specialized effector proteins into host cells. Characterization of these proteins and their interaction with host's R proteins could greatly help to limit crop losses due to diseases. Prediction of effector proteins by combining the transcriptome analysis and multiple in-silico approaches is gaining importance in revealing the pathogenic mechanism. The present study involved identification of 13 Puccinia triticina (Pt) coding sequences (CDSs), through transcriptome analysis, that were differentially expressed during wheat-leaf rust interaction; and prediction of their effector like features using different in-silico tools. NCBI-BLAST and pathogen-host interaction BLAST (PHI-BLAST) tools were used to annotate and classify these sequences based on their most closely matched counterpart in both the databases. Homology between CDSs and the annotated sequences in the NCBI database ranged from 79 to 94% and with putative effectors of other plant pathogens in PHI-BLAST from 24.46 to 54.35%. Nine of the 13 CDSs had effector-like features according to EffectorP 3.0 (≥0.546 probability of these sequences to be effector). The qRT-PCR expression analysis revealed that the relative expression of all CDSs in compatible interaction (HD2329) was maximum at 11 days post inoculation (dpi) and that in incompatible interactions (HD2329 + Lr28) was maximum at 3 dpi in seven and 9 dpi in five CDSs. These results suggest that six CDSs (>0.8 effector probability as per EffectorP 3.0) could be considered as putative Pt effectors. The molecular docking and MD simulation analysis of these six CDSs suggested that candidate Lr28 protein binds more strongly to candidate effector c14094_g1_i1 to form more stable complex than the remaining five. Further functional characterization of these six candidate effectors should prove useful for a better understanding of wheat-leaf rust interaction. In turn, this should facilitate effector-based leaf rust resistance breeding in wheat.

Genome-wide survey and characterization of microsatellites in cashew and design of a web-based microsatellite database: CMDB.

The cashew is an edible tree nut crop having a wide range of food and industrial applications. Despite great economic importance, the genome-wide characterization of microsatellites [simple sequence repeats (SSRs)] in cashew is lacking. In this study, we carried out the first comprehensive genome-wide microsatellites/SSRs characterization in cashew and developed polymorphic markers and a web-based microsatellite database. A total of 54526 SSRs were discovered in the cashew genome, with a mean frequency of 153 SSRs/Mb. Among the mined genome-wide SSRs (2-6 bp size motifs), the dinucleotide repeat motifs were dominant (68.98%) followed by the trinucleotides (24.56%). The Class I type of SSRs (≥20 bp) were 45.10%, while Class II repeat motifs (≥12-<20 bp) were 54.89% of the total genomic SSRs discovered here. Further, the AT-rich SSRs occurred more frequently in the cashew genome (84%) compared to the GC-rich SSRs. The validation of the in silico-mined genome-wide SSRs by PCR screening in cashew genotypes resulted in the development of 59 polymorphic SSR markers, and the polymorphism information content (PIC) of the polymorphic SSR markers ranged from 0.19 to 0.84. Further, a web-based database, "Cashew Microsatellite Database (CMDB)," was constructed to provide access to the genome-wide SSRs mined in this study as well as transcriptome-based SSRs from our previous study to the research community through a user-friendly searchable interface. Besides, CMDB provides information on experimentally validated SSRs. CMDB permits the retrieval of SSR markers information with the customized search options. Altogether, the genome-wide SSRs characterization, the polymorphic markers and CMDB database developed in this study would serve as valuable marker resources for DNA fingerprinting, germplasm characterization, genetic studies, and molecular breeding in cashew and related Anacardium species.

Virulence and genetic analysis of Puccinia graminis tritici in the Indian sub-continent from 2016 to 2022 and evaluation of wheat varieties for stem rust resistance.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), has re-emerged as one of the major concerns for global wheat production since the evolution of Ug99 and other virulent pathotypes of Pgt from East Africa, Europe, Central Asia, and other regions. Host resistance is the most effective, economic, and eco-friendly approach for managing stem rust. Understanding the virulence nature, genetic diversity, origin, distribution, and evolutionary pattern of Pgt pathotypes over time and space is a prerequisite for effectively managing newly emerging Pgt isolates through host resistance. In the present study, we monitored the occurrence of stem rust of wheat in India and neighboring countries from 2016 to 2022, collected 620 single-pustule isolates of Pgt from six states of India and Nepal, analyzed them on Indian stem rust differentials, and determined their virulence phenotypes and molecular genotypes. The Ug99 type of pathotypes did not occur in India. Pathotypes 11 and 40A were most predominant during these years. Virulence phenotyping of these isolates identified 14 Pgt pathotypes, which were genotyped using 37 Puccinia spp.-specific polymorphic microsatellites, followed by additional phylogenetic analyses using DARwin. These analyses identified three major molecular groups, demonstrating fewer lineages, clonality, and long-distance migration of Pgt isolates in India. Fourteen of the 40 recently released Indian wheat varieties exhibited complete resistance to all 23 Pgt pathotypes at the seedling stage. Twelve Sr genes were postulated in 39 varieties based on their seedling response to Pgt pathotypes. The values of slow rusting parameters i.e. coefficient of infection, area under disease progress curve, and infection rates, assessed at adult plant stage at five geographically different locations during two crop seasons, indicated the slow rusting behavior of several varieties. Six Sr genes (Sr2, Sr57, Sr58, Sr24, Sr31, and Sr38) were identified in 24 wheat varieties using molecular markers closely linked to these genes. These findings will guide future breeding programs toward more effective management of wheat stem rust.

De novo assembly and characterization of the draft genome of the cashew (Anacardium occidentale L.).

Cashew is the second most important tree nut crop in the global market. Cashew is a diploid and heterozygous species closely related to the mango and pistachio. Its improvement by conventional breeding is slow due to the long juvenile phase. Despite the economic importance, very little genomics/transcriptomics information is available for cashew. In this study, the Oxford nanopore reads and Illumina reads were used for de novo assembly of the cashew genome. The hybrid assembly yielded a 356.6 Mb genome corresponding to 85% of the estimated genome size (419 Mb). The BUSCO analysis showed 91.8% of genome completeness. Transcriptome mapping showed 92.75% transcripts aligned with the assembled genome. Gene predictions resulted in the identification of 31,263 genes coding for a total of 35,000 gene isoforms. About 46% (165 Mb) of the cashew genome comprised of repetitive sequences. Phylogenetic analyses of the cashew with nine species showed that it was closely related to Mangifera indica. Analysis of cashew genome revealed 3104 putative R-genes. The first draft assembly of the genome, transcriptome and R gene information generated in this study would be the foundation for understanding the molecular basis of economic traits and genomics-assisted breeding in cashew.

Genetic Diversity and Population Structure Reveal Cryptic Genetic Variation and Long Distance Migration of Puccinia graminis f. sp. tritici in the Indian Subcontinent.

Stem rust caused by Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of wheat worldwide since time immemorial. Several wheat stem rust outbreaks have been reported worldwide including India. Approximately 7 mha wheat area in central and peninsular India is highly vulnerable to stem rust epidemics. In this study, a repository of 29 single genotype uredospore pathotypes, representing five geographical regions, was characterized by investigating their virulence phenotype and simple sequence repeat (SSR) genotypes using 37 reproducible polymorphic SSR markers, 32 of which had ≥ 0.50 polymorphic information content (PIC) value. Virulence phenotypes were used to evaluate the virulence frequency (VF) and construct a hypothetical evolutionary hierarchy of these pathotypes. We projected seven lineages to explain the evolutionary pattern of the Pgt population. The VF of these pathotypes ranged between 0% and 100%. The virulence-based neighbor-joining (NJ) cluster analysis grouped Pgt pathotypes into five virulence groups. Likewise, five molecular groups were categorized using molecular genotypes. The molecular grouping was supported by principal coordinate analysis (PCoA), which revealed 25% of the cumulative variance contributed by the first two axes. Analysis of molecular variance (AMOVA) revealed 8 and 92% of the variation among and within the populations, respectively. The Mantel test confirmed a positive but weak correlation (R 2 = 0.15) between virulence phenotypes and SSR genotypes. The highest and lowest values of different genetic diversity parameters (Na, Ne, I, He, uHe, and %P) revealed maximum and minimum variability in the Pgt population from Maharashtra and Uttar Pradesh, respectively. The population structure analysis clustered 29 Pgt pathotypes into two subpopulations and an admixture. Our results demonstrated that there was significant genetic diversity among Pgt pathotypes resulting from their long-distance dispersal ability complemented by gene flow. These findings provide insights into the virulence patterns, genetic variations, and possible evolution of Pgt pathotypes, which would support strategic stem rust resistance breeding.

Advances in genomics and genome editing for breeding next generation of fruit and nut crops.

Fruit tree crops are an essential part of the food production systems and are key to achieve food and nutrition security. Genetic improvement of fruit trees by conventional breeding has been slow due to the long juvenile phase. Advancements in genomics and molecular biology have paved the way for devising novel genetic improvement tools like genome editing, which can accelerate the breeding of these perennial crops to a great extent. In this article, advancements in genomics of fruit trees covering genome sequencing, transcriptome sequencing, genome editing technologies (GET), CRISPR-Cas system based genome editing, potential applications of CRISPR-Cas9 in fruit tree crops improvement, the factors influencing the CRISPR-Cas editing efficiency and the challenges for CRISPR-Cas9 applications in fruit tree crops improvement are reviewed. Besides, base editing, a recently emerging more precise editing system, and the future perspectives of genome editing in the improvement of fruit and nut crops are covered.

Integrated genomic selection for rapid improvement of crops.

An increase in the rate of crop improvement is essential for achieving sustained food production and other needs of ever-increasing population. Genomic selection (GS) is a potential breeding tool that has been successfully employed in animal breeding and is being incorporated into plant breeding. GS promises accelerated breeding cycles through a rapid selection of superior genotypes. Numerous empirical and simulation studies on GS and realized impacts on improvement in the crop yields are recently being reported. For a holistic understanding of the technology, we briefly discuss the concept of genetic gain, GS methodology, its current status, advantages of GS over other breeding methods, prediction models, and the factors controlling prediction accuracy in GS. Also, integration of speed breeding and other novel technologies viz. high throughput genotyping and phenotyping technologies for enhancing the efficiency and pace of GS, followed by its prospective applications in varietal development programs is reviewed.

The progress of leaf rust research in wheat.

Leaf rust (also called brown rust) in wheat, caused by fungal pathogen Puccinia triticina Erikss. (Pt) is one of the major constraints in wheat production worldwide. Pt is widespread with diverse population structure and undergoes rapid evolution to produce new virulent races against resistant cultivars that are regularly developed to provide resistance against the prevailing races of the pathogen. Occasionally, the disease may also take the shape of an epidemic in some wheat-growing areas causing major economic losses. In the recent past, substantial progress has been made in characterizing the sources of leaf rust resistance including non-host resistance (NHR). Progress has also been made in elucidating the population biology of Pt and the mechanisms of wheat-Pt interaction. So far, ∼80 leaf rust resistance genes (Lr genes) have been identified and characterized; some of them have also been used for the development of resistant wheat cultivars. It has also been shown that a gene-for-gene relationship exists between individual wheat Lr genes and the corresponding Pt Avr genes so that no Lr gene can provide resistance unless the prevailing race of the pathogen carries the corresponding Avr gene. Several Lr genes have also been cloned and their products characterized, although no Avr gene corresponding a specific Lr gene has so far been identified. However, several candidate effectors for Pt have been identified and functionally characterized using genome-wide analyses, transcriptomics, RNA sequencing, bimolecular fluorescence complementation (BiFC), virus-induced gene silencing (VIGS), transient expression and other approaches. This review summarizes available information on different aspects of the pathogen Pt, genetics/genomics of leaf rust resistance in wheat including cloning and characterization of Lr genes and epigenetic regulation of disease resistance.

Rust pathogen effectors: perspectives in resistance breeding.

MAIN CONCLUSION: Identification and functional characterization of plant pathogen effectors promise to ameliorate future research and develop effective and sustainable strategies for controlling or containing crop diseases. Wheat is the second most important food crop of the world after rice. Rust pathogens, one of the major biotic stresses in wheat production, are capable of threatening the world food security. Understanding the molecular basis of plant-pathogen interactions is essential for devising novel strategies for resistance breeding and disease management. Now, it has been established that effectors, the proteins secreted by pathogens, play a key role in plant-pathogen interactions. Therefore, effector biology has emerged as one of the most important research fields in plant biology. Recent advances in genomics and bioinformatics have allowed identification of a large repertoire of candidate effectors, while the evolving high-throughput tools have continued to assist in their functional characterization. The repertoires of effectors have become an important resource for better understanding of effector biology of pathosystems and resistance breeding of crop plants. In recent years, a significant progress has been made in the field of rust effector biology. This review describes the recent advances in effector biology of obligate fungal pathogens, identification and functional analysis of wheat rust pathogens effectors and the potential applications of effectors in molecular plant biology and rust resistance breeding in wheat.