Fine mapping of a new common bean anthracnose resistance gene (Co-18) to the proximal end of Pv10 in Indian landrace KRC-5.

KEY MESSAGE: Mapping and fine mapping of bean anthracnose resistance genes is a continuous process. We report fine mapping of anthracnose resistance gene Co-18 which is the first anthracnose gene mapped to Pv10. The discovery of resistance gene is a major gain in the bean anthracnose pathosystem research. Among the Indian common bean landraces, KRC-5 exhibit high levels of resistance to the bean anthracnose pathogen Colletotrichum lindemuthianum. To precisely map the anthracnose resistance gene, we used a Recombinant Inbred Line (F2:9 RIL) population (KRC-5 × Jawala). The inheritance test revealed that KRC-5 carries a dominant resistance gene temporarily designated as Co-18. We discovered two RAPD markers linked to Co-18 among 287 RAPD markers. These RAPD markers were eventually developed into SCARs (Sc-OPR15 and Sc-OPF6) and flank Co-18 on chromosome Pv10 at a distance of 5.3 and 4.2 cM, respectively. At 4.0-4.1 Mb on Pv10, we detected a SNP (single-nucleotide polymorphism) signal. We synthesized 58 SSRs and 83 InDels from a pool of 135 SSRs and 1134 InDels, respectively. Five SSRs, four InDels, and two SCARs were used to generate the high-density linkage map, which led to the identification of two SSRs (SSR24 and SSR36) that are tightly linked to Co-18. These two SSRs flank the Co-18 to 178 kb genomic region with 13 candidate genes including five NLR (nucleotide-binding and leucine-rich repeat) genes. The closely linked markers SSR24 and SSR36 will be used in cloning and pyramiding of the Co-18 gene with other R genes to develop durable resistant bean varieties.

Mapping of quantitative trait loci for scab resistance in apple (Malus × domestica) variety, Shireen.

BACKGROUND: Scab caused by Venturia inaequalis (Cke.) Wint. is the most important fungal disease of apple. Fungicide application is a widely practiced method of disease control. However, the use of chemicals is costintensive, tedious, and ecologically unsafe. The development of genetic resistance and the breeding of resistant cultivars is the most reliable and safest option. One such source of scab resistance happens to be the variety 'Shireen', released from SKUAST-Kashmir. However, to date, the nature of resistance and its genetic control have not been characterized. Objective This research aimed to elucidate the genetic basis of scab resistance in Shireen. METHODS: Genetic mapping of quantitative trait loci (QTL) for resistance to apple scab disease was performed using an F1 cross developed between the susceptible cultivar 'StarKrimson' and the resistant cultivar 'Shireen'. The population was evaluated for two consecutive years. Further, six candidate genes were analyzed via quantitative real-time PCR, to determine their expression level in response to the pathogen infestation. RESULTS: Genotyping and disease phenotyping of populations led us to identify two quantitative trait loci (QTLs), namely qRVI.SS-LG2.2019 and qRVI.SS-LG8.2019 on chromosomes 2 and 8 with LOD-values of 7.67 and 4.99 respectively, and six potential CDGs for the polygenic resistance in 'Shireen'. The genomic region corresponding to the mapped QTLs in LG 2 and LG 8 of 'Shireen' was examined for candidate genes possibly related to scab resistance using in silico analysis. CONCLUSION: The QTLs mapped in the genetic background of Shireen are the novel QTLs and may be transferred to desirable genetic backgrounds and provide opportunities for isolation and cloning of genes apart from their utility to achieve durable resistance to scab.

DNA marker based diversity across rice genotypes and advanced breeding lines bred for temperate regions of North-West India.

BACKGROUND: Characterization and evaluation of plant genetic resources play an important role for their utilization in the crop improvement programmes. METHODS AND RESULTS: This study involves the agro-morphological and cooking quality besides, molecular characterization of 51 genotypes/advance breeding lines of rice from Kashmir Himalayas. Significant variability was observed for all agro-morphological and cooking quality traits among all the studied genotypes. Cluster analysis using UPGMA method divided the genotypes into two major clusters having 15 and 36 genotypes. Thirty eight genotypes screened using 24 SSR markers detected 48 alleles with 2.0 alleles for each locus with average polymorphism information content (PIC) of 0.37. High polymorphism information content (PIC) values was observed for the primers RM263 (0.67), RM159 (0.59) and RM333 (0.50). Furthermore, out of 38 SSR markers screened on 192 temperate rice germpalsm lines, R4M17 accurately differentiated indica and temperate japonica genotypes amplifying 220 bp and 169 bp, respectively. Accordingly, 15 genotypes were reported as indica and 28 temperate japonica in addition to 149 genotypes as intermediate types. CONCLUSION: The information on marker-based diversity and performance based on cooking quality and agronomic traits helped to select the most divergent lines for crossing. Also the analysis was useful to classify the temperate germplasm into indica and temperate japonica. The classification could be helpful to devise a strategy for inter-sub species hybridization to breed for improved rice varieties.

Molecular phylogeny, pathogenic variability and phytohormone production of Fusarium species associated with bakanae disease of rice in temperate agro-ecosystems.

Bakanae is the emerging disease threating the rice cultivation globally. Yield reduction of 4-70% is recorded in different parts of the world. A total of 119 Fusarium isolates were collected from rice plants at different geographical locations and seeds of different rice cultivars. The isolates were evaluated for morphological, biochemical and pathogenic diversity. The amplification of TEF-1α gene was carried out for exploring the species spectrum associated with the cultivated and pre-released rice varieties. The production of gibberellin varied from 0.53 to 2.26 µg/25 ml, while as that of Indole acetic acid varied from 0.60 to 3.15 µg/25 ml among the Fusarium isolates. The phylogenetic analysis identified 5 different species of the genus Fusarium viz. Fusarium fujikuroi, F. proliferatum, F. equiseti, F.oxysporum and F. persicinum after nucleotide blasting in NCBI. Only two Fusarium spp. F. fujikuroi and F. proliferatum were found to be pathogenic under virulence assays of the isolates. The isolates showed a considerable variation in morphological and pathogenic characters. The isolates were divided into different groups based on morphology and pathogenicity tests. The isolates showed a considerable variation in morphology, phytohormone profile and virulence indicative of population diversity. Three species F. equiseti, F.oxysporum and F. persicinum which have not been reported as pathogens of rice in India were found to be associated with bakanae disease of rice, however their pathogenicity could not be established.

Development and evaluation of near-isogenic lines for major blast resistance gene(s) in Basmati rice.

KEY MESSAGE: A set of NILs carrying major blast resistance genes in a Basmati rice variety has been developed. Also, the efficacy of pyramids over monogenic NILs against rice blast pathogen Magnaporthe oryzae has been demonstrated. Productivity and quality of Basmati rice is severely affected by rice blast disease. Major genes and QTLs conferring resistance to blast have been reported only in non-Basmati rice germplasm. Here, we report incorporation of seven blast resistance genes from the donor lines DHMASQ164-2a (Pi54, Pi1, Pita), IRBLz5-CA (Pi2), IRBLb-B (Pib), IRBL5-M (Pi5) and IRBL9-W (Pi9) into the genetic background of an elite Basmati rice variety Pusa Basmati 1 (PB1). A total of 36 near-isogenic lines (NILs) comprising of 14 monogenic, 16 two-gene pyramids and six three-gene pyramids were developed through marker-assisted backcross breeding (MABB). Foreground, recombinant and background selection was used to identify the plants with target gene(s), minimize the linkage drag and increase the recurrent parent genome (RPG) recovery (93.5-98.6 %), respectively, in the NILs. Comparative analysis performed using 50,051 SNPs and 500 SSR markers revealed that the SNPs provided better insight into the RPG recovery. Most of the monogenic NILs showed comparable performance in yield and quality, concomitantly, Pusa1637-18-7-6-20 (Pi9), was significantly superior in yield and stable across four different environments as compared to recurrent parent (RP) PB1. Further, among the pyramids, Pusa1930-12-6 (Pi2+Pi5) showed significantly higher yield and Pusa1633-7-8-53-6-8 (Pi54+Pi1+Pita) was superior in cooking quality as compared to RP PB1. The NILs carrying gene Pi9 were found to be the most effective against the concoction of virulent races predominant in the hotspot locations for blast disease. Conversely, when analyzed under artificial inoculation, three-gene pyramids expressed enhanced resistance as compared to the two-gene and monogenic NILs.

Advances and opportunities in unraveling cold-tolerance mechanisms in the world's primary staple food crops.

Temperatures below or above optimal growth conditions are among the major stressors affecting productivity, end-use quality, and distribution of key staple crops including rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mays L.). Among temperature stresses, cold stress induces cellular changes that cause oxidative stress and slowdown metabolism, limit growth, and ultimately reduce crop productivity. Perception of cold stress by plant cells leads to the activation of cold-responsive transcription factors and downstream genes, which ultimately impart cold tolerance. The response triggered in crops to cold stress includes gene expression/suppression, the accumulation of sugars upon chilling, and signaling molecules, among others. Much of the information on the effects of cold stress on perception, signal transduction, gene expression, and plant metabolism are available in the model plant Arabidopsis but somewhat lacking in major crops. Hence, a complete understanding of the molecular mechanisms by which staple crops respond to cold stress remain largely unknown. Here, we make an effort to elaborate on the molecular mechanisms employed in response to low-temperature stress. We summarize the effects of cold stress on the growth and development of these crops, the mechanism of cold perception, and the role of various sensors and transducers in cold signaling. We discuss the progress in cold tolerance research at the genome, transcriptome, proteome, and metabolome levels and highlight how these findings provide opportunities for designing cold-tolerant crops for the future.

Genome-wide SNP discovery and genotyping delineates potential QTLs underlying major yield-attributing traits in buckwheat.

Buckwheat (Fagopyrum spp.) is an important nutritional and nutraceutical-rich pseudo-cereal crop. Despite its obvious potential as a functional food, buckwheat has not been fully harnessed due to its low yield, self-incompatibility, increased seed cracking, limited seed set, lodging, and frost susceptibility. The inadequate availability of genomics resources in buckwheat is one of the major reasons for this. In the present study, genome-wide association mapping (GWAS) was conducted to identify loci associated with various morphological and yield-related traits in buckwheat. High throughput genotyping by sequencing led to the identification of 34,978 single nucleotide polymorphisms that were distributed across eight chromosomes. Population structure analysis grouped the genotypes into three sub-populations. The genotypes were also characterized for various qualitative and quantitative traits at two diverse locations, the analysis of which revealed a significant difference in the mean values. The association analysis revealed a total of 71 significant marker-trait associations across eight chromosomes. The candidate genes were identified near 100 Kb of quantitative trait loci (QTLs), providing insights into several metabolic and biosynthetic pathways. The integration of phenology and GWAS in the present study is useful to uncover the consistent genomic regions, related markers associated with various yield-related traits, and potential candidate genes having implications for being utilized in molecular breeding for the improvement of economically important traits in buckwheat. Moreover, the identified QTLs will assist in tracking the desirable alleles of target genes within the buckwheat breeding populations/germplasm.

Characterization of Pre-Breeding Wheat (Triticum aestivum L.) Germplasm for Stripe Rust Resistance Using Field Phenotyping and Genotyping.

Wheat is highly affected by stripe rust disease, particularly under cooler environments, and the losses can reach up to 100 percent depending on the intensity of infection and the susceptibility of the genotype. The most effective method to manage this disease is the use of resistant varieties. In the present study, 192 wheat genotypes were evaluated for stripe rust resistance under field conditions and also in a laboratory using molecular markers. These lines included pre-breeding germplasm developed for rust resistance and some high-yielding commercially grown wheat varieties. Out of 192 genotypes, 53 were found to be resistant, and 29 showed moderate resistance reaction under field conditions, whereas the remaining genotypes were all either moderately susceptible or susceptible. Under controlled conditions, out of 109 genotypes, only 12 were found to be resistant to all the six virulent/pathogenic pathotypes. Additionally, a selection of 97 genotypes were found resistant in field screening and were subjected to molecular validation using the markers linked to major R-genes, viz., Yr5, Yr10, Yr15 and Yr17. Nine genotypes possessed the Yr5 gene, twelve had the Yr10 gene, fourteen had the Yr15 gene and thirty-two had the Yr17 gene. The resistance genes studied in the current study are effective in conferring resistance against stripe rust disease. The genotypes identified as resistant under both field and controlled conditions can be used as sources in stripe rust resistance breeding programs.

Explicating genetic architecture governing nutritional quality in pigmented rice.

Rice is one of the most important staple plant foods that provide a major source of calories and nutrients for tackling the global hunger index especially in developing countries. In terms of nutritional profile, pigmented rice grains are favoured for their nutritional and health benefits. The pigmented rice varieties are rich sources of flavonoids, anthocyanin and proanthocyanidin that can be readily incorporated into diets to help address various lifestyle diseases. However, the cultivation of pigmented rice is limited due to low productivity and unfavourable cooking qualities. With the advances in genome sequencing, molecular breeding, gene expression analysis and multi-omics approaches, various attempts have been made to explore the genetic architecture of rice grain pigmentation. In this review, we have compiled the current state of knowledge of the genetic architecture and nutritional value of pigmentation in rice based upon the available experimental evidence. Future research areas that can help to deepen our understanding and help in harnessing the economic and health benefits of pigmented rice are also explored.

Combination of Strobilurin and Triazole Chemicals for the Management of Blast Disease in Mushk Budji -Aromatic Rice.

Rice blast is considered one of the most important fungal diseases of rice. Although diseases can be managed by using resistant cultivars, the blast pathogen has successfully overcome the single gene resistance in a short period and rendered several varieties susceptible to blast which were otherwise intended to be resistant. As such, chemical control is still the most efficient method of disease control for reducing the losses caused due to diseases. Field experiments were conducted over two successive years, 2018 and 2019, in temperate rice growing areas in northern India. All the fungicides effectively reduced leaf blast incidence and intensity, and neck blast incidence under field conditions. Tricyclazole proved most effective against rice blast and recorded a leaf blast incidence of only 8.41%. Among the combinations of fungicides, azoxystrobin + difenoconazole and azoxystrobin + tebuconazole were highly effective, recording a leaf blast incidence of 9.19 and 10.40%, respectively. The chemical combination mancozeb + carbendazim proved less effective in controlling the blast and it recorded a disease incidence of 27.61%. A similar trend was followed in neck blast incidence with tricyclazole, azoxystrobin + difenoconazole, and azoxystrobin + tebuconazole showing the highest levels of blast reductions. It is evident from the current study that the tested fungicide combinations can be used as alternatives to tricyclazole which is facing the challenges of fungicide resistance development and other environmental concerns and has been banned from use in India and other countries. The manuscript may provide a guideline of fungicide application to farmers cultivating susceptible varieties of rice.

Phylogeny and Optimization of Trichoderma harzianum for Chitinase Production: Evaluation of Their Antifungal Behaviour against the Prominent Soil Borne Phyto-Pathogens of Temperate India.

Trichoderma is the most commonly used fungal biocontrol agent throughout the world. In the present study, various Trichoderma isolates were isolated from different vegetable fields. In the isolated microflora, the colony edges varied from wavy to smooth. The mycelial forms were predominantly floccose with hyaline color and conidiophores among all the strains were highly branched. Based on morphological attributes, all the isolates were identified as Trichoderma harzianum. The molecular identification using multilocus sequencing ITS, rpb2 and tef1α, genes further confirmed the morphological identification. The average chitinase activity varied from 1.13 units/mL to 3.38 units/mL among the various isolates, which increased linearly with temperature from 15 to 30 °C. There was an amplified production in the chitinase production in the presence of Mg+ and Ca2+ and Na+ metal ions, but the presence of certain ions was found to cause the down-regulated chitinase activity, i.e., Zn2+, Hg2+, Fe2+, Ag+ and K+. All the chitinase producing Trichoderma isolates inhibited the growth of tested pathogens viz., Dematophora necatrix, Fusarium solani, Fusarium oxysporum and Pythium aphanidermatum at 25% culture-free filtrate concentration under in vitro conditions. Also, under in vivo conditions, the lowest wilt incidence and highest disease control on Fusarium oxysporum was observed in isolate BT4 with mean wilt incidence and disease control of 21% and 48%, respectively. The Trichoderma harzianum identified in this study will be further used in formulation development for the management of diseases under field conditions.

Improved protocol for efficacious in vitro androgenesis and development of doubled haploids in temperate japonica rice.

DH (Doubled haploid) is the immortal mapping population and an outcome of single meiotic cycle, contributed from male partner. An improved procedure was developed for high frequency androgenesis in japonica genotypes, K-332 and GS-88 and their F1s. A total of 207 fertile, green, di-haploid plants were generated from K-332 × GS-88 hybrids using the improved anther culture protocol. The investigation was carried out to evaluate callus induction potential and regeneration response for the genotypes and the derived F1s on N6 media and modified N6 media (N6M). Whereas, N6 failed to induce callusing, agarose solidified N6M media supplemented with 4% maltose, growth regulators; NAA (2 mg/l), 2, 4-D (0.5 mg/l), Kinetin (0.5 mg/l), and silver nitrate induced high calli percentage of 27.6% in F1s, 9.5% and 6.7% in GS-88 and K-332 respectively. Murashige and Skoog (MS) media supplemented with 3% sucrose, and the hormonal combination BAP (2 mg/l), Kinetin (1 mg/l) and NAA (1 mg/l) induced high green shoot regeneration rates (0-60.0%). The effect of cold pre-treatment at 4°C and the stage of anther collection and their interaction was studied. The effect of cold pre-treatment (CP) of collected boots at 4°C (for CP2: 2, CP4: 4, CP6: 6 and CP8: 8 days) at different stages of panicle emergence (BES4-6: 4-6, BES7-10: 7-10, BES11-13: 11-13, BES>13: more than 13 inches was worked out in relation to the effect on response of calli induction, albino regeneration, green plant regeneration and number of shoots/green calli. CP referred to the number of days for which the collected boots were incubated before they were inoculated. BES was the length (inches) between flag leaf and penultimate leaf at the time of boot collection. We concluded that CP6 and BES7-10 showed better response to callus proliferation and regeneration of plantlets across genotypes. The appropriate pre-treatment, stage of anther collection and favourable media composition resulted in high calli induction and green plant regeneration rates in recalcitrant japonica genotypes. The modified N6 media resulted into efficient callus induction and is expected to be useful for studies which aim at rapid generation of mapping populations for genetic studies.

Harnessing Genome Editing Techniques to Engineer Disease Resistance in Plants.

Modern genome editing (GE) techniques, which include clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and LAGLIDADG homing endonucleases (meganucleases), have so far been used for engineering disease resistance in crops. The use of GE technologies has grown very rapidly in recent years with numerous examples of targeted mutagenesis in crop plants, including gene knockouts, knockdowns, modifications, and the repression and activation of target genes. CRISPR/Cas9 supersedes all other GE techniques including TALENs and ZFNs for editing genes owing to its unprecedented efficiency, relative simplicity and low risk of off-target effects. Broad-spectrum disease resistance has been engineered in crops by GE of either specific host-susceptibility genes (S gene approach), or cleaving DNA of phytopathogens (bacteria, virus or fungi) to inhibit their proliferation. This review focuses on different GE techniques that can potentially be used to boost molecular immunity and resistance against different phytopathogens in crops, ultimately leading to the development of promising disease-resistant crop varieties.