Characterization and identification of sources of rust resistance in Triticum militinae derivatives.

Triticum militinae (2n = 4X = 28, AtAtGG), belonging to the secondary gene pool of wheat, is known to carry resistance to many diseases. Though some disease resistance genes were reported from T. timopheevii, the closest wild relative of T. militinae, there are no reports from T. militinae. Twenty-one T. militinae Derivatives (TMD lines) developed at the Division of Genetics, IARI, New Delhi, were evaluated for leaf and stripe rusts at seedling and adult plant stages. Eight TMD lines (6-4, 6-5, 11-6, 12-4, 12-8, 12-12, 13-7 and 13-9) showed seedling resistance to both leaf and stripe rusts while six TMD lines (7-5, 7-6, 11-5, 13-1, 13-3 and 13-4) showed seedling resistance to leaf rust but adult plant resistance to stripe rust and three TMD lines (9-1, 9-2 and 15) showed seedling resistance to leaf rust but susceptibility to stripe rust. Three TMD lines (2-7, 2-8 and 6-1) with adult plant resistance to leaf and stripe rusts were found to carry the known gene Lr34/Yr18. Ten TMD lines (7-5, 7-6, 9-1, 9-2, 11-5, 11-6, 12-12, 12-4, 12-8, and 15) with seedling resistance to leaf rust, showing absence of known genes Lr18 and Lr50 with linked markers requires further confirmation by the test of allelism studies. As not a single stripe rust resistance gene has been reported from T. militinae or its close relative T. timpopheevii, all the 8 TMD lines (6-4, 6-5, 11-6,12-4, 12-8, 12-12, 13-7 and 13-9) identified of carrying seedling resistance to stripe rust and 3 TMD lines (13-1, 13-3 and 13-4) identified of carrying adult plant resistance to stripe rust are expected to carry unknown genes. Also, all the TMD lines were found to be cytologically stable and thus can be used in inheritance and mapping studies.

Simple cryopreservation protocol for Luffa pollen: enhancing breeding efficiency.

This study aimed to develop a long-term pollen storage protocol for Luffa species (L. acutangula, L. cylindrica, L. echinata, and L. graveolens) and assess its potential for crop improvement. The optimal medium for in vitro pollen germination varied by species, with Brewbaker and Kwack (BK) medium with 10% sucrose suitable for L. acutangula, L. cylindrica, and L. echinata, and BK medium with 3% sucrose ideal for L. graveolens. Overestimation in staining tests compared to in vitro pollen germination was observed. The best results for cryopreservation were achieved with desiccation periods of 20, 30, and 40 min, maintaining moisture content between 14.04% and 18.55%. Pollen viability was negatively correlated with storage temperature (25, 4, and -20°C) and duration. Cryopreserved pollen at -196°C exhibited the highest viability over a prolonged period (2 months) and was comparable to fresh pollen in terms of germination, ovule fertilization, and fruit and seed set. This study presents a simple and reproducible pollen cryopreservation protocol applicable across Luffa species, facilitating long-term storage and its use in crop improvement efforts.

Leaf rolling in bread wheat (Triticum aestivum L.) is controlled by the upregulation of a pair of closely linked/duplicate zinc finger homeodomain class transcription factors during moisture stress conditions.

Zinc finger-homeodomain (ZF-HDs) class IV transcriptional factors (TFs) is a plant-specific transcription factor and play a key role in stress responses, plant growth, development, and hormonal signaling. In this study, two new leaf rolling TFs genes, namely TaZHD1 and TaZHD10, were identified in wheat using comparative genomic analysis of the target region that carried a major QTL for leaf rolling identified through multi-environment phenotyping and high throughput genotyping of a RIL population. Structural and functional annotation of the candidate ZHD genes with its closest rice orthologs reflects the species-specific evolution and, undoubtedly, validates the notions of remote-distance homology concept. Meanwhile, the morphological analysis resulted in contrasting difference for leaf rolling in extreme RILs between parental lines HD2012 and NI5439 at booting and heading stages. Transcriptome-wide expression profiling revealed that TaZHD10 transcripts showed significantly higher expression levels than TaZHD1 in all leaf tissues upon drought stress. The relative expression of these genes was further validated by qRT-PCR analysis, which also showed consistent results across the studied genotypes at the booting and anthesis stage. The contrasting modulation of these genes under drought conditions and the available evidenced for its epigenetic behavior that might involve the regulation of metabolic and gene regulatory networks. Prediction of miRNAs resulted in five Tae-miRs that could be associated with RNAi mediated control of TaZHD1 and TaZHD10 putatively involved in the metabolic pathway controlling rolled leaf phenotype. Gene interaction network analysis indicated that TaZHD1 and TaZHD10 showed pleiotropic effects and might also involve other functions in wheat in addition to leaf rolling. Overall, the results increase our understanding of TaZHD genes and provide valuable information as robust candidate genes for future functional genomics research aiming for the breeding of wheat varieties tolerant to leaf rolling.

Genome-wide characterization and identification of cyclophilin genes associated with leaf rust resistance in bread wheat (Triticum aestivum L.).

Cyclophilins (CYPs) are a group of highly conserved proteins involved in host-pathogen interactions in diverse plant species. However, the role of CYPs during disease resistance in wheat remains largely elusive. In the present study, the systematic genome-wide survey revealed a set of 81 TaCYP genes from three subfamilies (GI, GII, and GIII) distributed on all 21 wheat chromosomes. The gene structures of TaCYP members were found to be highly variable, with 1-14 exons/introns and 15 conserved motifs. A network of miRNA targets with TaCYPs demonstrated that TaCYPs were targeted by multiple miRNAs and vice versa. Expression profiling was done in leaf rust susceptible Chinese spring (CS) and the CS-Ae. Umbellulata derived resistant IL "Transfer (TR). Three homoeologous TaCYP genes (TaCYP24, TaCYP31, and TaCYP36) showed high expression and three homoeologous TaCYP genes (TaCYP44, TaCYP49, and TaCYP54) showed low expression in TR relative to Chinese Spring. Most of the other TaCYPs showed comparable expression changes (down- or upregulation) in both contrasting TR and CS. Expression of 16 TaCYPs showed significant association (p < 0.05) with superoxide radical and hydrogen peroxide abundance, suggesting the role of TaCYPs in downstream signaling processes during wheat-leaf rust interaction. The differentially expressing TaCYPs may be potential targets for future validation using transgenic (overexpression, RNAi or CRISPR-CAS) approaches and for the development of leaf rust-resistant wheat genotypes.

Identification of Novel Broad-Spectrum Leaf Rust Resistance Sources from Khapli Wheat Landraces.

Wheat leaf rust caused by Puccinia triticina Eriks is an important disease that causes yield losses of up to 40% in susceptible varieties. Tetraploid emmer wheat (T. turgidum ssp. Dicoccum), commonly called Khapli wheat in India, is known to have evolved from wild emmer (Triticum turgidum var. dicoccoides), and harbors a good number of leaf rust resistance genes. In the present study, we are reporting on the screening of one hundred and twenty-three dicoccum wheat germplasm accessions against the leaf rust pathotype 77-5. Among these, an average of 45.50% of the germplasms were resistant, 46.74% were susceptible, and 8.53% had mesothetic reactions. Further, selected germplasm lines with accession numbers IC138898, IC47022, IC535116, IC535133, IC535139, IC551396, and IC534144 showed high level of resistance against the eighteen prevalent pathotypes. The infection type varied from ";", ";N", ";N1" to ";NC". PCR-based analysis of the resistant dicoccum lines with SSR marker gwm508 linked to the Lr53 gene, a leaf rust resistance gene effective against all the prevalent pathotypes of leaf rust in India and identified from a T. turgidum var. dicoccoides germplasm, indicated that Lr53 is not present in the selected accessions. Moreover, we have also generated 35K SNP genotyping data of seven lines and the susceptible control, Mandsaur Local, to study their relationships. The GDIRT tool based on homozygous genotypic differences revealed that the seven genotypes are unique to each other and may carry different resistance genes for leaf rust.

Marker-assisted transfer of leaf and stripe rust resistance from Triticum turgidum var. durum cv. Trinakria to wheat variety HD2932.

A marker-assisted backcrossing program initiated to transfer leaf rust resistance gene LrTrk from Triticum turgidum cv. Trinakria to hexaploid wheat variety HD2932 cotransferred a stripe rust resistance gene, YrTrk, along with LrTrk. The cross of hexaploid recurrent parent HD2932 with tetraploid donor parent Trinakria produced pentaploid F1 plants. F1s were backcrossed with recurrent parent HD2932 to produce BC1F1 generation. Foreground and background selection was conducted in each backcross generation to identify plants for backcrossing or selfing. While foreground selection for LrTrk was carried out with linked and validated molecular marker Xgwm234, for background selection, 86 polymorphic SSR markers from the A and B genomes were used. Single selected plants from BC1F1 and BC2F1 generations backcrossed and selfed to produce BC2F1and BC2F2 generations, respectively. Background selection resulted in 83.72%, 91.86%, and 98.25% of RPG recovery in BC1F1, BC2F1, and BC2F2 generations, respectively. A total of 27 plants with LrTrk in homozygous state were identified in BC2F2 generation and selfed to produce 27 BC2F3 NILs. All the NILs were tested for leaf and stripe rust resistance at the seedling stage using seven Puccinia triticina and one Puccinia striiformis f.sp. tritici rust pathotypes. All the 27 NILs were found to be resistant to both leaf and stripe rust pathotypes. So, these NILs are designated to carry leaf and stripe rust resistance genes LrTrk/YrTrk.

Marker-Assisted Improvement of Bread Wheat Variety HD2967 for Leaf and Stripe Rust Resistance.

The mega wheat variety HD2967 was improved for leaf and stripe rust resistance by marker-assisted backcross breeding. After its release in 2011, HD2967 became susceptible to stripe rust and moderately susceptible to leaf rust. The leaf rust resistance gene LrTrk was transferred into HD2967 from the durum wheat genotype Trinakria. Then, HD2967 was crossed with Trinakria to produce F1 plant foreground selection for LrTrk and background selection for the recurrent parent genotype was carried out in BC1F1, BC2F1 and BC2F2 generations. Foreground selection was carried out with the linked marker Xgwm234, while polymorphic SSR markers between parents were used for background selection. Background selection resulted in the rapid recovery of the recurrent parent genome. A morphological evaluation of 6 near isogenic lines (NILs)-2 resistant to leaf and stripe rust, and 4 resistant to leaf rust only-showed no significant differences in yields among NILs and the recurrent parent HD2967. All of the 6 NILs showed the presence of 2NS/2AS translocation, carrying the linked genes Lr37/Sr38/Yr17 present in HD2967 and the targeted leaf rust resistance gene LrTrk. Two NILs also showed additional resistance to stripe rust. Therefore, these NILs with rust resistance and an at par yielding ability of H2967 can replace the susceptible cultivar HD2967 to reduce yield losses due to disease.

Genome-Wide Identification and Expression Analysis of the Thioredoxin (Trx) Gene Family Reveals Its Role in Leaf Rust Resistance in Wheat (Triticum aestivum L.).

Bread wheat (Triticum aestivum L.; Ta) is the staple cereal crop for the majority of the world's population. Leaf rust disease caused by the obligate fungal pathogen, Puccinia triticina L., is a biotrophic pathogen causing significant economic yield damage. The alteration in the redox homeostasis of the cell caused by various kinds of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to pathogenic infections is controlled by redox regulators. Thioredoxin (Trx) is one of the redox regulators with low molecular weight and is thermostable. Through a genome-wide approach, forty-two (42) wheat Trx genes (TaTrx) were identified across the wheat chromosome groups A, B, and D genomes containing 12, 16, and 14 Trx genes, respectively. Based on in silico expression analysis, 15 TaTrx genes were selected and utilized for further experimentation. These 15 genes were clustered into six groups by phylogenetic analysis. MicroRNA (miRNA) target analysis revealed eight different miRNA-targeted TaTrx genes. Protein-protein interaction (PPI) analysis showed TaTrx proteins interact with thioredoxin reductase, peroxiredoxin, and uncharacterized proteins. Expression profiles resulting from quantitative real-time PCR (qRT-PCR) revealed four TaTrx genes (TaTrx11-5A, TaTrx13-5B, TaTrx14-5D, and TaTrx15-3B) were significantly induced in response to leaf rust infection. Localization of ROS and its content estimation and an assay of antioxidant enzymes and expression analysis suggested that Trx have been involved in ROS homeostasis at span 24HAI-72HAI during the leaf rust resistance.

Analysis of NIA and GSNOR family genes and nitric oxide homeostasis in response to wheat-leaf rust interaction.

Nitric oxide (NO) modulates plant response to biotic and abiotic stresses by S-nitrosylation-mediated protein post-translational modification. Nitrate reductase (NR) and S-nitrosoglutathione reductase (GSNOR) enzymes are essential for NO synthesis and the maintenance of Nitric oxide/S-nitroso glutathione (NO/GSNO) homeostasis, respectively. S-nitrosoglutathione, formed by the S-nitrosylation reaction of NO with glutathione, plays a significant physiological role as the mobile reservoir of NO. The genome-wide analysis identified nine NR (NIA) and three GSNOR genes in the wheat genome. Phylogenic analysis revealed that the nine NIA genes +were clustered into four groups and the 3 GSNORs into two groups. qRT-PCR expression profiling of NIAs and GSNORs was done in Chinese spring (CS), a leaf rust susceptible wheat line showing compatible interaction, and Transfer (TR), leaf rust-resistant wheat line showing incompatible interaction, post-inoculation with leaf rust pathotype 77-5 (121-R-63). All the NIA genes showed upregulation during incompatible interaction in comparison with the compatible reaction. The GSNOR genes showed a variable pattern of expression: the TaGSNOR1 showed little change, whereas TaGSNOR2 showed higher expression during the incompatible response. TaGSNOR3 showed a rise of expression both in compatible and incompatible reactions. Before inoculation and after 72 h of pathogen inoculation, NO localization was studied in both compatible and incompatible reactions. The S-nitrosothiol accumulation, NR, and glutathione reductase activity showed a consistent increase in the incompatible interactions. The results demonstrate that both NR and GSNOR plays significant role in defence against the leaf rust pathogen in wheat by modulating NO homeostasis or signalling.

Molecular mapping of a new recessive wheat leaf rust resistance gene originating from Triticum spelta.

TSD276-2, a wheat genetic stock derived from the cross Agra Local/T. spelta 276 showed broad spectrum resistance against leaf rust pathogen. Genetic analysis was undertaken using F1, F2, F2:3 and BC1F1 generations derived from the cross TSD276-2/Agra Local. The results revealed a single recessive gene for leaf rust resistance, tentatively named as LrTs276-2, in TSD276-2. Molecular mapping of leaf rust resistance gene LrTs276-2 in TSD276-2 was done using SNP-based PCR and SSR markers. For Bulked Segregant Analysis (BSA), two bulks viz. resistant bulk and susceptible bulk, and the parents TSD276-2 and Agra Local were genotyped for SNPs using AFFYMETRIX 35K Wheat Breeders' AXIOM array. T. spelta 276 was also genotyped and used as a check. BSA indicated that the gene for leaf rust resistance in TSD276-2 is located on chromosome arm 1DS. Putatively linked SNPs on chromosome arm 1DS were converted into PCR-based markers. Polymorphic SSR markers on chromosome arm 1DS were also identified. Final linkage map was constructed using one SNP-based PCR and three SSR markers. The rust reaction and chromosomal location suggest that LrTs276-2 is a new leaf rust resistance gene which may be useful in broadening the genetic base of leaf rust resistance in wheat.

QTL detection and putative candidate gene prediction for leaf rolling under moisture stress condition in wheat.

Leaf rolling is an important mechanism to mitigate the effects of moisture stress in several plant species. In the present study, a set of 92 wheat recombinant inbred lines derived from the cross between NI5439 × HD2012 were used to identify QTLs associated with leaf rolling under moisture stress condition. Linkage map was constructed using Axiom 35 K Breeder's SNP Array and microsatellite (SSR) markers. A linkage map with 3661 markers comprising 3589 SNP and 72 SSR markers spanning 22,275.01 cM in length across 21 wheat chromosomes was constructed. QTL analysis for leaf rolling trait under moisture stress condition revealed 12 QTLs on chromosomes 1B, 2A, 2B, 2D, 3A, 4A, 4B, 5D, and 6B. A stable QTL Qlr.nhv-5D.2 was identified on 5D chromosome flanked by SNP marker interval AX-94892575-AX-95124447 (5D:338665301-5D:410952987). Genetic and physical map integration in the confidence intervals of Qlr.nhv-5D.2 revealed 14 putative candidate genes for drought tolerance which was narrowed down to six genes based on in-silico analysis. Comparative study of leaf rolling genes in rice viz., NRL1, OsZHD1, Roc5, and OsHB3 on wheat genome revealed five genes on chromosome 5D. Out of the identified genes, TraesCS5D02G253100 falls exactly in the QTL Qlr.nhv-5D.2 interval and showed 96.9% identity with OsZHD1. Two genes similar to OsHB3 viz. TraesCS5D02G052300 and TraesCS5D02G385300 exhibiting 85.6% and 91.8% identity; one gene TraesCS5D02G320600 having 83.9% identity with Roc5 gene; and one gene TraesCS5D02G102600 showing 100% identity with NRL1 gene were also identified, however, these genes are located outside Qlr.nhv-5D.2 interval. Hence, TraesCS5D02G253100 could be the best potential candidate gene for leaf rolling and can be utilized for improving drought tolerance in wheat.

A novel leaf rust resistance gene introgressed from Aegilops markgrafii maps on chromosome arm 2AS of wheat.

KEY MESSAGE: A novel leaf rust resistance gene, LrM, introgressed from Aegilops markgrafii and mapped on chromosome 2AS using SSR- and SNP-based PCR markers will aid in broadening the genetic base of rust resistance in wheat. A new leaf rust resistance gene tentatively named LrM was introgressed from the diploid non-progenitor species Ae. markgrafii (2n = 2x = 14, genome CC) into common wheat using the nulli-5B mechanism. The introgression line ER9-700 showed a high degree of resistance against a wide spectrum of Puccinia triticina pathotypes. Genetic analysis was performed using the F1, F2, F2:3 and BC1F1 generations derived from the cross ER9-700/Agra Local. The results showed a single dominant gene for leaf rust resistance. The resistance gene LrM was mapped on chromosome arm 2AS using SSR- and SNP-based PCR markers. Preliminary mapping with SSR markers in the F2:3 population from the cross ER9-700/Agra Local identified two SSR markers flanking the LrM. SNPs were identified in the genomic region flanked by SSR markers, and SNP-based PCR markers were developed to construct the final map. Three SNP-based PCR markers co-segregated and mapped closest to the resistance gene at a distance of 2 cM. The gene LrM was distinguished from all the other genes designated and mapped on chromosome arm 2AS by molecular markers and rust reaction. All five markers used in the mapping amplified identical alleles in the donor Ae. markgrafii accession and introgression line ER9-700. The chromosomal location and rust reaction suggest that LrM is a novel leaf rust resistance gene that may be useful in broadening the genetic base of leaf rust resistance in wheat.

Cytogenetic analysis and mapping of leaf rust resistance in Aegilops speltoides Tausch derived bread wheat line Selection2427 carrying putative gametocidal gene(s).

Leaf rust (Puccinia triticina) is a major biotic stress affecting wheat yields worldwide. Host-plant resistance is the best method for controlling leaf rust. Aegilops speltoides is a good source of resistance against wheat rusts. To date, five Lr genes, Lr28, Lr35, Lr36, Lr47, and Lr51, have been transferred from Ae. speltoides to bread wheat. In Selection2427, a bread wheat introgresed line with Ae. speltoides as the donor parent, a dominant gene for leaf rust resistance was mapped to the long arm of chromosome 3B (LrS2427). None of the Lr genes introgressed from Ae. speltoides have been mapped to chromosome 3B. Since none of the designated seedling leaf rust resistance genes have been located on chromosome 3B, LrS2427 seems to be a novel gene. Selection2427 showed a unique property typical of gametocidal genes, that when crossed to other bread wheat cultivars, the F1 showed partial pollen sterility and poor seed setting, whilst Selection2427 showed reasonable male and female fertility. Accidental co-transfer of gametocidal genes with LrS2427 may have occurred in Selection2427. Though LrS2427 did not show any segregation distortion and assorted independently of putative gametocidal gene(s), its utilization will be difficult due to the selfish behavior of gametocidal genes.

Genetics and mapping of a new leaf rust resistance gene in Triticum aestivum L. × Triticum timopheevii Zhuk. derivative 'Selection G12'.

A Triticum timopheevii-derived bread wheat line, Selection G12, was screened with 40 pathotypes of leaf rust pathogen, Puccinia triticina at seedling stage and with two most commonly prevalent pathotypes 77-5 and 104-2 at adult plant stage. Selection G12 showed resistance at both seedling and adult plant stages. Genetic analysis in F1, F2and F2.3 families at the seedling stage revealed that leaf rust resistance in Selection G12 is conditioned by a single incompletely dominant gene. The leaf rust resistance gene was mapped to chromosome 3BL with SSR markers Xgwm114 and Xgwm547 flanking the gene at a distance of 28.3 cM and 6 cM, respectively. Based on the nature of resistance and chromosomal location, it is inferred that Selection G12 carries a new gene for leaf rust resistance, tentatively named as LrSelG12.

Molecular and Morpho-Agronomical Characterization of Root Architecture at Seedling and Reproductive Stages for Drought Tolerance in Wheat.

Water availability is a major limiting factor for wheat (Triticum aestivum L.) production in rain-fed agricultural systems worldwide. Root architecture is important for water and nutrition acquisition for all crops, including wheat. A set of 158 diverse wheat genotypes of Australian (72) and Indian (86) origin were studied for morpho-agronomical traits in field under irrigated and drought stress conditions during 2010-11 and 2011-12.Out of these 31 Indian wheat genotypes comprising 28 hexaploid (Triticum aestivum L.) and 3 tetraploid (T. durum) were characterized for root traits at reproductive stage in polyvinyl chloride (PVC) pipes. Roots of drought tolerant genotypes grew upto137cm (C306) as compared to sensitive one of 63cm with a mean value of 94.8cm. Root architecture traits of four drought tolerant (C306, HW2004, HD2888 and NI5439) and drought sensitive (HD2877, HD2012, HD2851 and MACS2496) genotypes were also observed at 6 and 9 days old seedling stage. The genotypes did not show any significant variation for root traits except for longer coleoptiles and shoot and higher absorptive surface area in drought tolerant genotypes. The visible evaluation of root images using WinRhizo Tron root scanner of drought tolerant genotype HW2004 indicated compact root system with longer depth while drought sensitive genotype HD2877 exhibited higher horizontal root spread and less depth at reproductive stage. Thirty SSR markers were used to study genetic variation which ranged from 0.12 to 0.77 with an average value of 0.57. The genotypes were categorized into three subgroups as highly tolerant, sensitive, moderately sensitive and tolerant as intermediate group based on UPGMA cluster, STRUCTURE and principal coordinate analyses. The genotypic clustering was positively correlated to grouping based on root and morpho-agronomical traits. The genetic variability identified in current study demonstrated these traits can be used to improve drought tolerance and association mapping.