Success in seed multiplication and delivery efforts at UAS, Dharwad
University of Agricultural Services, India
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Seed is a basic, vital and central input in agriculture and in all farming systems. Timely availability of quality seeds of varieties/hybrids adapted to to different agro-climatic conditions and in sufficient quantity at affordable prices is a measure of the strength and health of an agricultural economy. Sustained increase in agricultural production requires a continuous development of improved crop varieties/hybrids, an efficient system of production, and a means of distribution to farmers. India is one of the few countries where the seed sector has advanced in parallel with the agricultural production. However, the availability of quality seed of improved varieties and hybrids is grossly inadequate and is a major constraint to enhanced production. Studies made by several workers (Gadwal 2003, Patil et al 2004, Hanchinal et al. 2007) clearly indicate that with high-volume low-value seeds, such as wheat, groundnut, soybean and chickpea, 80% of the cropping area is sown with farm-saved seeds of old and obsolete varieties During last few decades, a number of high yielding disease and pest resistant varieties/hybrids in different crops had 10 to 40% yield superiority over local cultivars. With the exception of high-value low-volume seeds, seed production of low-value high-volume crops is generally left to public sector agencies. The bulky nature of most self pollinated crops, and lack of adequate investment on infrastructure means low remuneration. Although there is enough breeder seed production in most of the high volume crops, further seed multiplication through the foundation and certified seed stages are major constraints to the availability of quality seed. The present rate of seed replacement (SRR) for such crops is 6 to 8%. There is a need to increase SRR to 25 to 30% in varieties and obviously 100% for hybrids. To increase the productivity of low-value high-volume crops farmers need to have access to improved seeds of the right type, at the right time, at the right place and at a reasonable price. For supply of such seeds, both the informal seed sector (farmer managed seed systems) and the formal seed system (seed enterprises) need to be engaged. The informal seed sector is often highly effective in reaching isolated, inaccessible, small holder areas and is a sound opportunity for entrepreneurs to gradually evolve into the formal enterprises Wheat, the most important food crop of world and backbone of global food security, belongs to the highvolume low-value seed group. Of the total area sown to both hexaploid bread wheat and tetraploid durum and emmer wheat worldwide, 44% (95 m ha) is in Asia. Of this,62 m ha are located in just three countries viz. China, India, and Pakistan (Table 1 and Figure 1). Food security and production stability are of paramount importance in most Asian countries, given that the majority of farmers are poor. The wheat rusts have historically been major biotic constraints both in Asia and the rest of the world. Stem rust has been under control since the beginning of the green revolution in South and West Asia in the 1960s. Leaf rust and stripe rust continue to be major threats to production over approximately 60 (63%) and 43 (46%) m ha, respectively, in Asia. Although, the timely application of fungicides can provide adequate control, their use adds to production costs and they are considered environmentally unsafe. Growing resistant cultivars is thus the most effective and efficient control strategy, as it has no cost to farmers and is environmentally safe. Rapid evolution of races with new virulences, or combinations of virulences, dictate a need for discovery and deployment of new resistance genes and/or resistance gene combinations.
Development and characterization of wheat lines carrying stem rust resistance gene Sr43 derived from tall wheatgrass
USDA-ARS, Northern Crop Science Laboratory
Stem rust resistance gene Sr43, derived from tall wheatgrass (Thinopyrum ponticum), is effective against Ug99 lineage Pgt races. Previous studies indicated that Sr43 was located on large Th. ponticum 7el2 chromosome segments in 7D/7el2 translocation stocks KS10-2 and KS24-1. In the present work, we applied a recently-established chromosome engineering procedure to reduce the size of the alien chromosome carrying Sr43. KS10-2 was crossed and backcrossed to the Chinese Spring (CS) ph1b mutant. BC1F1 plants were screened for stem rust response and Ph1- associated molecular markers. Resistant BC1F1 plants homozygous ph1bph1b were further backcrossed to CS. The resulting population of 706 BC2F1 plants was screened for stem rust response and with six co-dominant SSR markers. Wheat lines RWG33 and RWG34 carry Sr43 on shortened alien segments that are about 15% of that in KS10-2. Two molecular markers closely linked to Sr43 were identified; one was an SSR marker and the other a STS marker based on sequences of deletion bin-mapped expressed sequenced tags in wheat. The two new wheat lines with Sr43 and closely-linked markers may provide new resources for combating the threat of race Ug99 and derivatives.
Cracking the codes: genetic basis of nonhost resistance of barley to heterologous rust fungi
Rients E. Niks
Wageningen University, Laboratory of Plant Breeding
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Full nonhost resistance can be defined as immunity, displayed by an entire plant species against all genotypes of a plant pathogen. The genetic basis of (non)host-status of plants is hard to study, since identification of the responsible genes would require interspecific crosses that suffer from sterility and abnormal segregation. There are some plant/potential pathogen combinations where only 10% or less of the accessions are at most moderately susceptible. These may be regarded as marginal host or near-nonhost, and can provide insights into the genes that determine whether a plant species is a host or a nonhost to a would-be pathogen. Barley (Hordeum vulgare L.) is a near-nonhost to several rust pathogens (Puccinia) of cereals and grasses. By crossing and selection we developed an experimental line, SusPtrit, with high susceptibility to at least nine different heterologous rust taxa such as the wheat and Agropyron leaf rusts (caused by P. triticina and P. persistens, respectively). On the basis of SusPtrit and several regular, fully resistant barley accessions, we developed mapping populations. We established that the near-nonhost resistance to heterologous rusts inherits polygenically (QTLs). The QTLs have different and overlapping specificities. In addition, an occasional R-gene is involved. In each population, different sets of loci were implicated in resistance. Very few resistance genes were common between the populations, suggesting a high redundancy in barley for resistance factors. Selected QTLs have been introduced into near-isogenic lines to be fine-mapped. Our results show that the barley- Puccinia system is ideal to investigate the genetics of host-status to specialized plant pathogens.
Rpg1-mediated durable stem rust resistance: mechanisms of action
Department of Crop and Soil Sciences, Washington State University, USA
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Stem rust, caused by Puccinia graminis f. sp. tritici, is a devastating disease on wheat and barley. A single barley gene, Rpg1, has provided durable resistance since its commercial introduction in the 1940s. The cloned Rpg1 gene encodes a protein with two tandem protein kinase domains, one an active kinase (pK2) and one a pseudokinase (pK1). Function of both domains is required for resistance. The gene is constitutively expressed in all tissues with elevated levels in the epidermis. It is mostly cytoplasmic with small, but significant amount associated with the cell membrane. We have been studying this gene and protein to try to understand how it works and why it has been so durable. Here we report our most recent results showing that RPG1 is phosphorylated within 5 min after urediniospores from avirulent, but not virulent, races land on the leaf surface. Two effector proteins were isolated from the ungerminated spores and shown to work cooperatively to induce RPG1 phosphorylation and eventual degradation. The proteins were identified as a hypothetical protein (PGTG10537.2) with a fibronectin type III and BRCA1 C-terminal domains and vacuolar protein sortingassociated protein 9 (PGTG_16791). The rapidity of the effector function and the nature of the two protein effectors indicate that a unique mechanism for effector entry and signaling in the host cell is involved. This hypothetical mechanism may be similar to what is observed in animal cells where fibronectin proteins with an RGD-binding domain act to mediate communications between the extracellular matrix and plasma membrane.
Towards an understanding of the molecular mechanisms of durable and non-durable resistance to stripe rust in wheat
USDA-ARS Wheat Genetics, Quality, Physiology and Disease Research Unit, Pullman, WA
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Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, continues to cause severe damage worldwide. Durable resistance is a key for sustainable control of the disease. High-temperature adult-plant (HTAP) resistance, which expresses when the weather becomes warm and plants grow old, has been demonstrated to be durable. We have conducted numerous of studies for understanding molecular mechanisms of different types of stripe rust resistance using a transcriptomics approach. Through comparing gene expression patterns with racespecific, all-stage resistance controlled by various genes, we found that a greater diversity of genes is involved in HTAP resistance. The genes involved in HTAP resistance are induced more slowly and their expression induction is less dramatic than genes involved in all-stage resistance. The high diversity of genes and less dramatic expression induction may explain the durability and incomplete level of HTAP resistance. Identification of transcripts may be helpful in identifying resistance controlled by different genes and in selecting better combinations of genes for pyramiding to achieve adequate and more durable resistance.
Unraveling the entry mechanism of oomycete and fungal effector proteins into host cells
Shiv D. Kale
Virginia Bioinformatics Institute, Virginia Tech University, USA
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Oomycetes and fungi facilitate pathogenesis via secretion of effector proteins that have apoplastic and intracellular localizations. These effector proteins have a diverse array of functions that aid in pathogenesis, including modification of defense responses. In the oomycetes, well characterized effector proteins that can translocate into the host cells share a pair of conserved N-terminal motifs known as RXLR and dEER. The RXLR motif has been shown to mediate translocation of the oomycete avirulence proteins Avr1b and Avr3a into host cells. Detailed mutagenesis of the RXLR motif of Avr1b revealed that the motif is tolerant to several amino acid substitutions while retaining functional translocation activity, resulting in the definition of a broadened RXLR-like motif, [R,K,H] X[L/M/I/F/Y/W]X. This motif has been used to identify functional translocation motifs in several fungal effector proteins, AvrL567, Avr2, and AvrLm6. Effectors with both RXLR and RXLR-like motifs bind phosphatidylinositol- 3-phosphate (PI-3-P) to mediate translocation via lipid raft mediated endocytosis. Mutations in RXLR or RXLRlike motifs result in loss of phospholipid binding and translocation by effectors. Effector entry into plant cells can be blocked by proteins and inositides that disrupt binding to PI-3-P, suggesting effector-blocking technologies that could be used in agriculturally important plant species.
Investigating rust resistance with the model grass Brachypodium
USDA-ARS Plant Science Research Unit and Department of Agronomy and Plant Genetics, University of Minnesota, USA
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The model plant Arabidopsis thaliana has provided unique opportunities to explore and unravel many key biological features of plant biology including disease resistance. However, the inability of rust fungi of the genus Puccinia to infect Arabidopsis has prevented its use in exploring grass-rust interactions. The model plant Brachypodium distachyon is a member of the same grass subfamily as the principal cool-season grain crops, and can be infected with various Puccinia species. We have focused our efforts on establishing Brachypodium as a model for exploring grass - Puccinia graminis interactions. Brachypodium can be successfully infected by different formae speciales of the stem rust pathogen, including P. graminis f. sp. tritici. A wide range of response to stem rust occurs in Brachypodium and efforts are underway to decipher the genetic basis for this variation using recombinant inbred populations from parents with differing levels of response. Similarly, induced mutants with compromised stem rust resistance have been identified and are now being employed within a program to understand the molecular biology of stem rust resistance and susceptibility. Our results to date suggest that Brachypodium holds promise as a model plant for advancing our understanding of stem rust resistance.
New tools for wheat genetics and breeding: Genome-wide analysis of SNP variation
Department of Plant Pathology, Kansas State University, USA
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Single nucleotide polymorphism (SNP) is one of the most broadly distributed types of molecular variation in a genome which, along with the availability of costand labor-effective genotyping platforms, make it the marker of choice for many crops. Our work is aimed at the development of a dense set of genetically mapped SNP markers for low-cost high-throughput genotyping of wheat germplasm. Next generation sequencing of normalized cDNA libraries was used for developing gene-associated SNPs in polyploid wheat. A total of 7.5 million 454 reads were generated from cDNA libraries of 10 wheat cultivars from US and Australia and processed for discovering SNPs using a bioinformatical pipeline specifically designed for variant discovery in polyploid transcriptomes. A total of 25,000 high-quality SNPs distributed among 14,500 EST contigs were identified. All these SNPs were validated by comparison with RNAseq data generated from an additional set of 17 U.S. and Australian cultivars. A total of 9,000 genome-wide common SNPs were selected for designing an Illumina iSelect assay. Preliminary testing showed that more than 95% of SNPs produce high-quality genotype calls with up to 70% being polymorphic in a diverse sample of U.S. and Australian cultivars with a minor allele frequency >0.05. The assay is currently being used for studying patterns of genetic diversity in a worldwide collection of wheat cultivars and for developing a high-density SNP map. A long term goal of this initiative is to advance wheat research and breeding by developing genetic and genomic tools for efficient analysis of agronomic traits using high-resolution linkage and association mapping and deploying SNP markers in breeding programs
High yielding CIMMYT spring wheats with resistance to Ug99 and other rusts developed through targeted breeding
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Targeted breeding to develop high yielding wheat germplasm resistant to Ug99 and other rusts initiated at CIMMYT in 2006. Ug99 resistant materials, especially those with adult plant resistance (APR), were used in crossing. F3 and F4 populations from simple, BC1 and top crosses were grown for two generations under high rust pressures at Njoro, Kenya in a Mexico-Kenya shuttle breeding scheme. Parallel populations were also grown in Mexico for comparison. Approximately 5,000 advanced lines were tested for grain yield performance at Ciudad Obregon, Mexico in 2009/10 season, and phenotyped for resistance to Ug99 and other rusts. The 728 retained lines were evaluated for grain yield performance in five environments during the 2010/11 season in Mexico. About 68% of the 728 lines had nearimmune (16.5% entries) to adequate APR to Ug99. An additional 13.6% lines carried one of the six (Sr25, Sr26, SrTmp, SrHuw234, SrSha7, and an unidentified gene) race-specific resistance genes often in combination with APR gene Sr2. About 80% entries were highly resistant to yellow rust in Kenya and Mexico, and 90% entries to leaf rust in Mexico. Yield distribution of lines derived from Mexico-Kenya shuttle breeding was similar to lines selected only in Mexico. Sufficient lines with >5% superior yields than the Mexican checks varieties in 2 years testing were identified. Our results indicate that targeted crossing and shuttle breeding are powerful tools for a simultaneous improvement of grain yield potential and resistance to rusts.
Mapping of durable adult plant stem rust resistance in six CIMMYT wheats to Ug99 group of races
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Durable resistance to wheat stem rust fungus can Be achieved by developing and deploying varieties that have race-nonspecific, adult plant resistance (APR) conferred by multiple minor, slow rusting genes. Wheat lines ‘Kingbird, ‘Kiritati’, ‘Huirivis#1’, ‘Juchi’, ‘Muu’ and ‘Pavon 76’ showed high levels of APR to Ug99 races of stem rust fungus when tested in Kenya. The F5 and F6 generation recombinant inbred line (RIL) populations developed from the crosses of moderately susceptible ‘PBW343’ with five resistant parents were used in mapping. The non-Sr26 fraction of the ‘Avocet’ x Pavon 76 RIL population, developed earlier for leaf rust and stripe rust resistance studies, was also included. Field phenotyping of the parents and RILs were conducted at Njoro, Kenya for at least two years with Ug99+Sr24 (TTKST) race under high stem rust pressures. The continuous variation of APR in each RIL population and genetic analyses indicated quantitative nature of resistance that was likely governed by 3 or 4 minor genes. Single and joint year analyses by Inclusive Composite Interval Mapping (ICIM) using informative DArT and/or SSR markers identified consistent APR QTLs on chromosomes 1AL, 3BS, 5BL, 7A and 7DS in Kingbird; 2D, 3BS, 5BL and 7DS in Kiritati; 2B, 3BS, 4A, 5BL and 6B in Juchi; 2B, 3BS, 7B in Huirivis#1; 2B, 3BS and 5BL in Muu; and 1BL, 3BS, 5A and 6B in Pavon 76. QTLs on each genomic regions explained 10- 46% of the phenotypic variation for APR. Pseudo-black chaff phenotype associated with APR gene Sr2 on chromosome 3BS in all six resistant parents and identification of an APR QTL in the same region in all mapping populations confirmed the role of Sr2 in reducing stem rust severity. The 1BL QTL in Pavon 76 was in the same region where pleiotropic APR gene Lr46/Yr29/Pm39 is located. Similarly a 7DS QTL in Kingbird and Huirivis#1 was in the chromosomal region where pleiotropic APR gene Lr34/Yr18/Pm38 is located. These results indicate that the above two pleiotropic resistance genes confer APR to stem rust in addition to leaf rust, yellow rust and powdery mildew. Further studies are underway to saturate the genomic regions harboring new APR QTLs with additional molecular markers.