Resistance Gene Analogues (RGA) Markers (small)

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Resistance Gene Analogues: An Important Tool For Disease Resistance In Crop Plants:

Resistance Gene Analogues: An Important Tool For Disease Resistance In Crop Plants Pratiksha Mishra and AK Chhabra

Disease resistance:

Disease resistance Disease:- It is an abnormal condition in the plant produced by an organism or an environmental factor. What are R-Genes? 1. These are Resistance Genes 2. Plant R -genes follow gene-for-gene interactions with pathogens 3. These co-evolve with the pathogen virulence gene. 4. Leucine-rich repeats (LRR) which is a region of R -genes is involved in recognizing pathogens and they evolve very fast.

Gene-for-gene relationship:

Gene-for-gene relationship The gene-for-gene relationship was discovered by Harold Henry Flor who was working with rust ( Melampsora lini ) of flax ( Linum usitatissimum ). Flor was the first scientist to study the genetics of both the host and parasite and to integrate them into one genetic system Gene-for-gene relationships are a widespread and very important aspect of plant disease resistance . A total of 149 R genes are potentially expressed in the Arabidopsis genome,

Gene-for-gene relationship:

Gene-for-gene relationship Flor showed that the inheritance of both resistance in the host and parasite ability to cause disease is controlled by pairs of matching genes. One is a plant gene called the resistance ( R ) gene. The other is a parasite gene called the avirulence ( Avr ) gene. Plants producing a specific R gene product are resistant towards a pathogen that produces the corresponding Avr gene product. R VIR 1 VIR 2 VIR 3 R 1 R 2 R 3 Pathogen Host

Classes of Resistance Gene:

Classes of Resistance Gene There are several different classes of R Genes. The major classes are the NBS-LRR genes and the cell surface pattern recognition receptors (PRR). The protein products of the NBS-LRR R genes contain a nucleotide binding site (NBS) and a leucine rich repeat (LRR).

Classes of Resistance Gene:

Classes of Resistance Gene The protein products encoded by this class of resistance gene are located within the plant cell cytoplasm.

Importance of disease resistance:

Importance of disease resistance DAMAGE BY DISEASES: In order of there importance (based on the damage caused by them) the patogen may be listed as Fungi > bacteria > viruses > nematodes = insect LOSSES BY DISEASE: The losses due to diseases may range from a few to 20 or 30 per cent,in case of severe infection , the total crop may be lost. The estimated global loss due to insect pests in the potential yields of all the crops is ~14%. In India losses due to insect pests ranges from 10 to 20 %.

Slide 8:

DISEASE DEVELOPMENT

NBS-LRR Family:

Disease resistance (R) genes have been isolated in numerous plant species and the R genes with domains of nucleotide binding sites (NBS) and leucine rich repeats (LRR) represent the largest R gene family. NBS-LRR Family Award Abstract #0822393 Arabidopsis 2010: Functional Genomics of NBS-LRR Mediated Resistance

NBS-LRR Complexes:

NBS-LRR Complexes These are very complex structures, large protein molecules, less understood

Research Overview:

Research Overview Plants have developed sophisticated systems to recognize many proteins secreted into the plant cell by bacterial pathogens. Recognition of these effector molecules and subsequent defense induction involve several plant proteins and is often accompanied by rapid cell death , the hypersensitive response (HR). In order to understand the co-evolution of bacterial effectors and plant disease resistance R-genes, this group is conducting parallel studies of phenotypic and genetic diversity in the reaction between bacterial pathogens and multiple crop species.

Slide 12:

Plant proteins belonging to the nucleotide-binding site– leucine -rich repeat (NBS-LRR) family are used for pathogen detection. (R-PROTEIN) Harmful organism Recognition by resistance protein Signal to cell nucleus Genetic material Defense Response Defense protein (R-PROTEIN) Outside plant cell Inside plant cell Diagram of a plant disease resistance protein in action. A portion of the protein (MAROON) lies outside the cell and specifically recognises the harmful organism. The remaining portion of the protein (RED) resides inside the cell and communicates a signal to the plant’s genetic material, which in turn stimulates a defense response against the invading organism. R-Gene in Action © A.K. Chhabra NBS-LRR PROTEIN (R-GENE)

Slide 13:

Horizontal vs. Vertical Resistance Plant: Homozygous Heterozygous Population: Homogeneous Heterogeneous AABB AABB AABB AABB AABB AABB AaBb, AaBb, AaBb, AaBb, AaBb, AaBb, AaBb, AaBb, aaBB, AAbb AABB, aabb, AABB. aaBB AABB aaBB AAbb aabb AaBB AaBb VERTICAL REST. VERTICAL REST. HORIZONTAL REST. Homo-Homo Homo-Hetero Hetero-Hetero Hetero-Homo Same R-Gene Same R-Gene Different R-Genes © A.K. Chhabra

Slide 14:

Horizontal vs. Vertical Resistance VARIETIES Type of Rest. Pureline varieties VR Hybrid varieties VR Synthetic variety HR Composite variety HR Clones VR GENERATIONS Parents VR F1 VR F2 HR F3 HR F4 HR F5 F6 F7 F8 F9 VR Etc. Population Structure Homogeneous Homogeneous Heterogeneous Same R-Gene Different R-Genes © A.K. Chhabra

Conventional method of incorporating disease resistance:

Conventional method of incorporating disease resistance For monogenic and oligogenic traits: back cross method is use for develop disease resistance even if traits are dominant or recessive back crosses is used for disease resistance If disease resistance is polygenic trait than we have to identify the major QTLs.

Slide 16:

Dominant gene to transfer One extra year Another extra year Another extra year Almost double time taken © A.K. Chhabra

Limitations of using conventional methods:

Limitations of using conventional methods It is time consuming procedure. We may loss intensity of resistance. Linkage Drag

Slide 18:

Conventional vs. Non conventional TRADITIONAL PLANT BREEDING DNA is a strand of genes, much like a strand of pearls. Traditional plant breeding combines may genes at once Desired Gene Traditional Donor X Commercial Plant Variety Many genes are transferred New Variety PLANT BIOTECHNOLOGY Using plant biotechnology a single gene may be added to the strand Desired Gene Desired Gene Donor + Commercial Plant Variety A single gene is transferred Improved Commercial Plant Variety

These can be overcome by: :

Marker assisted selection Resistance gene analogues These can be overcome by:

RGAs ?????:

RGAs ????? Resistance Gene Analogs (RGA) in plants are related to the NBS-LRR class of disease resistance genes which are characterized by Nucleotide Binding Site (NBS) and Leucine Rich Repeat (LRR) regions. The NBS, in particular, contains several sequence motifs which are highly conserved among disease resistance genes and RGA from distantly related plants, thus facilitating their use for the isolation of new RGA and perhaps actual disease resistance genes.

Resistance gene analogues/resistance based marker:

Resistance gene analogues/resistance based marker These are fairly recent class of markers that are very important for disease resistance. these are some sequences in a disease resistance gene that are conserved over the species. This property of these sequences which are actually not the resistance gene but are resistance gene analogues,help breeders to fish out resistance gene over species and genera. These sequences are used as probes to identify similar sequences in other plant/ variety/ species/ genera.

Slide 22:

LRR Leucine Rich Repeat (LRR) regions Resistance Gene Analogs (RGA) in plants are related to the NBS-LRR class of disease resistance genes RGA NBS CLASSES Candidate gene approach Nucleotide Binding Site (NBS) contains several sequence motifs which are highly conserved among disease resistance RGAs Disease Resistance Gene Conserved region Conserved region WHEAT Can be used to isolate disease resistance genes in other crops like: RICE MAIZE SORGHUM FRUITS VEGETABLES etc. A GCT TTAAATCGAT TCG GATT

Slide 23:

a b WHEAT Disease 1 b a Disease 2 Disease 3 Disease 4 c c RICE MAIZE COTTON Crop Disease Genes And conserved regions RGAs Indicates conserved regions How to use RGAs Probe / primers © A.K. Chhabra

Slide 24:

Markers Target loci (gene dr ) Closest markers RAPD RFLP AFLP EST SSR ISSR SNP CAP a b c d e f g h i j k l m n o p q s t u v w x y z 1.3 cM 110 cM Anonymous markers with no known functions Anonymous markers provide different information Techniques that pinpoint genes proteins genomic DNA RFLPs c DNA EST RGAs etc. ISOENZYMES RFLPs SSR RAPDs etc. RGAs can be used in combination with other marker systems RGA-SSR RGA-AFLP RGA-ISSR Best Combination to increase specificity of R-Genes © A.K. Chhabra

Slide 25:

Pyramiding of genes Backcross 1 Backcross 2 Backcross 3 Backcross 4 Gene 1 Gene 2 Gene 3 Gene 4 Variety A x Source 1 source 2 source 3 source 4 6-8 years x x Variety A 1+2 Variety A 3+4 6-8 years x Variety A 1+2+3+4 6-8 years Time consuming, sanctity of variety may be lost, new varieties would come MULTIPLE RESISTANT VARIETY Different R-Genes

Slide 26:

Backcross 1 Backcross 2 Backcross 3 Backcross 4 Gene 1 Gene 2 Gene 3 Gene 4 HHB 67 x Downy mildew resistant races Race 1 Race 2 Race 3 Race 4 6-8 years x x HHB 67-1 HHB 67-2 6-8 years Example: HHB 67 ( Pearlmillet hybrid) Time reduced to 2-3 years Used MAS approach Version 1 Version 2 Different R-Genes

Slide 27:

Backcross 1 Backcross 2 Backcross 3 Backcross 4 Gene 1 Gene 2 Gene 3 Gene 4 Variety A x Source 1 source 2 source 3 source 4 6-8 years MULTILINE VARIETY Mixture of many pure lines x x Variety A 1+2 Variety A 3+4 6-8 years x Variety A 1+2+3+4 6-8 years Time consuming, sanctity of variety may be lost, new varieties would come Line 1 Line 2 Line 3 Line 4 ……………. Line n Uniform in all aspects except for disease resistant genes Mix Seed in equal quantities MULTILINE VARIETY Clean crop approach : i.e. same variety background in all the multilines Clean crop approach Different R-Genes

Slide 28:

Variety A x Source 1 source 2 source 3 source 4 Backcross 1 Backcross 2 Backcross 3 Backcross 4 Gene 1 Gene 2 Gene 3 Gene 4 6-8 years x x Variety A 1+2 Variety A 3+4 6-8 years x Variety A 1+2+3+4 6-8 years Time consuming, sanctity of variety may be lost, new varieties would come MULTILINE VARIETY Mixture of many pure lines Mix seed in equal quantity MULTILINE VARIETY Not as uniform as that of the variety developed through clean crop approach The variety creates horizontal resistance and limits the spread of the disease. DIRTY crop approach Different R-Genes Dirty crop approach : i.e. different varieties’ background in all the multilines

Slide 29:

EXAMPLES (Multiline varieties): important where new races of diseases are continuously developed three multiline varieties have been released in India in wheat Kalyansona was used as a recurrent parent in all these varieties KSML 3 8 lines MLKS 11 8 lines KML 7406 9 lines Robin, Ghanate, K1, Rend, Gabato, Blue bird, Tobari etc. E 6254, E 6056, E 5868, Frecor, HS 19, E 4894, etc. Wheat Rust Resistant Varieties INDIA USA Miramar wheat Oat multiline variety

Slide 30:

EXAMPLES WHERE RGAs HAVE BEEN SUCCESSFULLY USED

PLANT DISEASE RESISTANCE GENE ANALOGUES IN COTTON:

PLANT DISEASE RESISTANCE GENE ANALOGUES IN COTTON The objectives of the present study were: to develop and evaluate a genome-wide R gene profiling system; to map the disease resistant gene analogues (RGA); and to study the expression of the RGA in cotton tissues 2006 Beltwide Cotton Conferences, San Antonio, Texas - 2006 Yingzhi Lu Chen Niu New Mexico State University Las Cruces, NM Jinfa Zhang Las Cruces, NM COTTON

PLANT DISEASE RESISTANCE GENE ANALOGUES IN COTTON:

PLANT DISEASE RESISTANCE GENE ANALOGUES IN COTTON 2006 Beltwide Cotton Conferences, San Antonio, Texas - 2006 Yingzhi Lu Chen Niu New Mexico State University Las Cruces, NM Jinfa Zhang Las Cruces, NM COTTON

Mapping of RGAs in cotton:

Mapping of RGAs in cotton Pima 3–79 X NM24016. 88 ( G. barbadense) (G. hirsutum) Upland cotton RILs RGA markers were amplified by 8 pairs of RGA primers , while 131 polymorphic RGA-AFLP markers were produced using 6 RGA and AFLP primer pairs. Based on SSR markers with known chromosomal locations, 217 RGA and RGA-AFLP markers were assigned to 18 chromosomes using programs Mapmaker and JoinMap. COTTON

Slide 34:

Interestingly, these RGA and RGA-AFLP markers were not evenly distributed among chromosomes in that 189 markers (87%) of them were placed on three “giant” linkage groups (C6, C12, and C15). C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 The results confirm that RGA in cotton tend to cluster together. This demonstrated that RGA-AFLP is a powerful DNA marker system for identifying resistance gene analogous with more sequence specificity. Mapping of RGAs in cotton COTTON

Future plan in Cotton on RGA Research:

Future plan in Cotton on RGA Research Using more RGA and AFLP primer combinations, most, if not all, RGA can be amplified and identified genome-wide in cotton . The identification and mapping of RGA and RGA-AFLP markers would provide a framework to facilitate marker-assisted selection (MAS) for disease resistance in cotton breeding and to understand the genome organization of R genes in cotton. C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 COTTON

Slide 36:

Genetic Diversity Assessment Across Different Genotypes Of Mungbean And Urdbean Using Molecular Markers Mungbean Electronic Journal of Plant Breeding, 1(4): 379-383 (July 2010) to assess the molecular diversity so that a suitable mapping population can be developed to identify and validate markers related to resistant loci. Objective MYMV (Mungbean Yellow vein Mosaic Virus) is a virus transmitted by whitefly, Bemesia tabaci , the most serious disease of Mungbean and Urdbean In this study, six each of MYMV resistant and susceptible genotypes in Mungbean and Urbean respectively were selected for the diversity analysis using molecular markers. R R R R R R S S S S S S

Mungbean:

Mungbean a b Cowpea a MYMV c Mungbean Probe / primers Twenty four RGA primers from cowpea were used to screen the twenty four genotypes. Twenty four RGA primers R R R R R R S S S S S S RGA Twenty four RGA primers of cowpea which were designed using WEBSAT programme by searching in public data bases and downloading in fasta format from ncbi site, were used for the present study.

Sugarpine:

Sugarpine Isolation of a full-length CC–NBS–LRR resistance gene analog candidate from sugar pine showing low nucleotide diversity K. D. Jermstad . L. A. Sheppard . B. B. Kinloch .A. Delfino -Mix . E. S. Ersoz . K. V. Krutovsky .D. B. Neale Tree Genetics & Genomes (2006) 2: 76–85, DOI 10.1007/s11295-005-0029-6 a b Lettuce a Probe / primers Sugar pine Fungal pathogen sugar pine cone

Pearlmillet:

Pearlmillet Isolation, Cloning and Characterization of Resistance Gene Analogues in Pearl Millet Based on Conserved Nucleotide-binding Sites Sarosh Bejai Ramachandra 1 , Niranjan Raj Sathyanarayana 2 , Sivaramakrishnan , Subramonium 3 , Shekar Hunthrike Shetty 2 Article first published online: 24 DEC 2010 Journal of Phytopathology Specific primers designed from the conserved NBS regions, 22 RGAs were cloned and sequenced from pearl millet ( Pennisetum glaucum L. Br.). Phylogenetic analysis of the predicted amino acid sequences grouped the RGAs into nine distinct classes. GenBank database searches with the consensus protein sequences of each of the nine classes revealed their conserved NBS domains and similarity to other known R genes of various crop species.

Pearlmillet:

Pearlmillet Isolation, Cloning and Characterization of Resistance Gene Analogues in Pearl Millet Based on Conserved Nucleotide-binding Sites Sarosh Bejai Ramachandra 1 , Niranjan Raj Sathyanarayana 2 , Sivaramakrishnan , Subramonium 3 , Shekar Hunthrike Shetty 2 Article first published online: 24 DEC 2010 Journal of Phytopathology One RGA 213 was mapped onto LG1 and LG7 in the pearl millet linkage map. This is the first report of the isolation and characterization of RGAs from pearl millet, which will facilitate the improvement of marker-assisted breeding strategies. LG1 LG2 LG3 LG4 LG5 LG6 LG7 RGA 213 RGA 213

Slide 41:

RGAs can be used to cluster the genotypes based upon the kind of resistance gene these have.

Maize :

Maize Title Disease resistance identification and RGA analysis of new maize cultivars in Yunnan regional test. Authors Ma Rong ; Wu JingZhi ; Sha BenCai ; Wu YiXin; He YueQiu Journal Acta Agriculturae Universitatis Jiangxiensis 2009 Vol. 31 No. 2 pp. 208-213 ISSN 1000-2286 URL http://xuebao.jxau.edu.cn In 2006 and 2007, 42 new cultivars were identified for their resistances to gray leaf spot (GLS), northern leaf blight (NLB), southern leaf blight (SLB), Curvularia leaf spot (CLS), sheath blight (SB), stem rot (SR ear rot (ER), head smut (HS) rust by artificial inoculation.

Maize :

Maize Title Disease resistance identification and RGA analysis of new maize cultivars in Yunnan regional test. Authors Ma Rong ; Wu JingZhi ; Sha BenCai ; Wu YiXin; He YueQiu Journal Acta Agriculturae Universitatis Jiangxiensis 2009 Vol. 31 No. 2 pp. 208-213 ISSN 1000-2286 URL http://xuebao.jxau.edu.cn By using phenotypic data, the 42 cultivars were clustered into following groups. Hai 97-4 (2.38%) was resistant to 7 diseases, 7 cultivars (16.67%) to 6 diseases, 13 cultivars (30.96%) to 5 diseases, 20 cultivars (47.62%) to more than 5 diseases. By using RGA data, the 42 cultivars were clustered into 6 groups. Group 1 was resistant to CLS, NLB, SLB and HS; Group 2 to GLB, NLB, SLB and HS; Group 3 to CLS, NLB, SLB, SR and HS; Group 4 to HS, NLB and SLB; Group 5 to CLS, NLB and SLB and Group 6 to NLB and SLB. RGAs classified the genotypes in the more or less same way as the phenotypic classification revealed

Maize :

Mol Plant Microbe Interact. 1998 Oct;11(10):968-78. The isolation and mapping of disease resistance gene analogs in maize. Collins NC , Webb CA , Seah S , Ellis JG , Hulbert SH , Pryor A . Division of Plant Industry, Commonwealth Scientific and Industrial Research Organisation, Canberra, Australia. Maize

Maize :

Maize These maize resistance gene analogs (RGAs) were used as probes to map 20 restriction fragment length polymorphism (RFLP) loci in maize. Some RFLPs were shown to map to genomic regions containing virus and fungus resistance genes. Perfect co-segregation was observed between RGA loci and the rust resistance loci rp1 and rp3. The RGA probe associated with rp1 also detected deletion events in several rp1 mutants. These data strongly suggest that some of the RGA clones may hybridize to resistance genes

Slide 46:

RGAs in Comparative Genomics

Slide 47:

Flooding of Data AGCTTTATTACTTAG AGCTTTATTACTTAG AGCTTTATTACTTAG AGCTTTATTACTTAG Squencing Linkage maps AGCTTT CTTAGAA CTTATTATA AGCTTT Research Area The rapid accumulation of genome sequence data sparked an array of functional genomics tools that are being employed to understand the complex pathways involved in host plant-pathogen interaction.

RGA & Comparative Genomics :

RGA & Comparative Genomics Structural analysis of plant R proteins revealed that most R genes encode homologous proteins with similar domains. The existence of such conserved domain led to comparative genomics approach to discover candidate disease resistance genes in many plant species.

RGA & Comparative Genomics :

RGA & Comparative Genomics The technique involves three major steps: 1) amplification of the RGA region 2) cloning and sequencing 3) sequence analysis and similarity search . BLAST Search

RGA & Comparative Genomics :

Such homology-based identification of RGAs has been successfully utilized as short-cut method of resistance gene tagging and genome-wide survey for a complete set of genes containing NBS motif and other features. RGA technique has been used in cassava resulting in several novel sequences some of which were found to be similar to previously reported resistance gene analogs . Based on known domains of resistance genes, more than 900 wheat RGAs were mined from public databases. RGA & Comparative Genomics

RGAs for wheat diseases:

RGAs for wheat diseases Bacterial diseases Fungal diseases

RGAs for pearl millet diseases:

RGAs for pearl millet diseases

No. of RGAs identified:

No. of RGAs identified Sr. No. C8rop No. of RGAs identified in the genome using BLAST data base 1 Wheat 900 2 Maize 786 3 Rice 1012 4 Urdbean 342 5 Cowpea 453 6 Sugarcane 789 7 Jojoba 121 Source: http://www.springerimages.com/Images/LifeSciences/1-10.1007_s00122-003-1525-4-5

Conclusions :

Conclusions RGAs are Resistance Gene Analogs which are conserved regions in the disease resistance genes. These are present in all crop species and their domains are homologous in structure and sequences. These homologous domains are used to fish out disease resistant genes from different crop plants. RGAs have been successfully used in cotton, maize, pearl millet, vegetable crops, Brassica , forages etc. These have been used to fish out disease resistant genes, for comparative genomics, diversity analysis and to cluster the genotypes based on the kind of R-genes they have. Gene tagging for R-genes help in MAS and also development of multiline / multiple resistant genotypes and varieties.

Slide 55:

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