Marker assisted breeding of biotic stress resistance in Rice


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A marker is a DNA sequence which serves as a signpost/flag post
linked to the trait/gene of interest and is co-inherited along with
the trait
Presence of specific allele of marker = Presence of specific allele of target gene based on the concept the MAS practiced -R.M. Sundaram
Directorate Rice of Research, Hydrabad , July 3rd 2009, CPMB&B, TNAU presentation

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Marker assisted breeding of biotic stress resistance in Rice

  1. 1. Marker-assisted introgression of biotic stress resistance genes in rice Rice R.M. Sundaram Directorate of Rice Research Hyderabad 500030Bacterial blight Blast
  2. 2. Outline of the presentation…… Need for application of biotechnological tools in rice, particularly for biotic stress resistance Molecular marker assisted selection: An introduction Application of MAS for developing bacterial blight resistant rice: Success stories Scope of MAS: some thoughts
  3. 3. Rice - Indian perspective Staple food for two thirds of the population contributing 20-25% of GDP Coverage ~44 m.ha 22% of cropped area Accounts for 43% of food production Current production ~ 95 Mt Demand ~ 130 Mt by 2025 AD. How can we meet this demand?
  4. 4. Biotic stresses of rice: A major threat to production Biotic stresses limit rice yields upto 30% Major biotic stresses Insects - BPH and Gall midge Diseases - Bacterial leaf blight and blast Even though resistance breeding is successful, breakdown of single gene conferred resistance is a major concern Changing pest and disease scenario: Need to deploy „suitable‟ genes or gene combinations
  5. 5. Changing pest and disease scenarionecessitates an active breedingprogramme for introgression of bioticstress resistance genes into elite ricevarieties and hybrids…
  6. 6. Need for Biotechnological applications Conventional breeding – led to green revolution era But, can conventional breeding alone meet future rice production targets? NO Need for judicious and pragmatic application of biotechnological tools for rice improvement Biotechnology can be helpful to (i) increase productivity levels beyond yield barriers (ii) to sustain productivity (by limiting damage due to biotic and abiotic stresses)
  7. 7. Biotechnology can enhance breeding efficiency throughi. Precise manipulation of target trait without any disruption (or with minimal disruption) to non-target sites of the genome (i.e., increasing the efficiency of selection through MAS)ii. Transfer of economically important traits/genes across species and genera into the rice gene pool (i.e., broadening the genetic base through genetic engineering)
  8. 8. What is a molecular marker? A marker is a DNA sequence which serves as a signpost/flagpost linked to the trait/gene of interest and is co-inherited along with the trait. - Gene of interest 1 cM - MarkerPresence of specific allele of marker = Presence of specific allele of target gene
  9. 9. Marker on one side of the gene (Ideal: 1-2 cM from the gene) - Gene of interest 1 cM - Marker
  10. 10. Flanking markers (Ideal: 5-10 cM on either side of the gene) - Gene of interest 5 cM 5 cM - Marker
  11. 11. Application of DNA markers Seed purity testing Varietal fingerprinting and patenting Tagging, mapping and pyramiding major genes for pest and disease resistance Pyramiding QTL‟s of agronomic importance like Yield, abiotic stress tolerance etc. Map based cloning Testing of GMO‟s and GMO products through PCR based markers
  12. 12. Marker assisted breeding can reduce the time taken for breeding significantly Traditional breeding marker assisted breeding 10,000 breeding 10,000 breeding lines lines Molecular 500 -----Year 1----- 500 Marker analysisField testing 100 -----Year2---- 20 Field testing 20 Seed increaseSeed increase ----Year3----- Commercial launch ----Year4---- Of 4 or 5 varieties Commercial launch Of 4 or 5 varieties ----Year5----
  13. 13. Pyramiding multiple resistance genes in a single cultivar Bacterial blight resistance gene Blast resistance gene BPH resistance gene Gall midge Elite cultivar resistance gene Elite cultivarDesired allele of with multiple„target‟ gene biotic stresslinked to resistancemolecular marker
  14. 14. Usefulness of MAS – Pyramiding multiple genesIndian Success stories Bacterial leaf blight (BLB) resistance genes - Xa21, xa13, xa5 (DRR, PAU & IARI) Blast resistance genes - Pi1, Pi2 (CRRI & UAS) Multiple resistance genes – Gall midge and BLB (DRR) Seed purity analysis – Hybrids (DRR) and Inbreds (TNAU)
  15. 15. Success storyMarker assisted introgression of Bacterial blight resistance into Samba Mahsuri and Triguna (A DRR-CCMB Collaboration)
  16. 16. Bacterial blight (BB) BB caused by Xanthomonas oryzae pv. oryzae (Xoo) is wide spread in irrigated and lowland rice ecosystem. Growing resistant varieties is the only possible way to tackle the disease. More than 30 genes (Xa1, Xa2……..Xa30) characterized. Genotypes carrying single resistant gene have shown susceptibility at different locations Introgression of multiple R genes into the genetic background of elite rice varieties with the help of closely linked markers can ensure durable resistance
  17. 17. Disease symptomsAn Xoo colony Lesion Control Infected
  18. 18.  Effective bactericides are not available for controlling the disease Bacterium can overcome rice cultivars containing single genes for resistance against the disease Rice cultivars should be developed that contain multiple genes for Bacterial Leaf Blight Resistance
  19. 19.  What are the rice resistance genes that are effective against Indian strains of Xoo? What is the genetic diversity within the population of Xoo strains in India?
  20. 20. Xoo strains were collected from rice plants growing inmany different locations in IndiaThese were subjected to DNA fingerprinting andpathotype analysisThe analysis revealed presence of two main groups –Group I and Group II among Indian pathotypes of Xoo.Group I predominant in India
  21. 21. Rice resistance genes xa13 and Xa21 are effective against both groups of Indian Xoo strains_____________________________________________________ Xoo Rice resistance genes Strain Xa3 Xa4 xa5 Xa7 xa13 Xa21 TN-1_____________________________________________________ Group I S S R/S S R R S Group II S S S S R R S_____________________________________________________ R= Resistant; S= Susceptible
  22. 22.  A single lineage of Xoo (Group I) is widely distributed in India. Xa21 and xa13 rice resistance genes are effective against both groups of Indian Xoo strains The xa5 resistance gene provides a moderate level of resistance against strains within the widely distributed Xoo lineage
  23. 23.  A rice line called SS1113 that contains Xa21, xa13 and xa5 genes in the genetic background of PR106 has been developed at Punjab Agricultural University, Ludhiana SS1113 is resistant to Bacterial Blight disease but scores low on other characteristics that are favored by farmers and consumers SS1113 – Can serve as a donor for BB resistance into elite high yielding but Bacterial blight susceptible rice cultivars
  24. 24. Samba Mahsuri (BPT5204) Samba Mahsuri (also called BPT5204) is a leading rice variety of Andhra Pradesh and has been developed by scientists at the Acharya N.G. Ranga Agricultural University, Hyderabad This variety is being cultivated in many other parts of India because of its exceptional yield and quality characteristics. But Samba Mahsuri is susceptible to several diseases including Bacterial Blight (BB) Need for introgression of effecive BB resistance genes into Samba Mahsuri background without loss of its unique quality and yield characteristics
  25. 25. Marker assisted introgression of BB resistance genes into Samba Mahsuri and Triguna  Donor SS1113 (with Xa21, xa5 & xa13)  Recipient(s) Samba Mahsuri, Triguna  Linked markers (used for foreground selection) Xa21 - pTA 248 (ALP marker)- 0.1 cM – Chr. 11L xa13 - RG136 (CAPS marker)- 2.0 cM – Chr. 8L xa5 - RG556 (CAPS marker)- 0.1 cM – Chr. 5S  For accelerated recovery of recurrent parent genome parental polymorphic rice microsatellite markers used (background selection)
  26. 26. Approach for marker-assisted introgression Marker-assisted breeding/backcross breeding involving both foreground and background selection Foreground selection: Selection for backcross plants possessing target genes. Done using gene linked/functional markers to select backcross plants possessing target genes (viz., Xa21, xa13 & xa5) Background selection: Selection for „foreground selection positive‟ backcross plants which possess maximum recurrent parent genome introgression. Done using ~90 parental polymorphic SSR markers distributed uniformly throughout the rice genome Backcrossing continued till BC4 generation and further selections were done based on agromorphological traits
  27. 27. Schematic illustration of bacterial blight resistance gene transfer programme using molecular markers SS 1113 Samba Mahsuri (xa5 xa13 & Xa21) X (AABBcc) (aabbCC) 2000 F1 (AaBbCc) aa = xa5 Marker-assisted bb = xa13 Backcrossing BC1F1 CC = Xa21 (AaBbCc) BC2F1 (AaBbCc) BC3F1 (AaBbCc) BC4F1 (AaBbCc) BC4F2 (aabbCC) BC4F3; Evaluation of 2003-04 Resistance, yield, etc.
  28. 28. Foreground selection of BC1F1 progeny for target trait Selection for Xa21 44/145 Selection for xa13 Selection for xa5 23/44 11/23
  29. 29. Number of triple „R‟ gene heterozygotes amongst backcross progenyCross Progeny # of plants # of triple stage scored heterozygotesSS1113 X BC1F1 145 9BPT5204 BC2F1 156 11 BC3F1 160 12 BC4F1 148 10
  30. 30. Markers used for background selection1 2 3 4 5 6 7 8 9 10 11 12 RM3340 RM60 RM551 RM7029 RM508 RM295 RM337 RM316 RM6340 RM481 RM407 RM7545 RM485 RM589 xa5 RG556 RM3309 RM244 RM428 RM110 RM231 RM524 RM335 RM279 RM413 RM204 RM332 RM13 RM5556 RM402 RM320 RM247 RM261 RM6051 RM491 RM7488 RM174 RM346 RM490 RM3859 RM25 Xa21 RM536 RM511 RM583 RM169 RM434 RM271 pTA248 RM72 RM5590 RM324 RM7 RM331 RM269 RM287 RM339 RM242 RM304 RM229 RM463 RM561 RM541 xa13 RM24 RM119 RM163 RG136 RM107 RM5961 RM3628 RM294A RM18 RM478 RM215 RM5919 RM206 RM17 RM5473 RM6070 RM591 RM246 RM470 RM178 RM248 RM5545 RM458 RM411 RM340 RM264 RM212 RM497 RM280 RM250 RM7324 RM186 RM482 RM207 RM442 RM85 Foreground selection: 3 markers Background selection: ~ 90 SSR markers
  31. 31. Background selection of foreground selected BC2F1 plants using parental polymorphic SSR markers 1 2 3 4 5 6 7 8 9 10 11 RM19 RM201 1 = SS1113; 2 = Samba Mahsuri; 3 to 11 = BC2F1 lines
  32. 32. Estimation of recurrent parent genomecontribution to selected backcross progenyCross Progeny Cumulative stage contribution of recurrent parent genomeSS1113 BC1F1 72%X BPT5203 BC2F1 80% BC3F1 89% BC4F1 96%
  33. 33. Number of lines with multiple „R‟ gene combinations Ser. Gene combinations SS1113 X Samba Mahsuri # BC4F2 1 Xa21/Xa21 xa13/xa13 xa5/xa5 5 2 Xa21/Xa21 xa13/xa13 Xa5/Xa5 5 3 Xa21/Xa21 Xa13/Xa13 xa5/xa5 4 4 xa21/xa21 xa13/xa13 xa5/xa5 2 5 Xa21/Xa21 Xa13/Xa13 Xa5/Xa5 4 6 xa21/xa21 xa13/xa13 Xa5/Xa5 4 7 xa21/xa21 Xa13/Xa13 xa5/xa5 2
  34. 34.  At BC4F1, a line possessing the BB resistance genes along with maximum recurrent parent genome introgression was selfed to generated homozygous BC4F2 lines The lines were evaluated for their morphology, yield, quality and BB resistance from BC4F2 generation onwards Four homozygous lines possessing high BB resistance, yield and grain quality similar to Samba Mahsuri were nominated for All India Coordinated Rice Improvement Programme (AICRIP) trials - IET 19026 - IET 19045 - IET 19046 - IET 19590
  35. 35. Samba “The geneMahsuri pyramid lines are resistant to bacterial blight (BB)” 2 gene 1 gene 3 gene
  36. 36. The gene pyramided line IET19046 has shown high BB resistance under AICRIP trials Year Proposed Susceptible check Resistance check National entry (TN1) (SS1113) resistance check (Ajaya) (IET 19046)Total Number of locations tested 2005 13 13 13 13Number of locations where the 9 0 9 8entry showed high BLB resistance(score < 5)Number of locations where the 4 0 13 5entry showed moderate BLBresistance (score – 5)Severity Index value 3.8 8.0 2.5 3.9Total Number of locations tested 2006 18 18 18 18Number of locations where the 14 0 14 11entry showed high BLB resistance(score < 5)Number of locations where the 4 0 4 3entry showed moderate BLBresistance (score – 5)Severity Index value 3.4 7.7 3.4 4.2
  37. 37. The three gene pyramided line IET19046 is highly resistant to BB
  38. 38. The gene pyramided line IET19046 is highly resistant to BB under field conditionsVery small lesion length under No damage under BB infestation BB infestation
  39. 39. Yield levels of IET19046 was identical to Samba Mahsuri under AICRIP trials Year of No. of Improved Check 1 Check 2 Check 3 Qual. Qual. Var. Qual. testing trials Samba (Samba (Donor (Local Var. 1 2 Var. 3 Mahsuri Mahsuri) parent) check) (IET (IET (IET (IET19046) 19026) 19045) 19590)Mean Yield(kg/ha) K-2005 10 4727 4739 4486 4887 4597 4667 4574 K-2006 10 4542 4529 4344 4936 4625 5025 4910 Mean - 4635 4634 4415 4912 4611 4846 4742% increaseor decrease K-2005 10 - -0.25 +5.37 -3.27 +2.83 1.29 3.34over thechecks &qualifyingvarieties K-2006 10 - +0.29 +4.56 -7.98 -1.79 -9.61 -7.49Frequencyin the topgroup Pooled 20 10/20 10/20 6/20 9/20 10/20 10/20 10/20(pooled for2 years)
  40. 40. Improved Samba Mahsuri IET19046 has since been named as Improved Samba Mahsuri and released by Central Varietal Release Committee as a possible replacement of Samba Mahsuri in BB endemic areas A success story of MAS in India Improved Samba Mahsuri doing very well in farmers field. More than 20 tonnes of seed sold by DRR in 2008 Kharif alone Excellent example of Inter- Institutional collaboration (DRR-CCMB)
  41. 41. Development of high yielding BB resistant gene pyramids of „Triguna‟ Two lines: Trg12-5- 4-9 and Trg136-22- 3-5 nominated for AICRIP trials (IVT- IME), possess high yield and BB resistance (Xa21 and xa13)
  42. 42. Thank you