Parental Lines improvement by new approaches
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1) The document discusses three studies on improving rice varieties using molecular breeding techniques. 2) The first study used marker-assisted backcrossing to develop a novel cytoplasmic male sterile line by backcrossing the donor parent into the recipient parent for three generations with the aid of molecular markers. 3) The second study used gene pyramiding to transfer bacterial blight, insect, and sheath blight resistance genes from multiple parents into a single variety. Marker-assisted selection was used to identify introgressed genes. 4) The third study combined an artificial microRNA and target mimicry to improve plant height and panicle exsertion in a new rice line and its hybrids. The modified lines
Parental Lines improvement by new approaches
- 1. 1 Presented by Balaji S. Thorat M. Sc. (Scholar) Department of Agril. Botany Dr. B. S. K. K. V., Dapoli
- 2. A new tool for an old scienceA new tool for an old science 2 NATIONAL SEMINAR
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- 5. INTRODUCTION Rice (Oryza sativa L.) is staple food of more than 60% of Indian population. Rice occupies pivotal place in Indian Agriculture. Cytoplasmic male sterility (CMS) is a cornerstone of hybrid production in rice. In three-line hybrid systems, use of CMS, maintainer, and fertility restorer lines is necessary for production of hybrid seeds. Limited sources and low variation of CMS lines cause genetic vulnerability to pathogens. Therefore, diversifying the CMS sources, maintainer lines and restorers is indispensible for a sustainable production system of hybrid seed. 5
- 6. Indirect selection for a desired plant phenotype based on banding pattern of linked molecular (DNA) markers Marker2 Marker1 chromosome Gene of no interestGene of interest Marker1 Marker2 Gene itself Marker 3 Marker3 6
- 8. Parental line improvement in rice by Marker- Assisted Backcrossing (MAB) crop Character Gene/QTL Marker Assisted Seletion Forground Background Rice (Oryza sativa L.) Bacterial leaf blight resistance xa21, xa5, xa13 STS, SSR or CAPS RFLP, AFLP Blast resistance Pil SSR ISSR Quality Waxy (6) RFLP AFLP Root traits and aroma QTL on chromosomes 2, 7, 8, 9 and 11 RFLP and SSR RFLP and SSR Submergence tolerance QTL Sub1 SSR and phenotyping SSR Sterility WA-CMS SSR SSR Fertility restoration Rf1 STMS STMS Collard and Mackill, 2008 Selected examples of MAS for gene transfer through Marker Assisted Backcrossing programme 8
- 9. Material and method Yosen B line (maintainer line) Recipient parent IR68897A (CMS) Donor parent IR24 and IR36 fertility restorer lines CASE STUDY -1 9
- 10. Molecular breeding scheme for converting Yosen B fertile line to a novel CMS line via marker-assisted backcrossing (from BC1F1 to BC4F1). 10
- 11. 11 Ahmadikhah et. Al., 2015
- 12. Ahmadikhah et. Al., 2015 12
- 13. INFERENCE Results showed that three backcrosses facilitated by SSR and ISSR markers were sufficient to recover the RPG with high efficiency. Thus MABC greatly accelerated the efficiency of CMS development program and could save three to four generations of backcrossing. Finally, developed a novel CMS line (Yosen A) comparable to the original maintainer counterpart maintainer line, which can be integrated in hybrid seed production program. 13
- 14. What Is GENE PYRaMIDING ? Process of combining two or more genes from multiple parents to develop elite lines and varieties. 14
- 15. GENE PYRaMIDING Widely used for combining multiple disease resistance genes for specific races of a pathogen. Important to develop ‘durable’ insect and pest resistance against different races. It also used for to improve the qualitative characters into those have absence that traits. - e.g. amylose content, aromatic fragrance etc Gene pyramiding is extremely difficult to achieve using conventional methods, - Consider: phenotyping a single plant for multiple forms of seedling resistance – almost impossible 15
- 16. Resistant Rice Plant GENE PYRaMIDING IN RICE BaCtERIal REsIstaNCE shEath BlIGht INsECt REsIstaNC Dattaet.Al.,2002 16
- 17. sElECtED ExaMPlEs oF GENE PYRaMIDING IN RICE. Crop Character Gene transferred DNA markers Parent 1 Parent 2 Rice (Oryza sativa L.) Bacterial leaf blight resistance xa5, xa13 Xa4, Xa21 RELP, STS Blast resistance Pil, Piz-5 Pil, Pita RFLP, STS Brown plant hopper resistance (Bpt12, Bpt15) Bph1 Bph2 STS Grain quality, fertility restoration Rf1, Rf3 - STMS, AELP Aromatic fragrance (badh 2.1) Mianhui 725 WH6725 SSR, STS Collard and Mackill, 2008 17
- 18. CasE stUDY - 2 OBJECTIVE To transfer of Bacterial Blight Resistance Genes. To transfer of insect Resistance Genes. To transfer of Sheath Blight Resistance Genes. 18
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- 20. 1. Different lines carrying the Xa21 and RC7 genes showing a variable response to their respective bioassays Datta et. Al., 2002a : Selected for next generation 20
- 21. INFERENCE Identified of F1 plants introgressed with RC7, Xa21 and Bt genes. All these PCR-positive F1 plants showed the HindIII/BamHI 1.6- kb RC7 fragment received from the male parent TT-9 (carrying the RC7 and Bt genes) was a female parent, PCR analysis of the F1 plants identified the presence of the RC7 gene (Fig. 2a). All the PCR-positive F1 plants showed the expected 3.8-kb EcoRV fragment inherited from the male parent TT-103. PCR- negative lanes in Fig. 2a and b represent the plants from the selfed seeds. 21
- 22. TRANSGENIC BREEDING / GENETIC ENGINEERING / TRANSFORMATION 22
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- 24. CASE STUDY -3 24
- 25. In this study, to improve plant height and panicle exsertion of the A line two components artificial micro-RNA (ami-RNA) artificial target mimicry A ami-RNA is a recently developed gene silencing method with high specificity, while target mimicry is a natural mechanism inhibiting the miRNA function that was also recently characterized. This approach provides a paradigm to tune the expression of endogenous genes to achieve the desired phenotype by combining ami-RNA and artificial target mimicry technologies. 25
- 26. ExpERIMENTAl pROCEDURES 1. Construction of ami-RNA and MIM expression vectors The ami-RNA precursor was released from the T vector with the digestion of BamHI and KpnI and cloned into the vector pC1300- Ubi-nos, between the maize ubiquitin promoter and the nos terminator, to form the final ami-RNA expression vector pEUI-ami- RNA. PC1300-Ubinos is a previously modified vector derived from the Ti binary vector pCAMBIA1300 by inserting the maize ubiquitin promoter and the nos terminator into its multiple cloning site. The artificial MIM genes were generated by modifying the native rice target mimic gene OsIPS1. The OsIPS1 had been isolated and cloned into the plasmid MT375. The MIMa and MIMb were finally cloned into pC1300-Ubi-nos between the maize ubiquitin promoter and the nos terminator. 26
- 27. Experimental procedures Collection of Parent Materials Construction of ami-RNA and MIM expression vectors Rice transformation Southern blot analysis RT-PCR and qRT-PCR 27
- 28. Chen et. Al., 2013 28
- 29. Fig. (a) The design of the mimicry sequences of MIMa and MIMb mRNAs. Fig. (b) flow diagram of all transformations and cross combinations in this study. 29
- 30. Chen et. Al., 2013 30
- 31. The phenoType of TeZS97A Fig. (a) The teZS97A has greater plant height Fig. (b) Panicle exsertion 31
- 32. The phenoType of Te-hybridS And The relATive expreSSion level of eui1 in Te- hybridS. Fig. (a) The hybrid teZS97B/MH63 was significantly taller than wild-type ZS97B/MH63. Fig. (b) The plant height of teZS97B/MH63-MIMa recovered to normal level after MIMa was introduced. 32
- 33. inference 33 With the help of an ami-RNA and a target mimic (MIM) gene improved the Panicle exsertion of newly developed line and their hybrids also. The hybrid derived from the crossing of the te-A line and the MIM R line remains a semi-dwarf phenotype if the R line is semi-dwarf because MIM genes from the R line suppress the activity of amiRNA-eui1 from the te-A line.
- 34. concluSion Developed a novel CMS line (Yosen A) comparable to the original maintainer counterpart maintainer line, which can be integrated in hybrid seed production program by MAB. (Ahmadikhah et. Al., 2015) Identified of F1 plants introgressed with RC7, Xa21 and Bt genes by gene pyramiding. (Datta et. Al., 2002) With the help of an ami-RNA and a target mimic (MIM) gene i.e. Transgenic breeding improved the Panicle exsertion and plant height of newly developed line and their hybrids also. (Chen et. Al., 2013) 34
- 35. Cooperation between breeders and molecular biology scientists can enhance the PLANT BREEDING considerably. 35
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Editor's Notes
- #7: A marker is usually a DNA sequence located close to the gene of interest. It may occur within the gene itself. Actually, there are many genes on the chromosome. Maybe we are interested in this red one. The marker is a DNA fragment near to gene or inside the gene.