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2014 - 201618
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Subject: Biotechnology × End date: 2016 × Start date: 2014 × Type: Thesis × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    Marker Assisted Introgression of Pi1 and Pi2 Genes for Blast Resistance in Intan Variety of Rice
    (University of Agricultural Science, Dharwad, 2016-03) Hegde, Shrinidhi S.; Prashanthi S. K.
    The most devastating diseases that constrain rice production is rice blast, caused by Magnaporthe oryzae, ranking first in threat to high productivity. The use of resistant cultivars is the most effective and economical way to control rice blast disease. “Intan” is a medium slender indica variety, popular with farmers and consumers across Karnataka because of its high yield and excellent popping quality. However, this variety is highly susceptible to blast disease. The present study was aimed to improve Intan variety for blast resistance. Near isogenic lines (NILs) in BPT5204 background carrying blast resistant genes Pi1 and Pi2 were used as donor parent to introgress these genes into Intan variety using marker assisted backcrossing (MABC). Molecular markers genic/linked, flanking and unlinked to Pi1 and Pi2 gene were used for foreground, recombinant and background selection respectively in marker assisted selection (MAS). Genic/linked markers RM224 (0.0 cM), RM1233 (0.0 cM) for Pi1 gene and AP5930 (0.0 cM), AP5659-5 (0.10cM) for Pi2 gene were polymorphic and were used for confirmation of hybrids. Flanking markers RM144 (3.0 cM), RM206 (18.2 cM) and AP5413 (1.2 cM), RM6836 (1.5 cM) were polymorphic between the parents for Pi1 and Pi2 gene respectively. Among 63 genome wide markers, 26 markers were polymorphic between Intan and BPT5204 NIL possessing Pi2 gene. F1 plants were generated by independent cross for both the genes and hybridity was confirmed. One plant out of seven plants for Pi1 gene and ten plants out of thirty two plants for Pi2 gene were confirmed to be heterozygous. Wx marker linked to Wx gene responsible for amylase content was also used for hybrid confirmation. Further, these F1 plants were challenge inoculated with Magnaporthe oryzae isolates and F1s showed resistant reaction, confirming the hybridity. Confirmed F1 plants were backcrossed to generate BC1F1 plants.
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    ThesisItemOpen Access
    Marker Assisted Introgression of Qsbr11-1 qtl in Bpt5204 Backcross Lines Carrying Blast Resistance Gene Pi2 and Marker Trait Association for Sheath Blight Resistance in Rice
    (University of Agricultural Science, Dharwad, 2016-02) Lavale, Shivaji Ajinath; Prashanthi S. K.
    Sheath blight (ShB) caused by Rhizoctonia solani Kuhn. is one of the most important rice diseases worldwide. It has been difficult to generate ShB resistant rice varieties by relying on traditional breeding because resistance to rice ShB is a quantitative trait. Marker-assisted backcross breeding approach was adopted for introgression of ShB QTL qSBR11-1 into an elite rice variety Samba Mahsuri (BPT5204) which is already introgressed with Pi2 gene conferring blast resistance. Tetep was used as the donor line for QTL qSBR11-1. Tightly linked polymorphic markers viz. RM224 and Sbq33 flanking QTL region (~0.85 Mb) were used to confirm hybridity in F1 plants. These plants were screened against R. solani by artificial inoculation by following microchamber method. The F1 plants were resistant to ShB with less relative lesion height than the recipient parent. These plants were further backcrossed and BC1F1 plants were generated. Foreground selection for QTL qSBR11-1 was done using polymorphic RM224 and Sbq33 marker. Phenotypic evaluation of 134 rice landraces was carried out for identification of new resistance sources for sheath blight. Seven landraces were found to be resistant to sheath blight. Genotyping was done using 63 RM markers representing all the linkage groups of rice crop. Population structure analysis divided the mapping panel into two subgroups. Marker trait association study was carried using phenotypic and genotypic data of all the 134 rice landraces. Twenty one marker loci were identified to be significantly associated with the sheath blight resistance at significance level of 0.05 with phenotypic variation ranging from 3.02 per cent to 22.71 per cent. Thirteen new markers (viz. RM206, RM231, RM337, RM5556, RM295, RM72, RM3628, RM163, RM320, RM247, RM508, RM246 and RM583) were identified for sheath blight resistance. This study validated the previously identified markers (viz. RM5919, RM85, RM551, RM335, RM169, RM7488, RM287 and RM224) flanking different sheath blight resistance QTLs. Since, marker interval RM337 and RM5556 on chromosome 8 are not yet reported to posses any QTL for sheath blight resistance, this may be a novel region governing ShB resistance in Tetep.
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    ThesisItemOpen Access
    Evaluation of Advanced Backcross–Quantitative Trait Loci (Ab-Qtl) Lines of Icgs 76 and Dh 86 for Foliar Disease Resistance and Productivity Traits in Groundnut (Arachis hypogaea L.)
    (University of Agricultural Science, Dharwad, 2016-02) Madhumitha D. V.; Bhat, Ramesh S.
    Sheath blight (ShB) caused by Rhizoctonia solani Kuhn. is one of the most important rice diseases worldwide. It has been difficult to generate ShB resistant rice varieties by relying on traditional breeding because resistance to rice ShB is a quantitative trait. Marker-assisted backcross breeding approach was adopted for introgression of ShB QTL qSBR11-1 into an elite rice variety Samba Mahsuri (BPT5204) which is already introgressed with Pi2 gene conferring blast resistance. Tetep was used as the donor line for QTL qSBR11-1. Tightly linked polymorphic markers viz. RM224 and Sbq33 flanking QTL region (~0.85 Mb) were used to confirm hybridity in F1 plants. These plants were screened against R. solani by artificial inoculation by following microchamber method. The F1 plants were resistant to ShB with less relative lesion height than the recipient parent. These plants were further backcrossed and BC1F1 plants were generated. Foreground selection for QTL qSBR11-1 was done using polymorphic RM224 and Sbq33 marker. Phenotypic evaluation of 134 rice landraces was carried out for identification of new resistance sources for sheath blight. Seven landraces were found to be resistant to sheath blight. Genotyping was done using 63 RM markers representing all the linkage groups of rice crop. Population structure analysis divided the mapping panel into two subgroups. Marker trait association study was carried using phenotypic and genotypic data of all the 134 rice landraces. Twenty one marker loci were identified to be significantly associated with the sheath blight resistance at significance level of 0.05 with phenotypic variation ranging from 3.02 per cent to 22.71 per cent. Thirteen new markers (viz. RM206, RM231, RM337, RM5556, RM295, RM72, RM3628, RM163, RM320, RM247, RM508, RM246 and RM583) were identified for sheath blight resistance. This study validated the previously identified markers (viz. RM5919, RM85, RM551, RM335, RM169, RM7488, RM287 and RM224) flanking different sheath blight resistance QTLs. Since, marker interval RM337 and RM5556 on chromosome 8 are not yet reported to posses any QTL for sheath blight resistance, this may be a novel region governing ShB resistance in Tetep.
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    ThesisItemOpen Access
    Evaluation of Qtl Introgressed Lines of Jl 24 for Foliar Disease Resistance and Productivity Traits in Groundnut (Arachis hypogaea L.)
    (University of Agricultural Science, Dharwad, 2016-02) Meghashree H. M.; Bhat, Ramesh S.
    Five hundred and sixty eight advanced lines developed from the straight cross and marker-assisted backcrosses of an elite groundnut variety, JL 24 as the recurrent parent, and three donor parents (GPBD 4, ICGV 86699 and ICGV 99005) were evaluated for resistance to late leaf spot (LLS) and rust, and productivity traits during 2014 kharif. The lines differed significantly for the disease resistance and productivity traits. Moderate to high variability, high heritability and high genetic advance over mean were recorded for LLS score, rust score, pod number per plant, total pod weight per plot (g) and pod weight per plant (g). Significant positive association was found between LLS and rust scores. Pod number per plant showed significant and negative correlation with both LLS and rust severity. Pod weight per plant and total pod weight per plot exhibited positive correlation with LLS and rust response. Productivity traits were significantly and positively correlated with each other. Of the total 568 lines, 23 lines possessed significantly higher total pod weight per plot and disease resistance over JL 24. Most of these selected lines showed superiority over JL 24 for other productivity traits like pod number per plant, pod weight per plant, test weight, shelling percentage, sound mature kernel weight percentage, medium pod constriction, medium pod reticulation and slight pod beak. These lines differed for the number and combination of QTL governing LLS and rust resistance. Line 63 (W-61) carried a maximum of five QTL, line 112 (75-18) had a combination of 4 QTL and five lines had three QTL contributed by their respective resistant parent. Considering both phenotypic and genotypic superiority, four lines [63 (W-61), 112 (75-18), 84 (45-3) and 100 (54-10)] were selected as superior lines and were proposed for the next level of evaluation for variety development.
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    ThesisItemOpen Access
    Profiling of Avirulence Genes, Transposable Elements, Mating Type Alleles and Virulence Analysis in Magnaporthe oryzae Isolates
    (University of Agricultural Science, Dharwad, 2016-02) Fathy, Khaled; Prashanthi S. K.
    Magnaporthe oryzae (Couch & Khon) is one the devastating pathogen causing blast disease in rice crop. Ninety seven isolates of M. oryzae from different locations of south India collected from various varieties were characterized. Presence/Absence polymorphism for eight avirulence genes were analysed in 97 isolates. Avr-ACE1 was distributed in 74 isolates accounting highest frequency of 72.28 per cent, followed by Avr-PWL2 57 with 58.76 per cent and Avr-Piz-t with 53.60 per cent. Avr-Pi9 was present in 47 isolates accounting 48.45 per cent distribution. Avr-Pi9 partial gene coding for secretary protein was sequenced for nine isolates. Transposable elements distributed on chromosome seven were studied for polymorphism among 97 isolates. Mg-SINE was distributed more frequently in singletons than other families. Mg-SINE 7-125-M1 showed 57.73 per cent amplification whereas Mg-SINE 7-193-M3 showed least frequency. Mating type of all isolates was tested. Forty six isolates showed mating type MAT1-1 allele (male fertile), 42 isolates amplified mating type MAT1-2 allele (female fertile) and 7 isolates Mo-si-076a, Mo-si-084, Mo-si-007, Mo-si-215, Mo-si-15c, Mo-si-53 and Mo-si-98a were hermaphrodite in nature. Virulence frequency analysis of 97 isolates on LTH monogenic lines containing blast resistance genes indicated that Pik-p, Pik-s, Pik-h, Piz-t, Pita, Pish and Pi9 resistance genes had less matching virulences in the pathogen population studied in this investigation. Isolate Mo-si-206 containing Avr-Pi9, Mo-si-209 containing Avr-Pi20 avirulence gene and Mo-si-076b containing Avr-Pik-m. Two isolates Mo-si-100 and Mo-si-030 showed virulence reaction on all monogenic lines.
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    ThesisItemOpen Access
    Genetic Transformation for Fibre Quality Improvement in Cotton (Gossypium spp.)
    (University of Agricultural Science, Dharwad, 2016-01) Vishal Kumar; Vamadevaiah, H. M.
    A study was conducted during 2014-15 in Agriculture Research Station, Dharwad Farm, University of Agricultural Sciences, Dharwad to validate different concentrations of growth regulators for callogenesis from cotyledon and hypocotyl explants of Coker-312 cotton. Between explants, hypocotyls were found most prominent in callus growth. MS medium supplemented with 0.1 mgl-12, 4-D + 0.05 mgl-1 kinetin was found useful with respect to days of callus initiation (9.5 days), per cent callus induction (99 per cent) and callus weight (0.85g) in hypocotyls. Creamish friable callus observed higher in callus induction (94 per cent) in three per cent glucose. The per cent somatic embryogenesis was significantly higher (71 per cent) in MS medium with reduced concentration of 0.01 mg/l kinetin + 0. 1mg/l 2, 4-D followed by 0.1 mg/l kinetin (63 per cent) and 1.0 mg/l kinetin (58 per cent). Embryo maturation and plantlets development was highest in media devoid of PGR. MS with 4 week incubation followed by one week incubation in growth chamber showed significantly higher number of root and shoot growth. Agrobacterium strain EHA-105 carrying pCAMBIA2300 construct having E6, Expansion, Annexin+cry1Ac and Arabinogalactan genes were used for genetic transformation in Coker-312, BN and Sahana. Explants without Agrobacterium were highest when the colonization period of 10 minutes with 24 hours of co-cultivation. Cefotaxime at 1000 mg/l showed complete elimination of excess Agrobacterium from explant surface, Kanamycin at 100 mg/l showed complete death of untransformed explants. Pre-culture of 48 hours prior to transformation resulted in highest number of kanamycin resistant calli. Confirmation of putative transformants obtained via in planta method was done by PCR. Significant variation for halo disc length confirmed the expression in these transformed plants. Further study should be carried out with RT-PCR/qRT-PCR for expression analysis and then such plants from these events can thus be used in introgression breeding.
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    ThesisItemOpen Access
    Genetic Transformation Studies in Cotton (Gossypium spp.)
    (University of Agricultural Science, Dharwad, 2015-12) Pralhad, Jadhav Mangesh; Katageri, I. S.
    In the present genetic transformation study, both in vitro and in planta approaches were tried to transfer cry1Ac and cry1Acm genes. Coker-312 (Gossypium hirsutum L.) having in vitro regeneration ability, was used in in vitro transformation study. In planta transformation studies were carried out in Sahana (Gossypium hirsutum L.) and BCS 23-18-7 (Gossypium barbadanse L.) cotton varieties. In in vitro transformation studies, 230 embryogenic calli and 135 hypocotyl, (20mm) were used as explants source for each gene. In in planta transformation, eight to ten days old seedlings were used as explants. Each 90 of seedling of Sahana and 70 of BCS 23-18-7 were used in transfer of cry1Acm and cry1Ac respectively. The variable numbers of plants (26-37) of each combination were established. In these established plants, few plants from each combination (3-6 plants) were recorded as positive for PCR with gene of interest (cry1Ac and cry1Acm) and selectable marker gene (npt-II) through PCR. PCR positive plants from in vitro studies were also subjected for expression studies through RT-PCR and detection of Cry protein through Bt express strips (Amar immunodiagnostics). However absence of inheritance of these genes to next generation (T1) in both cases indicates transient expression. This may be due to non integration of foreign gene into host genome and its elimination during cell division.
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    ThesisItemOpen Access
    Selection of Parents for Developing the Mapping Populations And Validation of Markers for Studying Pod Shattering in Mungbean (Vigna radiata)
    (University of Agricultural Science, Dharwad, 2015-11) Nirmalbharathi M.; Bhat, Sumangala
    Seventy six mungbean (Vigna radiata) genotypes were screened for pod shattering and related traits during summer 2014, kharif 2015 and summer 2015 under field condition. Also laboratory method such as incubation at room temperature and alternate wetting and drying were standardized and the genotypes showed significant variation for the traits studied. Summer season was found to be more suitable for pod shattering. The percentage of pod shattering ranged from 0- 54%. Based on the pod shattering percentage, genotypes were grouped into five classes according to the scale given by AVRDC, (1979). Using single marker analysis fourteen already identified SSRs markers were used to validate the germplam. Of which 3 markers were found to be significantly associated with pod shattering and five showed association with yield traits. One marker (CEDG245) showed significant association with pod shattering in field, incubation at room temperature, alternate wetting and drying with 3.48%, 12.00%, and 3.90% of R2 value respectively. Chinamung and GPM-19 showed high susceptibility to shattering and Pantmung-2, SML-108, NM-1 and COGG912 showed high resistance. The maximum pod shattering observed during summer 2015 was in Chinamung (52%) and GPM-19 (54%) where as Pantmung-2, SML-108 and NM-1 did not show any shattering. These resistant and susceptible genotypes were screened with 34 SSR markers and total 12 polymorphic SSR makers were identified between Chinamung and Pantmung-2 and nine were between Chinamung and SML-108. Molecular diversity analysis showed resistant and susceptible parents come under different clusters. Also PIC value of 34 markers ranged from 0 (GMES4232 and CEDG012) to 0.89 (X-20 and X-21) with an average of 0.41. Two sets of crosses were made to develop the mapping population viz., Chinamung x Pantmung-2 and Chinamung x SML108. Using polymorphic markers true hybrid nature was confirmed in both the crosses and F2 seeds were collected.
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    ThesisItemOpen Access
    Molecular Characterization of Wheat Genotypes for Nitrogen use Efficiency
    (University of Agricultural Science, Dharwad, 2015-09) Issifu, Muazu; Krishnaraj, P.U.
    Molecular characterization and genetic diversity of Indian wheat germplasm were investigated using 20 polymorphic SSR markers. The selected cultivars were evaluated for yield and nitrogen use efficiency indices under zero and 120 kg ha-1 of nitrogen application in a simple Augmented Randomized Block Design during Rabi 2013/2014. The study was under taken to characterize wheat genotypes for nitrogen use efficiency using SSR markers associated with nitrogen use efficiency. Analysis of variance revealed highly significant differences among entries in respect of some of the characters studied. High PCV, GCV, heritability and genetic advances in T1 were recorded for grain yield, biomass, harvest index and NUE. While in T0, similar results were observed for number of tillers per meter row, biomass and NUE. The rest of the traits recorded moderate to low PCV, GCV, h2 (BS) and Genetic Advance over Mean (GAM). The strength of association between 20 markers and NUE traits was performed using single marker analysis. Results indicated that in T1, fourteen (14) markers showed significant association for NUE traits. Out of these, four markers, MO81, wmc612, wmc24 and Xwmc3918 exhibited the highest R2 value of phenotypic variation whereas in T0, nine (9) markers exhibited significant association with NUE traits with wmc24 and Xwmc3918 exhibiting highest R2 value. Four markers, M081, wmc24, wmc154 and wmc612 were found to be associated with various traits in both T1 and T0 and thus were considered as superior markers under both conditions. These markers could be considered as a potential for marker assisted breeding in wheat. Genetic similarity was calculated using the Jaccard’s similarity coefficient, and dendrogram obtained by clustering according to the Un-weighted Pair Group Method with Arithmetic Average (UPGMA) based on the SSR data. Most of the genotypes exhibited relatively good NUE. Therefore, they can be used as donor parents in wheat breeding program to combine NUE with high yield varieties.