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Professor Jayashankar Telangana State Agricultural University, Hyderabad (Telangana State)
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ThesisItem Open Access MAPPING OF GENE(S) FOR RESISTANCE TO POST FLOWERING STALK ROT IN MAIZE (Zea mays L.) CAUSED BY Macrophomina phaseolina (Tassi) Goid.(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD, 2016) SUNEETHA, P; ANURADHA, GAmong major cereal crops in production, maize (Zea mays L.) is the world’s third leading crop after wheat and rice grown in different agro-ecologies of the world. It has highest genetic yield potential amongst the cereal crops. 67% of maize is used for live stock feed and 25% of maize for human consumption, industrial purposes and the balance is used as seed. Diseases are one of the major constraints in realizing the potential yield of this crop. It suffers from number of diseases on a global basis. In India there are four downy mildews, four stalk rots, three foliar diseases, root rots and other diseases affecting kernel and other aerial parts. Disease spectrum varies in different agro climatic zones. More serious diseases are leaf blight, stalk rots, downy mildews and rusts. Post flowering stalk rot complex is one of the most serious, destructive and widespread group of diseases in maize and yield losses range from 10 to 42% and can be as high as 100% in some areas. Stalk rots take a heavy toll, among which stalk rots caused by Macrophomina phaseolina (Tassi) Goid. and Fusarium moniliforme results in 30-40% losses. Post flowering stalk rot (PFSR) is the complex disease caused by three fungi,viz., Cephalosporium acremonium, Macrophomina phaseolina (Tassi) Goid., Fusarium moniliforme and one bacterium Erwinia carotovora var zeae. Out of these, post flowering stalk rot caused by Macrophomina phaseolina (Tassi) Goid is the important disease of maize in the state of Andhra Pradesh. The most promising and cheapest way to control stalk rot disease might be through growing resistant lines. Application of molecular markers play an important role in identifying pest/disease resistant cultivar through already available designed marker (MAS). Though mapping of genes for resistance to PFSR caused by M.phaseolina was carried out, very small attempt was made to identify markers linked to resistance to PFSR caused by M.phaseolina (Tassi) Goid. through BSA in F2 population developed from cross involving BPPTI66 and BPPTI34.The present investigation was to confirm the marker identified involving different set of parents and further to map the gene for resistance to PFSR caused by M.phaseolina and also to validate the marker identified in different genetic backgrounds. . Selection of parents was the first step in present investigation. A PFSR resistant line JCY3-7-1-2-1-b-1 identified as resistant to PFSR caused by M.phaseolina at New Delhi and Ludhiana conditions was selected as resistant parent. This inbred was screened at Hyderabad during kharif, 2011 under artificial inoculation with M.phaseolina culture. It expressed a disease score of 1 under Hyderabad conditions. In order to study the genetics of resistance to post flowering stalk rot resistant maize inbred JCY3-7-1-2-1-b-1(resistant) and an inbred 5238 (Susceptible) were crossed to produce F1. F1s were selfed as well as back crossed to the susceptible parent to derive F2 and BC1F1 populations, respectively. Parents (P1 and P2), F1 and individuals in two mapping populations F2 (295) and BC1F1 (113) were artificially inoculated with the tooth pick dipped in M. phaseolina culture. F1s inoculated with the culture showed resistant reaction revealing resistance for post flowering stalk rot is governed by dominant gene. F2 population (295 individuals) segregated in 3:1 ratio i.e 214 resistant: 81 susceptible and BC1F1 population (113 individuals) segregated in the ratio of 1:1 i.e. 62 resistant: 51 susceptible confirming that resistance to post flowering stalk rot is governed by a single dominant gene For mapping of gene resistant to PFSR, a total of 413 microsatellite markers distributed over entire genome (10 chromosomes) were used to screen the parents. Of these, 84 SSR markers from ten chromosomes were found polymorphic in the parents. These eighty four markers were used to screen the bulk DNAs prepared from eight resistant plants and eight susceptible plants from F2 mapping population to find the markers linked to the resistance gene. The markers umc1269 and bnlg439 clearly distinguished resistant and susceptible bulks. Three additional markers umc2012, umc1977 and umc1071 present around umc1269 and bnlg439 were used for screening 295 F2 individual plants. Linkage analysis was carried with the marker data of umc1269, umc2012, umc1977, umc1071 and bnlg439. Linkage analysis revealed that umc1269, umc2012 and umc1977 were linked at a distance of 1.1 cM and 23cM. Single marker analysis was performed for umc1269, umc2012 and umc1977. Results of single marker analysis showed that umc1269 explained 61% (P < 0.0001) of the phenotypic variance of post flowering stalk rot resistance, which again confirms the hypothesis of single-gene inheritance of post flowering stalk rot resistance. umc1269 is the EST SSR of CSU454 gene (Glutathione S-transferase) which is already reported as the resistance governing gene. The marker umc1269 was validated for its association with disease resistance among eight resistant and three susceptible genotypes of maize. The primer umc1269 was able to distinguish the resistant and susceptible genotypes. From the above investigation this marker umc1269 which was found linked to the gene for PFSR resistance can safely be utilized in future molecular breeding programmes aimed at breeding resistant variety for post flowering stalk rot caused by M.phaseolina.ThesisItem Open Access MOLECULAR MAPPING OF ALTERNATE DWARFING GENE(S) TO WIDELY USED DEE-GEO-WOO-GEN ALLELE OF sd1 GENE IN RICE (Oryza sativa L.)(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY. HYDERABAD, 2015) SIDDHARTHA SWARUP JENA; DURGA RANI, CH. V.The short statured varieties of rice developed using Dee-Geo-Woo-Gen (DGWG), a spontaneous dwarf mutant as the donor, have enabled many countries to achieve selfsufficiency in rice production. Initial attempts to study the genetics of semi-dwarfism using crosses of traditional tall varieties and semi-dwarf varieties suggested that semi-dwarfism is controlled by a single recessive gene, sd1. The sd1 gene has been reported to reduce plant height by 25% through proportional reduction in top five internodal lengths without any penalty on panicle length. The success of DGWG- based varieties such as IR8 and Taichung (Native) 1 has made breeders all over to depend excessively on these two rice cultivars as source breeds for short stature trait. Genetic analysis of a large number of dwarfs of spontaneous and induced origin has revealed that dwarfs non-allelic to sd1 are rare. Recently dwarf accessions non-allelic to sd1 gene (putatively termed as asd1 (alternate semi-dwarf) gene(s)) have been identified from a set of 33 mutant dwarf accessions of rice employing a gene specific marker for sd1 gene and biochemical assay (Gibberellic acid (GA) response). Keeping the foregoing in view, the present study was initiated to find and map alternative dwarfing gene (asd1) from a F2 population derived from the cross of a mutant dwarf accession, LND384 and a tall landrace, INRC10192. The F2 population was grown and the phenotypic data of 14 traits related to important plant height and yield component traits has been recorded in parents i.e., LND384 and INRC10192 and 189 F2 individuals. The parents are significantly different in all the traits measured which are reflected in obtaining of many transgressive segregants ranging from 7.94% (plant height) to 100% (appearance of sixth internode) in F2 offspring. Though plant height is believed to be a quantitative trait, in the present investigation, its distribution is skewed towards tall parent type. Parental polymorphism study between the parents was done using 627 hypervariable SSR markers. Genotyping of F2 individuals followed by Linkage mapping and QTL analysis using 27 marker loci and 14 phenotypic traits data by MAPMAKER/QTL Cartographer revealed 20 QTLs, of which 9 QTLs were associated directly with plant height related traits. Two QTLs for plant height, five QTLs for culm height, one QTL for internode number and one QTL for internode length (INL-3) were detected on chromosome 6 with a phenotypic variance in the range of 10-77%. All the QTLs detected were found to cluster in six marker intervals on chromosome 6. The GO (Gene Ontology) terms enrichment of QTL confidence intervals was performed using the Plant Gene Set Enrichment Analysis (GSEA). In the interval of RM3353 and RM19291 one potent gene involved in plant structure, Os06g0110000- Similar to DWARF3 (Fragment) has already been reported earlier. The QTL, qch6.3 for the trait culm height was found tightly linked with the marker RM19291 in the present study with a genetic distance of 2.9 cM.ThesisItem Open Access MOLECULAR MAPPING OF QTLs GOVERNING WATER USE EFFICIENCY IN RICE(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY. HYDERABAD, 2015) ROJA VEERAGHATTAPU; LAKSHMI NARAYANA REDDY, VWater Use Efficiency (WUE) is especially an important consideration where available water resources are limited or diminishing. WUE is the strategy where, the crop can produce more biomass under limited supply of water. Therefore, breeding rice with high WUE can ameliorate water shortage through saving irrigation water. Hence, the present study has been undertaken with the objective of identifying chromosomal regions/QTLs that regulate the water use efficiency in rice crop. A set of 48 rice genotypes were grown under two moisture conditions i.e., control and treatment, screened for water use efficiency related traits viz., stomatal characteristics, relative water content, specific leaf area and carbon isotope discrimination as well as molecular diversity analysis for identification of the parents. Based on the above criteria INRC10192, Azucena, Soulmpiket, Lalnakanda and NL48 varieties have been chosen as high WUE. A total of 153 RILs in F8 generation derived from a cross between INRC10192 and IR64 was used to map QTLs governing water use efficiency and related traits under both control and treatment conditions. The two parents were screened for polymorphism using 652 rice microsatellite markers and 21 indel markers. Of these, 110 microsatellite markers (16.3%) were polymorphic and distributed all over 12 chromosomes. Out of 110 polymorphic markers used for screening the entire 153 population, 79 were employed for constructing the linkage map. The total length of the linkage map is 2544.2cM using the Kosambi mapping function, resulting in an average of one marker per every 32.2cM, using MAPMAKER/EXP v 3.0 and Map Disto v 1.7 softwares. QTL mapping was carried out employing both simple interval mapping (IM) and composite interval mapping (CIM) methods of the QTL Cartographer v 2.5. A total of 36 QTLs were detected commonly using both the methods under control as well as treatment conditions. Of the 36 QTLs identified in the present study, 17 were detected under control and 19 were detected under treatment conditions on the chromosomes 1, 2, 4, 8, 9, 10 and 11. Further, out of 36 QTLs, 15 QTLs were novel and 21 stable QTLs were identified under both control and treatment conditions. The QTLs that were detected only under treatment conditions qpnot1.1, qtyldt2.1, qtyldt4.1, qpnot8.1, qnfgt8.1, qrtlnt8.1, qtwtt11.1 and the QTL, qspf1.1 was detected only under control situation. In all, 20 major effect QTLs which accounted for more than 15% phenotypic variation were identified. Four markers namely, RM6703, RM404, RM257 and RM3662 are found to be tightly linked to water use efficiency and yield related traits can be used as potential markers in crop breeding through MAS. Nine QTL intervals harbouring major QTLs as well as QTL clusters were targeted for candidate gene identification using gene ontology (GO) and transcriptome based analysis. The percentage of significantly overrepresented (enriched) GO terms was ranged between 0 to 21.05%. The GO analysis revealed that the terms associated with water use efficiency and stress tolerance traits namely, Subtilase activity (GO: 0004289), Carbohydrate biosynthetic process (GO: 0016051), Protein serine/threonine kinase activity (GO: 0004674), Protein tyrosine kinase activity (GO: 0004713), Response to water stimulus (GO: 0009415), Cellular response to stress (GO: 0033554), and Response to stress (GO: 0006950) were overrepresented within the QTLs of water use efficiency related traits. Out of the 9 regions targeted, the QTL intervals, RM486-RM6703, RM6703-RM11484, RM404-RM447, RM24879-RM171 and RM229-RM332 on chromosome 1, 8, 10 and 11 were found to harbour the genes governing majority of water use efficiency related traits. Transcriptome analyses of differentially expressed genes within the QTLs and the PosMed database yielded candidate genes controlling water use efficiency related traits. The gene, auxin efflux carrier component - Os01g0818000 is found to be associated with carbon isotope discrimination QTL, qcid1.1. The genes identified within the three QTL intervals, RM6703-RM11484, RM404-RM447 and RM24879-RM171 controlling leaf related traits viz., specific leaf area, leaf width under control and treatment and panicle number under treatment were namely, calmodulin-binding protein, ABC transporter ATP-binding protein, brassinosteroid insensitive 1-associated receptor kinase 1 precursor, ethylene-responsive transcription factor, protein phosphatase 2C and zinc finger RING-type were found to be involved in improving water use efficiency by regulating stomatal related parameters. The gene “abscisic stress-ripening putative expressed- Os11g0167800” (ASR) was found to be responsible for maintaining water levels especially under stress within the region RM229-RM332 wherein a QTL for RWC under treatment was identified. Out of 9 QTL intervals targeted, RM335-RM518 on chromosome 4 and RM3395-RM3662 on chromosome 8 were found to contain no candidate genes for that particular trait. Hence, these QTL regions identified were good candidates for fine mapping and positional cloning studies and further exploited for progressive improvement through recombination breeding using concerned QTLs as selectable markers and recombinant DNA technology by transferring selectively cloned QTL flanking markers of promise into good agronomic basis.ThesisItem Open Access MOLECULAR DETECTION OF MYCOTOXIN PRODUCING GENES IN CONTAMINATED RICE (Oryza sativa L.) USING REAL-TIMEPCR METHOD(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY. HYDERABAD, 2015) SRIDEVI, R.N.V.S.; MANORAMA, KRice (Oryza sativa L.) is the staple food of over half the world's population. According to FAO, 25% of the world’s food crops are affected by mycotoxins. The major mycotoxin producing fungi are Aspergillus, Fusarium and Pencillium. Aflatoxins, fumonisins, trichothecenes, orchratoxins, cyclopiazonic acid, patulin, deoxynivalenol, zearalenone, citrinin, gliotoxin and sterigmatocystin are some of the important mycotoxins. Mycotoxin detection is important for quality control, especially while exporting food grains. Timely detection will help to alleviate consumption of afflicted food grains by population and susceptibility to health problems associated with their consumption. Detection systems like High Performance Liquid Chromatography (HPLC) are time consuming. Molecular detection methods using PCR can replace conventional methods due to their precision as well as speed. Mycotoxin producing gene fragments in fungal strains were amplified using PCR and realtime PCR along with Bioanalyzer in an attempt to develop a rapid assay for mycotoxin detection. As many as 120 samples of rice were collected from various storage areas like godowns, wholesale and retail shops and farmers’ fields in major rice growing districts in Andhra Pradesh (A.P). Fungal DNA was extracted using DNA extraction kits. DNA from fungal contaminated rice samples was extracted by the CTAB method. Forward and reverse primers were designed by identifying homologous regions from original gene sequences (obtained from GENBANK) of the three genes, aflQ, Tri13-DON and pks gene, which are key genes involved in the production of three mycotoxins viz., aflatoxin, deoxynivalenol (DON) and ochratoxin respectively. Sequences from different species of fungi were aligned using megalign software from DNASTAR Lasergene 8.0 version, and homologous regions were identified. Primer 3.0 software was used for designing primers from these homologous regions. Using these three sets of primers, a 167 base pair fragment of the aflQ gene was amplified from DNA of Aspergillus parasiticus and Aspergillus flavus, a 196 bp fragment of ochratoxin producing polyketide synthase gene (pks) was amplified from DNA of Aspergillus ochraceus, and a 236 bp fragment of Tri13-DON (deoxynivalenol) gene was amplified from DNA of Fusarium culmorum. PCR and realtime PCR assays were conducted to detect the presence of the genes encoding the production of three mycotoxins, viz., aflatoxin, deoxynivalenol (DON) and ochratoxin. Results showed that out of 120 rice samples tested for the detection of the genes producing aflatoxtin, ochratoxin and DON, 35 samples were positive for the aflatoxin producing aflQ gene and pks gene was detected in 7 samples. None of these samples showed amplification of the Tri13-DON band corresponding to the gene fragment of size of 236 bp. The fragments were resolved on the DNA 1000 LabchipR, for generating data of fragment sizes. Combination of PCR with the Bioanalyzer was advantageous compared to traditional agarose gel electrophoresis and staining methods, in terms of precision of the bands and band sizes. Amplification of these gene fragments was also achieved using RT-PCR with probes SYBRGreen, with Ct values ranging from 20 to 25 for the three genes. Fungal DNA was subjected to duplex PCR initially using two sets of primers of aflQ and Tri13-DON and aflQ and pks primer combinations. Simultaneous amplification of aflQ along with pks gene fragments was also achieved. However simultaneous amplification was not achieved in the assay where primers for pks and Tri13-DON were used in a single reaction. A combination of three sets of primers also did not successfully amplify all three gene fragments. Duplex PCR was attempted in contaminated rice samples using only aflQ and pks primers together in the same assay. Out of 120 samples, 35 samples amplified the 167 bp gene fragment aflQ, 07 samples amplified the 196 bp pks gene and no results were observed for Tri13-DON gene. Out of the samples that showed positive reaction to aflatoxin, only seven were positive for amplification of pks gene fragments. Sample numbers H3, R2, R11, EG3, EG18, WG2 and P3 were positive for both, aflQ and pks gene fragments. Hence, no rice samples showed contamination with two different mycotoxins in the same sample, therefore multiplex PCR did not show any positive results of multiple bands in samples.ThesisItem Open Access MOLECULAR DISSECTION AND FINE MAPPING OF MAJOR QTL GOVERNING DROUGHT TOLERANCE TRAITS IN RICE(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY. HYDERABAD, 2015) SANTOSH PATIL; LAKSHMI NARAYANA REDDY, VDrought is considered as one of the major obstacles to rice for its yields and stability, especially in rainfed conditions. Development of rice cultivars with inherent capacity to withstand drought stress would improve rainfed rice production. The genomic regions harbouring drought tolerant QTLs could be of great targets to identify the candidate genes by finemapping. To this end, a QTL region on Chromosome 8 with marker interval of RM38 and RM331 governing important drought tolerant traits viz., root dry weight, root shoot weight ratio have been identified in a recombinant inbred population (RIL) derived from the cross between a semi-dwarf variety IR64 and landrace INRC10192, was targeted for fine mapping. Saturating this region with more polymorphic markers helped to delimit the QTL cluster to manageable size and identification of candidate genes underlying this QTL region by employing combination of QTL mapping and RNA-Seq analyses were the objectives of present investigation. Parents IR64 and INRC10192 used in the present study exhibited significant differences for traits like shoot length (SL) and root dry weight (RDW). The mapping population consisted of 153 F10 RILs and 435 BC1F2 population developed from a cross between a semi dwarf variety IR64 and a landrace INRC10192 was used as material for finemapping. Transgressive segregants in RI population were ranged from 45.1 % (root length) to 91.5% (root to shoot weight ratio) under control conditions, while under stress, 35.29% (shoot length) to 98.69% (root to shoot weight ratio), whereas, the relative parameters showed the transgressive segregants in the range of 62.75% (shoot length) to 95.42% (root to shoot ratio). Out of 159 rice microsatellite markers, 25 (15.72%) showed polymorphism. RILs were genotyped using these polymorphic markers and linkage map was constructed from the genotypic and phenotypic data using MAPMAKER V 3.0. Total map length of 45.6 cM using the kosambi mapping function, resulted in an average of one marker per every 2.53 cM distance. QTL mapping using RIL population revealed, 3 QTLs namely qrdwrs8.1 for relative root dry weight, qrlrs8.1 for relative root length and qslrs8.1 for shoot length with phenotypic variance of 15.95%, 15.88% and 11.62%, respectively. QTL cluster RM38-RM331 on chromosome 8 was narrowed down from 10.17 Mb to 715.67 Kb with an interval RM6999-RM7080. Backcross segregating population was developed to further delimit the QTL region identified for root dry weight using RIL population, in the present study. To reduce the background noise of the donor parent, QTL introgression was followed, wherein, the complete introgression of QTL region in BC1F1 was assessed through foreground and recombinant selections with internal (RM7080) and flanking markers (RM38 and RM331) of the targeted QTL. The BC1F1 plant having maximum recovery of recurrent parent allele was selfed to generate BC1F2 population and was subjected to stress under field conditions. Transgressive segregants observed in BC1F2 population were in the range of 8.97 % (plant yield) to 84.83 % (root dry weight). All the examined traits for the BC1F2 individuals displayed a continuous variation with the skewness ranging from −0.05 to 1.32, indicating quantitative inheritance. Correlation studies in BC1F2 population for root dry weight showed significant positive correlation with traits, viz., relative water content (0.124**) and leaf rolling score (0.142**). QTL mapping using genotypic and phenotypic data generated using BC1F2 population showed the consensus QTL for root dry weight and region was further finemapped from 715.67 Kb to 366.75 Kb with new marker interval RM22483-RM7080. In silico analysis showed 33 annotated genes within this interval. RNA-Seq for two parental root tissues was done on next generation platform (Ion torrent) to identify differentially expressed genes (DEGs) within the QTL region between parents. Total number of reads obtained through RNA-Seq was 4.7 million in INRC10192 and 4.06 million in IR64. Through RNA seq, it was found that 9,795 genes were expressed exclusively in INRC10192 and 6,901 genes were expressed only in the parent IR64, whereas 2455 genes were expressed commonly in both the parents. Through RNA-Seq approach it was found that 18 genes were differentially expressed within the targeted QTL region. Semi-qPCR was performed for all shortlisted 18 genes, but out of which only five showed differential expression. Semi-qPCR results showed that the genes, LOC_Os08g06110 (MYB related Transcription factor), LOC_Os08g07060 (Choloro respiratory reduction 6 protein) and LOC_Os08g08090 (Wound inducible protein) were up-regulated under PEG induced stress compared to control in IR64, while the genes LOC_Os08g06500 (Pentacoripeptide repeats protein) and LOC_Os08g06430 (NADH ubiquinone oxidoreductase) showed very negligible/no change under both control and PEG induced stress in IR64. On the other side, LOC_Os08g06430 (NADH ubiquinone oxidoreductase) and LOC_Os08g06500 (Pentacoripeptide repeats protein) showed down-regulation in parent INRC10192 under stress conditions when compared to control. LOC_Os08g06110 (MYB related Transcription factor), LOC_Os08g07060 (Choloro respiratory reduction 6 protein), LOC_Os08g08090 (Wound inducible protein) showed up regulation in INRC10192 under PEG induced stress compared to control. Study of functional association of gene/s with target trait was performed for all 5 genes but it was confirmed only for LOC_Os08g06110 (MYB related transcription factor). The results from this study indicated that, an integrated strategy which combines QTL-mapping and RNA-seq analysis of parents was very successful at identifying candidate gene/s for root dry weight. In combination with them, a functional association study was a welcome addition for dissecting likely candidate genes for trait of interest. Marker-assisted introgression of this QTL region or candidate genes underlying in it into modern cultivars would pave the way for increasing the yield ceiling under rainfed conditions.ThesisItem Open Access IDENTIFICATION OF CANDIDATE GENE(S) UNDERLYING QTL CLUSTER FOR GRAIN SIZE TRAITS IN BASMATI RICE(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY. HYDERABAD, 2015) ANNE KITTY DEBORAH, D; ANURADHA, GOf the traits that determine the quality of Basmati rice, grain size is one of the important character not only from consumer's angle but more so from traders’ and millers’ angle. Though many genes governing grain size traits have been identified in indica and japonica, little work has been done in basmati rice. Earlier, a QTL cluster controlling grain size was identified on chromosome 5 using a population derived from Basmati370 and Jaya. In the present investigation, it was aimed for narrowing down the identified QTL cluster governing grain size traits in basmati rice employing association mapping and QTL mapping approaches besides identification of candidate gene(s) underlying it. The results obtained are presented below: In the association mapping study, a 96 diverse rice germplasm was used (aromatic (27), indica (45), japonica and javanica (19) and aus (5) groups) which differed significantly for grain size traits. The germplasm was screened with a total of 55 markers (21 SSR markers in the QTL cluster (10 Mb), 18 SSR markers covering other chromosomes to avoid spurious associations with an average number of markers per chromosome as 3.25 and 16 gene specific markers tightly linked to 9 genes reported earlier to govern grain size). Diversity analysis showed a total of 224 alleles with average number of alleles per locus as 4.2 and an average PIC value, 0.53. Phylogenetic tree constructed using DARWIN5.0 revealed, Cluster 1 consisting mainly of aromatic group, Cluster 2, indica group and Cluster 3, aus group in one subcluster and having japonica and javanica accessions in the separate subclusters with an admixture of indica varieties. Association mapping was done using TASSEL v 2.1. Out of six SSRs associated with grain size traits, three SSRs, RM 6024 (grain breadth), RM1237 and RM18582 (grain length breadth ratio) were ‘constitutive QTL’ markers as these were associated with same traits in RILs and association mapping panel across two years which covered a physical distance of 889kb. Thus, the QTL cluster was narrowed from 10Mb to 889kb. Of the nine earlier reported genes governing grain size, GS3, GW2, GS5, GW5, GS7, qSS7, QSS7, QGW8 and SRS5, five genes GW2,GS3, GW5, QSS7, QGW8 showed association with grain size traits in accordance with the earlier reports. To further narrow down the fine mapped QTL cluster, QTL mapping was employed in 410 F2 progeny of a cross, Jaya and Basmati370. To map QTLs for grain size in F2, 39 SSR markers were used for parental polymorphism study in the marker interval RM6024-RM18582. Of which, 7 markers showed polymorphism between Basmati370 and Jaya accounting for 18% of polymorphism. The QTLs for grain size, thousand grain weight and panicle number were clustered in the region RM6024-RM18550 with a physical distance of 268 kb. However, there were no QTLs found for single plant yield in this region. This region within the QTL cluster is novel as it was not reported earlier to govern grain size in basmati rice. With the help of RICE TOGO browser, 24 genes were found in this narrowed down QTL region of RM6024-RM18550. The candidate genes were predicted using three approaches viz., semiquantitative pcr, qTELLER and nonsynonymous SNPs. Employing semiquantitative PCR technique to find out DEGs (Differentially Expressed Genes) in the QTL cluster between parents, Basmati370 and Jaya, Zinc finger transcription factors (Os05g0389600), Cytochrome p450 (brassinosteroid signalling) (Os05g0372300) and tetratricopeptide like helical domain containing proteins (Os05g0374500) were involved in regulating grain length whereas, ubiquitin mediated protein degradation proteins (Os05g037060, Os05g0371200 and Os05g0372800) and Cytokinin Oxidase1 (Os05t0374200) were predicted to regulate grain breadth in Basmati rice. Besides candidate genes predicted in the fine mapped QTL cluster, earlier reported grain size regulating genes viz., AP2 (Os05g0389000) and Zinc finger, RING type domain (Os05389600) showed high expression in Basmati370 similar to expression pattern reported earlier. These genes were located nearly 1.7 Mb away from the present QTL cluster. Two genes, CaLB domain containing protein and protein kinase domain containing protein were found to be highly expressed at early inflorescence stage utilising qTELLER information. Unfortunately, there were no non-synonymous SNPs found in the genes underlying the fine mapped QTL cluster. However, a non-synonymous SNP was found in VQ domain (Os05g32460) which was 1 Mb far from the fine mapped QTL cluster. From the above investigation, association mapping along with QTL mapping is found to be an effective tool in narrowing down the QTL and the germplasm used for association mapping is an ideal population for diversity and association mapping studies. The associated markers in the association mapping study can be used for MAS. Marker-assisted introgression of this QTL region or candidate genes underlying it after further confirmation into modern cultivars would help us tailor varieties according to consumer preferences as the genes underlying this region are homologous to earlier reported grain size regulating genes.ThesisItem Open Access CONVERSION OF ELITE NON QPM INBREDS OF MAIZE TO QPM USING MARKER ASSISTED SELECTION(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY. HYDERABAD, 2015) SURENDER MADDALA; SOKKA REDDY, SDevelopment of QPM (Quality Protein Maize) with high lysine and tryptophan is foremost important task in enhancing nutritional quality in maize through breeding programme. Marker assisted selection is the most feasible way of developing QPM hybrids in short time. The present investigation deals with conversion of elite normal maize inbred lines BML6 and BML7 (parental lines of DHM117 hybrid) into QPM lines using marker assisted selection. The nutritional quality of maize is enhanced by introgression of the opaque2 (o2) gene along with numerous modifiers for kernel hardness. To improve the efficiency of QPM breeding, the utility of three simple sequence repeat (SSR) markers viz. umc1066, phi057 and phi112 were used in selection and introgression of the opaqaue2 gene. Polymorphism was detected between recipient parents (BML7 and BML6) and donor parent (CML181) with umc1066 SSR marker. Foreground selection was exercised in each generation using opaque2 specific marker umc1066 while background selection was carried out in BC1F1 and BC2F1 generations to recover the recurrent parent (RPG) genome using SSR markers distributed across the genome. In BC2F1 the recovery of recurrent parent was between 90 to 93% and the plants with highest recovery were selfed to generate advanced generations (BC2F2 and BC2F3). Kernels were screened for endosperm hardness using light box and kernels showing less than 25% opacity were selected. Rigorous phenotyping was done for plant characters and tryptophan was estimated using colorimetric method. Tryptophan content varied from 0.76% to 0.95% in BC2F3 derived population of BML6 and 0.72% to 0.92% in BC2F3 derived population of BML7. Normal looking converted inbreds (CBML6 and CBML7) with high tryptophan and high yield were used for reconstitution of the QPM version of DHM117 maize hybrid. Key words: QPM- Quality protein maize, opqaue2, tryptophan, recurrent parent genomeThesisItem Open Access MAPPING QTLs FOR Fe AND Zn CONCENTRATION IN RICE GRAINS(ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY, RAJENDRANAGAR, HYDERABAD, 2014) KIRANMAYI, S. L; MANORAMA, KRice is the staple food for more than half of the world’s population. Hence, even a small increase in micronutrient concentration would have a significant impact on the human health. Biofortification by using molecular breeding techniques in staple crops has been evolved as a new strategy to address micronutrient malnutrition. Hence, the present study has been undertaken using 126 Recombinant Inbred Lines (RILs / F6) obtained from a cross between a deep water rice cultivar, Jalmagna, which is high in iron (Fe) and zinc (Zn) concentrations, and a high yielding popular rice cultivar, Swarna (MTU7029) with the prime objective of mapping QTLs for grian Fe and Zn concentrations and yield and related traits in brown rice. One hundred and twenty six RILs along with the parents were analysed for grain Fe and Zn concentrations using Atomic Absorption Spectrometry (AAS). The Fe concentration recorded in the grains of rice cultivar Jalmagna was 40.05 ppm at Hyderabad and 38.95 ppm at Warangal while that of Swarna was 32.05 ppm at Hyderabad and 28.05 ppm at Warangal. The Zn concentration recorded in the grains of rice cultivar Jalmagna was 24.28 ppm at Hyderabad and 22.53 ppm at Warangal while that of Swarna was 14.88 ppm at Hyderabad and 13.33 ppm at Warangal. Grain Fe concentration in 126 RILs ranged from 4.13-187.93 ppm at Hyderabad and 5.83-270.15 ppm at Warangal. Grain Zn concentration varied from 14.20-28.13 ppm at Hyderabad and 13.58-30.85 ppm at Warangal. The top ten lines had high grain Fe concentration ranging from 29.85 to 186.93 ppm at Hyderabad and 28.30 to 270.15 ppm at Warangal while that of grain Zn concentration ranged from 23.85 to 28.13 ppm at Hyderabad and 27.08 to 30.85 at Warangal. Three RILs recorded high concentrations of both grain Fe and Zn among the top ten lines at Hyderabad whereas at Warangal none of the RILs among top lines were found to have high concentrations of both these micronutrients. Grain Fe and Zn concentrations showed significant positive correlation (r = 0.256) at Warangal and significant negative correlation (r = -0.193) at Hyderabad. Grain Zn concentration at Warangal showed non-significant correlation with grain Fe concentration at Hyderabad (r = 0.118), grain Zn concentration at Hyderabad showed significant negative correlation with grain Fe concentration at Warangal (r = -0.182). Grain Fe and Zn concentrations showed non-significant correlation with most of the yield and related traits at both the locations. Both grain Fe and Zn concentrations exhibited negative correlation with single plant yield and yield / plot. Single plant yield and yield per plot were significantly and positively correlated with most of the yield related traits at both the locations. Parental polymorphism survey was conducted to identify the polymorphic markers between the parents. Out of 800 rice micro satellite markers, 106 (13.25 %) markers were polymorphic and among 37 gene specific markers, 12 (32.43 %) markers were polymorphic. In all, 118 (14.1%) markers distributed over all the 12 chromosomes of rice were polymorphic between the parents. A molecular linkage map was constructed using only 112 microsatellite markers (as 6 out of 118 markers were found to be unlinked) spanning a total map length of 1169.9cM using the kosambi mapping function, resulting in an average of one marker every 10.44 cM. Single marker analysis revealed four linked markers for Fe, nine linked markers for Zn and 46 linked markers for yield and related traits. A total of 29 QTLs, 20 by CIM and 9 by IM methods governing grain Fe and Zn concentrations and yield and related traits were detected across two locations. These 29 QTLs included 18 and 11 QTLs detected at Hyderabad and Warangal locations, respectively. In all, 4 QTLs for grain iron and zinc concentrations and 25 QTLs for yield and related traits were identified. One QTL, qFe8.1 was detected for Fe concentration on chromosome 8 at Warangal. Three QTLs, qZn3.1, qZn5.1 and qZn8.1 were located on chromosomes 3, 5 and 8 for grain Zn concentration indicating the polygenic inheritance of a trait. A QTL, qZn5.1 located on chromosome 5 accounted largest phenotypic variance (11.78 %) compared to other QTLs identified for grain Fe and Zn concentration. These QTLs were spread over all the chromosomes except chromosomes 9, 10 and 11, and most of them were on chromosomes 4, 6 and 8. Trait enhancing favorable QTL alleles were largely contributed by Jalmagna for grain Fe and Zn concentration (75 %), while the trait enhancing favorable QTL alleles for yield and related traits were contributed by Swarna (76 %). The markers RM81, RM413 and RM1111, which were found to be closely linked to grain Fe and Zn concentration in the present investigation can be used in crop breeding through marker assisted selection (MAS). Co-localized QTLs were identified for grain Fe and Zn concentration on chromosome 8. QTLs for hundred grain weight and number of tillers at 30 days after transplanting were found to stable QTLs of yield related and related traits. The genomic region between the markers RM6999 and RM547 on chromosome 8 accounts for grain Zn and Fe concentrations and number of filled grains per / panicle and grain area traits, while the genomic region between the markers RM551 and RM518 on chromosome 4 accounts for kernel width, kernel length / width and grain width reflecting either pleiotropism of this locus or a complex locus with tightly linked non‐allelic genes.ThesisItem Open Access IMPROVEMENT OF MTU 1010 FOR BACTERIAL LEAF BLIGHT AND BLAST RESISTANCE THROUGH MARKER ASSISTED BREEDING(ACHARYA N. G. RANGA AGRICULTURAL UNIVERSITY, RAJENDRANAGAR, HYDERABAD, 2013) ARUNA KUMARI, K; DURGA RANI, Ch. VMTU 1010 (Cottondora Sannalu), is one of the popular rice varieties released by Andhra Pradesh Rice Research Institute (APRRI), Maruteru. It is a short duration, high yielding; long slender rice variety occupied maximum area in India particularly during Rabi season. It is susceptible to bacterial blight (BB) disease, which is endemic to many rice growing areas in India and is also susceptible to blast disease. The present investigation was attempted to introgress BB and blast resistance genes into MTU 1010 using marker assisted backcross breeding method. Improved Samba Mahsuri (ISM) or B95-1 was used as a donor for bacterial blight resistance genes, xa13 and Xa21, while NLR 145 (Swarnamukhi) was used as donor for blast resistance Pi-kh (renamed as Pi54) gene. Donor parents were validated for the target genes by using xa13-promo (functional marker) for xa13 gene, pTA248 (STS marker) for Xa21 gene and RM206 (SSR marker), Pi54 MAS (functional marker) for Pi54 gene and found that the resistant alleles were present in accordance with earlier reports. Recurrent parent and donor parents showed polymorphism for the selected target markers. Parental polymorphic study carried out between two donors and recurrent parent (MTU 1010) with 617 SSR markers. Out of 617 SSR markers, 82 markers showed polymorphism between MTU 1010 and ISM, while 83 were polymorphic between MTU 1010 and NLR 145. Fifty six markers in common showed polymorphism between recurrent parent and both the donor parents. Two crosses viz., MTU 1010 x ISM and MTU 1010 x NLR 145 were made during Rabi 2009-10 and confirmed hybrid plants were used for producing BC1F1 generation. At each backcross generation foreground as well as background analysis was carried out to identify the plant carrying target genes in heterozygous condition with maximum recurrent parent genome. Inter cross was made between two BC2F1s of MTU 1010 x ISM (female) and MTU 1010 x NLR 145 (male) to obtain ICF1. Out of 320 ICF1 plants, four plants having required three gene combination, viz., xa13, Xa21 and Pi54 in heterozygous condition were found. These four ICF1 plants were analysed to screen the recovery percent of recurrent parent genome by using parental polymorphic markers. ICF1-16th plant with recurrent parent genome (90%) was selected and selfed to produce ICF2 seed. A total of 880 F2 plants were screened and 11 triple gene homozygous plants identified. Phenotyping for BB was carried out at 55 days old seedling stage with DRR isolate. As compared to MTU 1010, BB gene introgressed plants (lines having xa13 and Xa21) exhibited very small lesion lengths indicating a very high level of resistance. In addition, the lines containing either Xa21 alone or xa13 alone also exhibited limited lesion lengths. The „triple positive‟ ICF2 plants (possessing xa13, Xa21 and Pi54 in homozygous condition) were screened with parental polymorphic SSR markers for selecting those „positive‟ plants possessing maximum recurrent parent genome. Highest value recorded in ICF2-16-59th (92%) plant. Chromosome wise analysis of the background showed complete recovery of chromosomes 3, 5, 6, 7, 9 and 10. Donor parent introgression was analysed using Graphical genotypes, in all individuals 1.0Mb region around the xa13 gene, 3.5 Mb region around the Xa21 and Pi54 gene was introgressed from the donor parents. All the ICF2 pyramided lines selected through marker assisted selection, which are having xa13xa13Pi54Pi54, Xa21Xa21Pi54Pi54 and triple positive xa13xa13Xa21Xa21Pi54Pi54 in homozygous condition were selfed and ICF3 families were screened for blast resistance at Agriculture Research Station, Nellore and APPRI, Maruteru which are hotspots for blast disease. The donor parent NLR145, which possessed Pi54 gene showed high level of resistance for rice blast with „3‟ disease score and the MTU 1010 showed a disease score „7‟ and all introgressed lines showed score between „1 and 3‟ (highly resistant). The families with three gene and two genes showed resistance to BB and blast (14 lines) were analysed for agro-morphological characters along with parent MTU 1010 was planted in RBD design. Replication data was subjected to statistical analysis by using window stat software for obtaining the CV, CD and ANOVA. ICF3-16-59 line showed statistically on par with respect to yield and yield related characters when compared with MTU 1010 besides showing resistance to both BB and blast. In this present study, ICF3-16- 235 line showed significant yield superiority over MTU 1010 coupled with BB and blast resistance can be backcrossed once with MTU 1010 to further improve the recurrent parent genome recovery as it is carrying 82% of recurrent parent background.