Browsing by Author "ANURADHA, G"
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ThesisItem Open Access CONFIRMATION AND FINE MAPPING OF MAJOR QTL FOR GRAIN SIZE IN BASMATI RICE(ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY, 2011) ANNE KITTY DEBORAH, D; ANURADHA, GBasmati rice is a unique varietal group that has gained wider acceptance as a speciality rice all over the world by virtue of its unique quality traits. Grain size plays a crucial role in determining the grain quality in Basmati rice. Genetic control of basmati grain quality traits is quite complex, but breeding of new varieties having Basmati grain quality characters can be greatly facilitated by the use of molecular markers tightly linked to these traits. Hence, in the present investigation 155 recombinant inbred lines (RILs) developed from a cross between Basmati370 and Jaya were used for confirmation and fine mapping of a major QTL for grain size in Basmati rice using SSR markers derived from inside and near the genomic region associated with grain size in the marker interval of RM289 and RM18600 on chromosome 5, that was already identified by Vemireddy (2008) using F2 population developed from the same cross. The parents used for mapping, Basmati370 and Jaya differed significantly with respect to all the three traits viz., grain length (GL), grain breadth (GB) and Length- Breadth ratio (LBR). Transgressive segregation of 45.8 % in grain length, 21.93 % in grain breadth and 12.25 % in LB ratio was observed in the RILs. All the three traits exhibited normal distribution in the RIL population indicating quantitative inheritance of these traits. Correlation analysis among the grain characters studied in RILs revealed a highly significant negative association of LB ratio with grain breadth and significant positive correlation with grain length. Out of 52 SSR and 1 INDEL markers used, 23 (43.40 %) were polymorphic, 27 (39.62 %) were monomorphic and 9 (16.98 %) were not amplified between Basmati370 and Jaya. The RILs were genotyped for these 23 polymorphic markers and the linkage map employing phenotypic and genotypic data of the RILs was constructed using MAPMAKER v 3.0. The QTLs were identified using interval mapping (IM) and composite interval mapping (CIM) methods of QTL Cartographer v 2.5 software with 2.5 as LOD threshold for detecting a QTL. A minor QTL for grain length qGL5.1 was identified by IM in the marker interval of RM6024 and RM1237 with Phenotypic Variance (PVE) of 3.7 %. A single QTL designated as qGB5.1 was detected for grain MAPMAKER v 3.0. The QTLs were identified using interval mapping (IM) and composite interval mapping (CIM) methods of QTL Cartographer v 2.5 software with 2.5 as LOD threshold for detecting a QTL. A minor QTL for grain length qGL5.1 was identified by IM in the marker interval of RM6024 and RM1237 with Phenotypic Variance (PVE) of 3.7 %. A single QTL designated as qGB5.1 was detected for grain breadth in the marker interval of RM1237 and RM18582 with PVE of 3.58 % in CIM and 4.51 % in IM and a QTL qGLB5.1 for Length- Breadth Ratio was identified with PVE of 11.8 %. The genetic distance of the flanking markers harbouring QTL cluster in the previous study conducted by Vemireddy (2008) was 26.5 cM whereas in the present study it was narrowed down to 15.7 cM. The physical distance also has come down from 11,128 kb to 685 kb. Comparision of rice genome database revealed that LOC_OS05g31920 (zinc ion binding protein), LOC_OS05g31930 (retrotransposon protein, putative, unclassified), LOC_OS05g31940 (retrotransposon protein, putative, unclassified), LOC_OS05g31950 (retrotransposon protein, putative, Ty3-gypsy subclass), LOC_OS05g31959 (hypothetical protein) and LOC_OS05g31970 (expressed protein) genes are present at this region. Interestingly, two predicted genes closure to this region viz., AP2 transcription factor and RING E3 ligase were reported to be involved in controlling the seed size and weight by earlier studies. In the present study, one microsatellite marker i.e., RM18582 showed close association with the grain size QTLs. This marker has potential to be used in markerassisted improvement of the grain size in Basmati rice. Though the present study was carried out in single environment, together with the results of F2 population of the same cross, it could be concluded that these three QTLs associated with grain size traits of Basmati could be considered as stable QTLs. These QTLs, apart from their suitability in improvement of the traits concerned, can also serve as potential candidates for fine mapping. These stable QTLs also facilitate development of Near‐isogenic lines and advanced breeding lines.ThesisItem Open Access FINE MAPPING OF DOMINANT RICE GALL MIDGE RESISTANCE GENE, Gm4 USING MICROSATELLITE MARKERS(ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD, 2004) VANI GUBBALA; ANURADHA, GRice gall midge is one of the serious insect pests of rice. Chemical control of the pest is difficult and sometimes impossible and cultivation of varieties possessing one or more genes conferring resistance against gall midge is the economically feasible and ecologically viable strategy for its management. Many varieties possessing single gene conferred resistance have become gall midge susceptible due to emergence of virulent biotypes of the insect. Hence, many researchers have suggested pyramiding 2-3 gall midge resistance genes in a single genetic background for enhancing the durability of resistance. The dominant resistance gene, Gm4 originally identified from Ptb10 is an ideal candidate for deployment in gene pyramiding programmes. Gm4 was originally tagged with RAPD based marker F43 and mapped to Chromosome 8 of rice using RFLP markers by Nair et al. (1996) and Mohan et al. (1997) respectively. But none of these markers have been validated in alternative populations nor used in any gall midge resistance breeding programmes. The present study was designed to fine map Gm4 with help of rice microsatellite markers specific for Chromosome 8 of rice using the progeny tested F2 mapping population (consisting of 98 individuals) derived from the cross TN1/Abhaya. About 15-18 F3 seedlings derived from each F2 line were screened in a glasshouse using biotype 1 of gall midge to identify the genotype of each F2 line. Resistant F3 seedlings were observed to show presence of additional tiller and on dissection, they possessed necrotic brown discoloration in the meristematic region. Susceptible F3 seedlings showed gall formation and emergence of adult insect after 20th day of infestation. Twenty-six microsatellite markers located on Chr. 8 were screened for molecular polymorphism between the parental lines (TN1 and Abhaya) and six (RM547, RM25, RM337, RM152, RM256, RM32) were observed to be parental polymorphic. These six markers were analyzed in the F2 population for co-segregation with trait phenotype. Of the six markers, only two, RM547 and RM25 were observed to exhibit clear cosegregation with trait phenotype (resistance/susceptibility). Based on the cosegregation analysis, the linkage distance between the two markers and Gm4 was calculated to be 18.6 and 30.6 cM respectively with respect to RM547 and RM25. The other markers did not show any clear co-segregation pattern. A linkage map of Chr. 8 consisting of the five (excluding RM32) parental polymorphic microsatellite markers has been constructed with a total linkage distance of about 133.1cM. The two microsatellite markers RM547 and RM25 after further validation in a larger sized mapping population can be used for marker-assisted selection of the gene.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 LARGE SCALE PRODUCTION OF Cry 1Aa PLANTS IN SORGHUM(ACHARYA N. G. RANGA AGRICULTURAL UNIVERSITY, RAJENDRANAGAR, HYDERABAD, 2005) KAVITHA, A; ANURADHA, GSorghum is an important dryland crop used as food, feed and fodder. The increasing shift in the demand for sorghum from staple food to industrially preferred commodity in poultry, bakery and brewing industries requires large scale production according to the needs of different sectors. Therefore, it is important to develop and deploy novel technologies such as genetic transformation to increase the grain quality and resistance to pests that are difficult to breed by conventional means. Two principal methods viz., Agrobacterium infection and particle bombardment were used for genetic transformation. In vitro response of sorghum was low, monocalcitrant and was highly genotype dependent. Tissue culture and regeneration response was low in sorghum when compared to other cereals. Agrobacterium method of transformation was preferred because of its cost efficacy and low copy number. But regeneration of explants was very low due to overgrowth of Agrobacterium. Hence during the course of study alternate methods of Agrobacterium infection were studied using two inbred lines AKMS 14B and 296B. Different media adjuvants were studied to control Agrobacterium growth. AgNO3 and CuSO4 were effective in controlling the bacterial growth. Gus expression was studied in cotransformation experiments using two individual genes viz., pDM805 and pCAMBIA 3300 cry 1Aa. Gus results revealed that they were independently transformed without influencing each other. A non tissue culture approach was done to produce transformants at large scale. Though transient gus expression was present in the immature embryos, mature seeds were not selected in basta screening. Putative transformants were evaluated through basta spray tests and PCR analysis using bar and Cry 1B primers. Results of basta swab test were in agreement with PCR results.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 MAPPING OF GENE(S) FOR RESISTANCE TO POWDERY MILDEW IN SESAMUM (Sesamum indicum L.)(ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY, 2011) SRAVANI, DINASARAPU; ANURADHA, GPowdery mildew is a serious disease of sesamum (Sesamum indicum L). It occurs in epidemics under heavy rainfall conditions followed by low night temperatures and high humid conditions. Powdery mildew causes yield loss of 25 to 50% depending upon the severity. Classical breeding for incorporation of resistance using backcrossing is difficult because consistent disease reaction in the field is hard to achieve due to environmental factors. Identification of molecular markers linked to powdery mildew resistant gene(s) is one approach to improve selection for resistant cultivars. The objective of this study was to identify the mode of inheritance of disease resistance and to use microsatellite (SSR) markers to identify markers linked to resistance gene(s) and map gene(s) resistance to powdery mildew. In the present study Swetha til, a high yielding popular white seeded variety but susceptible to powdery mildew and BB3-8 accession of Sesamum mulayanum resistant to powdery mildew from RARS, Jagtial were selected as parents for hybridization. F1 was developed by crossing the resistant parent ( BB3-8 accession of Sesamum mulayanum ) and the susceptible parent (Swetha til) during Late summer, 2009. F1 was selfed during Kharif 2010 to generate F2. Four populations including P1, P2 , F1, F2 were sown during Late summer, 2010 to screen powdery mildew under field conditions. The F1 of the cross between Swetha til and BB3-8 accession of S.mulayanum exhibited susceptible reaction to the disease suggesting resistance to be governed by recessive gene. On screening of 104 plants of F2 population, the plants segregated into 61 susceptible and 43 resistant plants. Chi-square analysis showed the observed ratio to confirm the expected ratio of 9:7 ratio (susceptible : resistant) and the resistance to disease was governed by two pairs of recessive genes contributed by S.mulayanum. The two parents were screened for parental polymorphism using 300 sesame microsatellite markers, of which 240 showed clear amplification pattern and 24 markers exhibited polymorphism (10%) and the same were used for genotyping. DNA from 104 F2 progenies were collected for genotyping with 24 Polymorphic SSR primers. Molecular linkage map was constructed with SSR markers SM10 178 and SM10 176 using the MAPMAKER/EXP version 3.0 Both the markers were mapped to one and the same linkage group at a distance of 14.2 cM. These two markers were found to be linked to the powdery mildew resistance gene.ThesisItem Open Access TAGGING OF GENE FOR RESISTANCE TO POST FLOWERING STALK ROT IN MAIZE(Zea mays) CAUSED BY Macrophomina phaseolina(ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD, 2010) SUNEETHA, P; ANURADHA, GMaize is one of the most important economic cereal crops and an ideal forage crop. It occupies a prominent position in global agriculture. 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.The productivity of maize is influenced by both biotic and abiotic factors. Maize suffers from about 110 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. Stalk rots take a heavy toll, among which stalk rots caused by Macrophomina phaseolina (Tassi) Goid and Fusarium moniliformae results in 30-40% losses. Post flowering stalk rot (PFSR) is the complex disease caused by three fungi,viz., Cephalosporium acremonium, Macrophomina phaseolina, Fusarium moniliformae and one bacterium Erwinia carotovora var zeae. Out of these, post flowering stalk rot caused by Macrophomina phaseolina is the important disease of maize in the state of Andhra Pradesh. Author : P.SUNEETHA Title of the thesis : “TAGGING OF GENE FOR RESISTANCE TO POST FLOWERING STALK ROT IN MAIZE(Zea mays) CAUSED BY Macrophomina phaseolina” Degree to which it is submitted : MASTER OF SCIENCE IN AGRICULTURE Faculty : AGRICULTURE Department : AGRICULTURAL BIOTECHNOLOGY Major Advisor : Dr. G.ANURADHA Principal Scientist (Breeding) Biotechnology Unit Agricultural Research Institute Rajendranagar, Hyderabad – 30. University : ACHARYA N. G. RANGA AGRICULTURAL UNIVERSITY Year of submission : 2010 In order to tag the post flowering stalk rot resistant gene maize inbred lines BPPTI-34 (resistant) and BPPTI -66(Susceptible), were crossed to produce F1. F1’s were selfed as well as back crossed to the susceptible parent to derive F2 and BC1F1 populations respectively. Parents P1 and P2, F1 and two mapping populations F2 and BC1F1 were artificially inoculated with the Macrophomina phaseolina culture. F1s inoculated with the culture showed resistant reaction revealing for that resistance for post flowering stalk rot is governed by dominant gene. F2 population segregated in 3:1 ratio i.e 87 resistant: 27 susceptible and BC1F1 population segregated in the ratio of 1:1 i.e 26 resistant: 24 susceptible showing that resistance to post flowering stalk rot is governed by single dominant gene. A total of 150 microsatellite markers distributed on entire genome were used to screen the parents. Of these, 54 SSR markers from ten chromosomes were found polymorphic in the parents. These fifty four markers were used to screen the bulk DNAs prepared from 10 plants each of resistant and susceptible plants from F2 and BC1F1 populations to find the markers linked to the resistance gene. By bulked segregant analysis (BSA) the marker umc 1269 clearly distinguished resistant and susceptible bulks as that of resistant and susceptible parents indicating that this marker is tightly linked to the gene for resistance to post flowering stalk rot.