1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)

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    Marker assisted selection in 3-R gene pyamided lines of rice variety uma for bacterial blight resistance
    (Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2023-04-03) Bharde Pranali Rajendra; Rose Mary Francis
    The tropical humid environment prevailing in Kerala serves as a conducive environment for development and spread of bacterial blight (BB) disease in rice. The level of crop loss due to BB disease has been found to vary from 6-60%, depending on the cultivar, stage of infection, and severity of the illness under natural conditions. Uma, the popularly grown elite rice variety of Kerala, is highly susceptible to the BB pathogen. Considering the recurrent incidence of BB disease and the loss incurred by the farmers, efforts were initiated at College of Agriculture, Vellanikkara, to impart durable resistance to the cultivar Uma against BB pathogen, by pyramiding three R genes (xa5+xa13+Xa21) into the variety from donor Improved Samba Mashuri (ISM). The present study was conducted during 2019-2021 and it aimed to delineate the 3-R gene pyramids among BC3F1s (10 Nos.), through marker assisted selection. In addition, pathotyping and selfing of BB resistant plants among backcross generations; BC1F3s (725 Nos.) and BC2F2s (107 Nos.) were also envisaged. The results obtained are discussed below. Good quality DNA isolated from BC3F1s (10 Nos.) and parents (ISM and Uma) were subjected to foreground selection. Profiling of the BC3F1 individuals using the xa5 gene linked STS marker RG556 and functional marker xa5SR/R resulted in monomorphic banding pattern. In case of marker xa5SR/R, a single amplicon was found to be present in the donor (ISM), recurrent parent (Uma) as well as the BC3F1s. Four amplicons of variable size were visible on visualisation of the restriction enzyme digested PCR amplified product of STS marker RG556 in all the experimental population. The results thus indicated the endogenous presence of R-gene xa5 in both the parents and BC3F1s in the homozygous state. Screening of BC3F1 individuals with STS marker RG136 and functional marker xa13-prom linked to R-gene xa13 revealed that BC3F1 Plant No. 8.3.9.10.3 was heterozygous at xa13 locus. Further, the absence of resistant allele of xa13 was evident in the other BC3F1 individuals. Foreground selection of the BC3F1s with STS marker pTA248 linked to dominant gene R-gene Xa21 indicated the absence of gene Xa21 in the BC3F1s individuals. The results of foreground selection thus pointed out that BC3F1 Plant No. 8.3.9.10.3 was a 2-R-gene pyramid homozygous at xa5 locus but heterozygous at xa13 locus. Background profiling of the 2-R-gene pyramided BC3F1 Plant No. 8.2.9.10.3 with 42 rice microsatellite (RM) markers revealed that it possessed amplicons similar to the donor parent w.r.to ten markers (RM11069, RM11554, RM85, RM251, RM17182, RM307, RM252, RM508, RM26868 and RM260). In case of RM589 and RM23087, monomorphic banding pattern was observed in the experimental individuals. Presence of amplicons identical to that found in the recurrent parent Uma was evident in all other cases. The results thus point out that the backcross progeny BC3F1 Plant No. 8.3.9.10.3, majorly resembled the recurrent parent i.e., at 30 out of the 42 marker loci analyzed. Graphical Genotyping indicated that the 2-R-gene introgressed BC3F1 Plant No. 8.3.9.10.3 registered 75.00 per cent recovery of recurrent parent genome. Characterization of the recurrent parent Uma, donor parent ISM and BC3F1s indicated wide variability for various morphological traits studied. It was evident that the 2-R-gene pyramided BC3F1 Plant No. 8.3.9.10.3 was short statured with short but broader leaves. It was found to flower earlier (109.00 days) than the donor parent ISM (112.00 days) but was found comparable with recurrent parent Uma (107 days). It possessed longer grains (7.20 mm) than the parents (ISM: 6.62 mm; Uma: 6.80 mm). However, although broader than ISM in width, the grains were found to be slender than Uma (3.10 mm). The identified 2-R-gene pyramid (BC3F1 Plant No. 8.3.9.10.3) was selfed resulting in production of BC3F2s (149 Nos.). Bioassay of BC1F3 population (725 Nos.) through leaf clipping method (IRRI, 1996) revealed that 5.52 per cent were resistant to bacterial blight. Moderate level of resistance was observed in 23.31 per cent of the population, while 57.79 per cent of the individuals were found to exhibit moderate susceptibility to BB pathogen. BC1F3s exhibiting resistance or moderate resistance to BB pathogen were selfed. This yielded 216619 seeds of BC1F4 generation. Pathotyping of BC2F2s indicated that out of 107 progenies, 11.21 per cent were resistant against the BB pathogen, while, 36.45 per cent were found to be moderately resistant. BC2F2 individuals exhibiting moderate resistance and resistance to BB pathogen were selfed. This resulted in the production of 40535 BC2F3s. Agronomic evaluation of both BC segregants that were 1 F 3 s and BC2F2s indicated the presence of better than the parental genotypes in most of the yield attributes as well as yield. In general, majority of the backcross individuals were short in stature and took longer period to flower than the parental genotypes. To summarize, MAS helped to deduce that BC3F1 Plant No. 8.3.9.10.3 was a 2-R gene pyramid (xa5xa5 + Xa13xa13). Backcrossing of the R-gene introgressed pyramid to the donor parent is necessary to recover the R-gene Xa21. Further evaluation through a combination of MAS and phenotypic evaluation will lead to development of BB resistant cultivar in the background of cultivar Uma. The novel gene combinations arising in the advanced breeding lines (BC1F3 and BC2F2), can serve as base population for future breeding programmes.
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    Marker assisted backross breeding in two-R gene pyramid lines of rice variety Jyothi for bacterial blight resistence
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2019) Nayana Nayak; Rose Mary Francies
    Rice variety Ptb 39, also known as Jyothi, is extremely popular amongst farmers and consumers of Kerala. Despite its popularity, the variety is highly susceptible to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo). The climatic conditions prevailing in the state enhances the occurrence and spread of the bacterial blight disease. In both rice growing seasons (Kharif and rabi), spraying of antibiotics is not much effective, as it is often washed-off during the monsoon showers. Moreover, both the researchers and the consumers are concerned about the food safety aspects as well as the negative impact of plant protection chemicals on the environment. Hence, the best alternative is to exploit host-plant resistance by pyramiding multiple R-genes to impart durable and broad- spectrum stable resistance to varieties against the pathogen. Considering the impact of the BB disease on food security and sustainability, efforts to introgress the three R-genes (xa5, xa13 and Xa21) into the elite cultivar Jyothi (Ptb 39) through Marker Assisted Selection (MAS) were initiated at College of Horticulture, Vellanikkara. Improved Samba Mahsuri (ISM) was used as the donor parent. The present study aimed to identify R-gene pyramided plants in BC1F₁ population and to develop stable pyramided BC2F1 lines of variety Jyothi with low linkage drag from the donor parent (ISM). It also aimed to evaluate the BC2F5 population for resistance to BB pathogen (pathotyping) and production of next-generation backcross populations (BC2F1s, BC1F2s and BC2F6). The good quality DNA, isolated from BC1F1s (24 Nos.) and parents (ISM and Jyothi) was subjected to foreground selection to identify the plants introgressed with the three R-genes, using markers RG556 and xa5 SR, RG136 and xa13 promoter, and pTA248 linked to xa5, xa13 and Xa21, respectively. Monomorphic banding pattern was observed in all the BC1F1s as well as the parents on analysis with markers RG556 and xa5 SR, pointing to the endogenous presence of R-gene xa5 in all the individuals tested. Screening of BC1F1 individuals with STS marker RG 136 linked to R-gene xa13 and functional marker xa13 promoter revealed that the three BC1F1s i.e., Plant No. 9.7, Plant No. 9.15 and Plant No. 9.17 were heterozygous at xa13 locus while, all other BC1F1 individuals possessed alleles similar to that of the recurrent parent (RP) Jyothi. The result thus pointed out that BC1F1 Plant No. 9.7, Plant No. 9.15 and Plant No. 9.17 were 2-R-gene pyramids (xa5xa5 + Xa13xa13). Foreground selection with STS marker pTA 248 to detect the presence of Xa21 gene revealed that the above mentioned three BC1F1s were heterozygous at Xa21 locus. Results of foreground selection, thus indicated that three BC1F1 plants, i.e., Plant No. 9.7, Plant No. 9.15 and Plant No. 9.17 were the only 3-R-gene pyramids (xa5xa5 + Xa13xa13 + Xa21xa21) among the 24 BC1F1 individuals investigated. The other BC1F1s possessed only a single recessive R- gene xa5. Background selection of the three BC1F1 3-R gene pyramids was done using 58 microsatellite markers. The profiles revealed that BC1F1 Plant No. 9.7, Plant No. 9.15 and Plant No. 9.17 were similar to recurrent parent Jyothi and donor parent ISM at 24 and 21 marker loci, 22 and 21 marker loci, and 34 and 12 marker loci, respectively and heterozygous at 13, 15 and 12 marker loci respectively. Dendrogram based on molecular data, further suggested that the 3-R-gene introgressed BC1F1s were more similar to the recurrent parent Jyothi than to the donor parent ISM. The recovery of the recurrent patent genome was found to be the highest in Plant No. 9.17 (93.00 %) while, it was 89.00 per cent each in Plant No. 9.7 and Plant No. 9.15. Morphological characterisation revealed that the 3-R-gene introgressed BC1F1s Plant No. 9.7 and Plant No. 9.15 and Plant No. 9.17 exhibited about 60 per cent similarity to the parents Jyothi and ISM. BC1F1 Plant No.9.17 was near similar to the recurrent parent Jyothi with respect to the days to flowering, plant height, seed length and width as well as kernel colour. However, the length of panicles and the number of spikelets per panicle in the selected BC1F1s were found to be less than that of the parents. The identified 3-R-gene pyramids (BC1F1 Plant No. 9.7; 9.15 and 9.17) were backcrossed to recurrent parent Jyothi as well as selfed resulting in production of BC2F1s (5 Nos.) and BC1F2s (220 Nos.). Wide variability was observed among the BC2F5 individuals (111 Nos.) for various morphological traits. Pathotyping of BC2F5 population through leaf clipping method, as suggested by IRRI (2002), revealed that 11.71 per cent (13 Nos.) of the progenies exhibited resistance reaction to BB infection, while, 10 (9.00 %) were moderately resistant, 24 (21.62 %) moderately susceptible, 21 (18.91%) susceptible and 43 (38.73 %) highly susceptible. Selfing of the BC2F5 individuals exhibiting resistance and moderate resistance to BB pathogen resulted in production of 1425 BC2F6 seeds.