Marker assisted backross breeding in two-R gene pyramid lines of rice variety Jyothi for bacterial blight resistence

Abstract

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.