Marker assisted improvement of super fine grain rice variety, VGD 1 for bacterial blight resistance

doi:10.21203/rs.3.rs-4716099/v1

Background

Super fine grain rice variety, VGD 1 (ADT 43 x Seeragasamba) is land mark variety in India for its grain quality traits. However, it is highly susceptible to bacterial blight (BB) disease. It is pertinent to improve the disease resistance without altering grain quality traits and high yielding capacity. We introgressed broad spectrum BB resistance genes, xa5, xa13 and Xa21 into VGD1 through marker assisted selection. Genome wide SSR markers were used to recover the recurrent genome contribution. Foreground selection was carried out at BC₁F₁, BC₂F₁, BC₂F₂ to BC₂F₄. Background screening was performed for RPG recovery at BC₁F₁, and BC₂F₁ generations.

Results

True breeding lines in BC₂F₄ were confirmed for targeted genes; screened for bacterial blight disease; and they were evaluated for agronomic and grain quality traits. Foreground selection of BC₁F₁ revealed four positive plants (Plant-7, plant-18, plant-36, and plant-58) for targeted bacterial blight R genes which were subjected to background selection and genotype (Plant-18) with high RPG (78.44%) was backcrossed to obtain BC₂F₁. In BC₂F₁ generation, two positive plants (Plant 10 and Plant 12) were screened for polymorphic SSR markers where plant-12 with RPG of 86.20% was advanced to BC₂F₂. Phenotypic screening of BC₂F₄ breeding lines for bacterial blight disease revealed that the seven lines with three genes pyramided were resistant showing the mean lesion length ranging from 0.76 ± 0.18 to 2.26 ± 0.04.

Conclusion

The superfine variety, VGD 1 was enhanced with xa5, xa13 and Xa21 genes for bacterial blight resistance. Seven pyramided lines were identified with high resistant to BB and grain quality similar to VGD1. The line 56 is promising with all desired agronomic and quality traits with improved resistance and higher yield than VGD1 was identified.

Rice

fine grain

bacterial blight

marker assisted selection

Rice is categorised as one of the major and essential staple food crop for more than 50% of world population. As the population is increasing at a faster rate, agricultural productivity also needs to be rapidly increased (Simon et al., 2023). Notably, rice plants are inevitably encountering pressing challenges from different types of biotic/abiotic stresses that cause significant rice grain yield reduction (Khush, 2005; Dixit et al., 2020). Biotic stresses caused by pests and diseases pose a significant risk to global rice yield production by 52%, of which approximately 30% of these damage are due to pathogen infection (Jamaloddin et al., 2021). Among the various biotic stresses, bacterial blight disease was caused by Xanthomonas oryzae pv. oryzae (Xoo), which is one of the most destructive serious diseases in rice which reduces the yield by 30% (Mishra et al., 2023). The extent of damage is determined by crop stage, environmental circumstances, and disease severity (Ou 1985). In order to reduce the damage and yield loss, host plant resistance has to be maintained which is very effective control of bacterial blight. Till date, 46 bacterial blight resistance (R) genes are identified from diverse sources. Out of them, eleven R genes have been characterised and cloned (Fiyaz et al., 2022). Both conventional and molecular breeding strategies for BB resistance attempt to improve the genetic foundations of rice cultivars and the BB pathogen for host plant resistance (Li et al., 2020). The level of resistance provided by a single gene is challenged by natural allelic differences in Xoo; therefore, the pyramiding of two or more efficient resistance genes is necessary for broad-spectrum and long-lasting resistance to Xoo under field circumstances. The popular broad spectrum BB resistance genes xa5, xa13 and Xa21 has already been introgressed into popular varieties across globe (Perumalsamy et al., 2010; Ramalingam et al., 2020; Sundaram et al., 2008; Singh et al., 2001) and parental line of hybrid rice (Ramalingam et al., 2017) and found very promising. The genotypes, PR106 and Jalmagna (Singh et al., 2001; Pradhan et al., 2015), and CMS lines (Ramalingam et al., 2017). Jyothi, IR50, Mahsuri, PRR78, KMR3, and Pusa6B had BB genes (Xa4, xa5, xa13, and Xa21) (Bharathkumar et al., 2008, Guvvala et al., 2013; Shanti et al., 2010) were improved for BB resistance using xa5, xa13, and Xa21 combinations through MAS (Ramalingam et al., 2020). The conventional methods are hindered by the masking effect has been occurred for dominant and recessive genes in combination while pyramiding of genes will helpful to overcome this issue.

Once the genetic factors conferring biotic stress resistance are identified, they can be easily and effectively transferred to the target background varieties by marker-assisted selection (MAS) (Simon et al., 2023). Although this technique has been proven as a potent tool to evaluate the disease resistance traits such as BB, sheath blight, and blast resistance, which are expensive and difficult to analyse in a short period of time (Tanksley et al., 1989). Marker assisted backcross breeding (MABB) is most effective for transferring BB resistance genes/QTLs into rice varieties (Basavaraj et al., 2009, Gopalakrishnan et al., 2008). MABB employs background selection that focuses on accelerating the genomic recovery of recipient parent genotype (RPG) with the help of markers distributed throughout the entire genome (Xu et al., 2012). Any crop improvement program’s key criterion is the selection of requisite genotypes with all desirable grain quality and yield contributing attributes.

Among the cloned genes, xa5, xa13 and Xa21 are being used in marker assisted breeding widely either alone for BB resistance or in combination with genes/ QTLs conferring tolerance to other stress, more than 70 varieties of rice or hybrid parental lines are improved (Fiyaz et al., 2022). xa13, a fully recessive gene, identified in cultivar BJ1 and mapped on chromosome 8 (Zhang et al., 1996); which was correlated to a plasma membrane protein, which confers recessive resistance to PXO99 (Chu et al., 2006). On the subtelomeric region of chromosome 5, xa5, abroad spectrum recessive resistant gene was mapped (Blair et al., 2003); this gene encodes for gamma transcription factor-like protein (TFIIAγ) and when it is pyramided with other dominant genes, it provides long-lasting resistance to Xoo (Huang et al., 1997). Xa21 is a dominant resistant gene which encodes a leucine-rich repeat (LRR) receptor kinase-type gene, mapped on the long arm of 11th chromosome (Song et al., 1995). A PCR -based highly efficient co-dominant molecular (pTA248) was developed for marker assisted selection of Xa12(Ronald et al., 1992). Overall to improve the genetic potential, the present study focussed on transferring bacterial blight resistant genes in to the local elite cultivar VGD1, a high-yielding fine grain variety preferred by people for biryani making with good market value through marker assisted backcross breeding and also to identify and evaluate true breeding lines against bacterial blight disease and grain quality traits.

Plant Materials

The recurrent parent is VGD1, a cross derivate of ADT43/ Seeragasamba is having superfine grain variety with high yield and high marketing value make this variety mostly preferred for biryani making (Mohan et al., 2021). Notably, VGD1 is highly substitute to bacterial blight. The donor, ADT55 is the first rice variety developed for bacterial blight (BB) resistance by TNAU using functional marker-assisted breeding. It harbours xa5, xa13, Xa21 bacterial blight resistance genes. The variety demonstrated strong resistance to almost all the 11 races of Xoo (Perumalsamy et al., 2010 and Sakthivel et al., 2017).

Introgression of bacterial blight resistance genes

VGD1 (female parent) and ADT 55 (male parent) were crossed to generate F₁ hybrid population. The genotypes of progenies were examined by deploying marker-assisted selection. After evaluation, true F₁’swere backcrossed to recurrent parent (VGD 1). In the present study after analysing for targeted genes, first backcross progenies (BC₁F₁) were subjected to background selection and selected individual was backcrossed to generate second backcross progenies. The BC₂F₁ progenies were genotyped and individuals with all three BB resistance genes (xa5, xa13 and Xa21) in heterozygous condition were sorted out and the plant with the highest parent genome was selfed to generate segregating BC₂F₂ population and forwarded to BC₂F₂-F_4.

Foreground selection

The identification of bacterial blight resistance genes (xa5, xa13 and Xa21) in each generation were utilized Functional/linked markers, pTA248, xa13 prom and xa5-1 for the genes xa5, xa13 and Xa21 respectively in foreground selection (Table 1).

Molecular analysis

Molecular analysis conducted for foreground selection includes a series of prescribed steps with proper protocols i.e., genomic DNA extraction, quantification and dilution, Polymerase chain reaction (PCR), Gel electrophoresis, and documentation. The DNA extraction was carried out by CTAB (Cetyl Trimethyl Ammonium Bromide) method (Dellaporta et al., 1983). The PCR protocol for marker-assisted selection of targeted genes/QTLs was followed according to the earlier reports (Channamallikarjuna et al., 2010; Ramkumar et al., 2011). Ten microliters of PCR reaction mixture contain 4 µl of Dream Taq green 2PCR master mix (Thermo scientific, United States), 4 µl of nuclease free water, 50 ng of template DNA, and 30 ng each of forward and reverse primers. To find the specific allelic pattern of xa5 allele, 5–10 µl of PCR product is digested with BsrI (5 units of enzyme) at 65°C for 4 h with 2 µl of 10× PCR buffer (IyerPascuzzi and McCouch, 2007). For xa5, after PCR, restriction digestion with BSR1 enzyme @ 65°C for 4 hours was done.

The cyclic conditions for PCR reaction for functional/linked markers for foreground selection xa5-1 involved an initial denaturation step at 94°C for 5 min, followed by 35 cycles of amplification at 94°C for 45 sec, 56°C for 1 min for primer annealing and 72°C for 1:30 min for extension. The final extension was carried out at 72°C for 7 min; whereas xa13-prom confers a 94°C for 5 min for initial denaturation, 94°C for 1 min for denaturation followed by 58°C for 1 min for primer annealing and 72°C for 1:30 min for extension, final extension was carried out at 72°C for 7 min for 35 cycles and pTA248 has initial denaturation at 94°C for 5 min, followed by 30 cycles of amplification at 94°C for 45 sec, 58°C for 1 min for primer annealing and 72°C for 1 min for extension. The final extension was carried out at 72°C for 10 min and finally infinite hold at 4°C.Electrophoresis was the technique used to separate amplified PCR products on 2.5 % agarose gel stained with ethidium bromide in 1x TBE buffer. The wells of the solidified agarose gel were loaded with 5 microliters of amplified PCR products and a 2µl of 100 bp DNA ladder. One and a half hours were spent running the gel at 100 volts. A gel documentation unit (Bio-Rad), UV transilluminator was used to visualise bands. The banding pattern was scored visually by comparing it with a 100bp DNA ladder.

Bioassay against bacterial blight disease resistance in BC₂F₄ generation

The selected BC₂F₄ breeding lines based on genotype and phenotype were screened for resistance. To test the efficacy of three-gene pyramided lines, two lines that are with single a gene and two genes of the same were examined. Three replications were maintained in the greenhouse and the leaf clipping method (Kauffman 1973) was followed for inoculating Xoo culture. As the blight disease progressed for each injected leaf up to 21 days post inoculation (dpi), the lesion length was determined. It was defined as the distance between the tip cutting edge and the leading edge of greyish to chlorotic symptoms. Finally, such mean lesion length was recorded, and disease score was given following International Rice Research Institute (IRRI), Standard evaluation system (SES) for rice

Evaluation of agronomic and quality traits

Major agronomic traits considered against yield for this study are plant height, number of productive tillers per plant, panicle length, 100-grain weight, and yield per plant. The BC₂F₄ breeding lines, which were opted by genotyping and phenotyping, has been evaluated for agronomic traits along with their parents. For the analysis randomly chosen five plants from every replication in each line were used to record the observations and the mean value was calculated in a randomized block design with three replications. The mean data collected was subjected to analysis for calculating variance, standard deviation, standard error, and critical difference using standard formulae. The identified homozygous resistant genotypes were examined to determine the grain type in BC₂F₂. The quality parameters studied are Kernel Length (KL), Kernel breadth (KB), and Kernel Length/breadth ratio (LBR), where KL and LBR are studied. Breeding lines in BC₂F₄ generation were also subjected to evaluation, for grain type along with evaluation of amylose content.

The introgression of three BB resistance genes into VGD1 was achieved via a crossing scheme (Figure 1) where in ADT55 (xa5, xa13 and Xa21 genes) was used as the donor parent. This cross made to develop a F₁ population. True F₁’s were confirmed for all the three bacterial blight R genes in heterozygous condition. They were crossed with recurrent parent to generate a backcross population (BC₁F₁) as well as selfed to raise segregating population (F₂). Notably, utilizing marker assisted backcross breeding (MABB) strategy, analysis of backcross generations (BC₁F₁, BC₂F₁, BC₂F₂, BC₂F₃andBC₂F₄). The results of the parental polymorphism survey revealed a significant level of polymorphism in BC₁F₁ and BC₂F₁ generations have high recovery parent genome (Table 2; Table S1).

In foreground selection, heterozygous true F₁’s identified from genotyping, using functional/linked markers for bacterial blight (BB) resistance genes, were backcrossed to VGD1, to develop the BC₁F₁ population. Foreground selection for targeted genes was taken up for 63 BC₁F₁ plants. Four plants (Plant-7, Plant-18, Plant-36, Plant-58) were heterozygous for each of the three genes (xa5, xa13 and Xa21); whereas in BC₂F₁ the Selected plant-18 was backcrossed with VGD1. Progenies (BC₂F₁) were grown, and they were put forth for positive selection. Only two plants (Plant-10 and Plant-12) were found to be heterozygous for all three (Xa21, xa13 and xa5) genes out of 22 BC₂F₁ plants genotyped.

Segregating population (BC₂F₂) was developed from plant-12 of BC₂F₁ generation. One hundred and forty-nine (149) plants were raised. All of them were initially subjected to positive selection for two genes i.e., Xa21, and xa13and results revealed 26 homozygous resistant plants. Only those 26 plants that displayed resistance to both the genes were subsequently evaluated for xa5 genes and fifteen plants were found resistant. In conclusion, a total of 15 plants out of 149 are homozygous for all the resistance genes of bacterial blight (Xa21, xa13 and xa5). Seven BC₂F₄ breeding lines were confronted to inspect for all three resistance genes (xa5, xa13 and Xa21) in homozygous conditions. Breeding lines were raised along with their parents in three replications using a randomised block design. Five random plants in each line were analysed using PCR based linked/functional markers (xa5-1, xa13-prom and pTA248) (Figure 2). Results unveiled the homozygous condition for the (xa5, Xa21 and xa13) three resistance genes for all the plants in each of the seven BC₂F₄ breeding lines

Screening of BC₂F₄ breeding lines for bacterial blight disease

Male parent ADT55 which was a resistant check exhibited a mean lesion length of 0.85 ± 0.35 with the disease score-2 (Resistant) and the female parent VGD 1, the susceptible check showed a mean lesion length of 17.5 ± 0.23 with the disease score 8 (Susceptible) (Figure 3; Table 3). All the seven lines with three genes (xa5, xa13 and xa21) pyramided were scored as resistant showing mean lesion length ranging between 0.76 ± 0.18 and 2.26 ± 0.04. In addition, the line with two genes pyramided i.e., Xa21 and xa13genes was scored as moderately resistant with a mean lesion length of 9.7 ± 2.05, given the disease score of 5. The line carrying the Xa21R gene alone was observed with an LL of 11.5 ± 0.80

In BC₁F₁ the background selection, 58 polymorphic markers were examined by performing parental polymorphic analysis by using 166 rice microsatellite markers between the parents VGD 1 and ADT 55. Plant-7, plant-18, plant-36, and plant-58 of BC₁F₁ generation, which are heterozygous were subjected to background analysis and recurrent parent genome (RPG) recovery percentage was calculated. The genome recovery percentage was ranged from 72.42% to 78.44% and Plant-18 showed the highest RPG recovery of 78.44%. Hereby, Plant-18 was adopted for backcrossing to develop BC₂F₁ generation. Furthermore, in BC₂F₁ Positive plants (Plant-10 and Plant-12), heterozygous for all three genes were subjected to background selection using 58 polymorphic SSR markers for recurrent parent genome recovery. The percentage of RPG was 82.75% for plant-10 and 86.20% for Plant-12. Plant-12 showed the highest recovered parent genome recovery (Figure 4; Table 4). Therefore, Plant-12 was tagged and selfed seeds were collected to generate BC₂F₂ segregating population.

Characterization of major agronomic traits and grain quality

Major agronomic traits were studied for seven breeding lines in comparison with recurrent and donor parents (Table 5). In BC₂F₄ plant height ranges from 82.6 ± 0.5 cm to 89.4 ± 0.9cm; whereas three lines (BC₂F₄-11, 56, and 112) were significantly taller than the recurrent parent, where plant, F₄-112, with mean PH of 89.4 ± 0.9 cm recorded as the tallest height. The highest number of productive tillers was recorded for the sixth F₄ breeding line (F₄-56) line. The panicle length of progenies ranged between 23.1 ± 0.8 and 25.2 ± 0.8. The lowest seed weight was recorded for the fourth F₄ breeding (F4 -56) line (9.50 ± 0.08) which was closely like the recurrent parent.Mean single plant yield was higher than the donor parent for all the seven homozygous lines, i.e., F₄-4 with 34.70 ± 0.34, F₄- 11 with 34.18 ± 0.38, F₄-12 with 32.60 ± 0.65, F₄-56 with 35.60 ± 0.40, F₄-94 with 32.30 ± 0.80, F₄-112 with 34.43 ± 0.92 and F₄-123 with 31.24 ± 0.31. The highest mean single plant yield was recorded for the F₄-56 breeding line (35.60 ± 0.40) which was superior to the recurrent parent (VGD 1).

Grain traits on kernel length, kernel breadth, kernel length/breadth ratio and amylose content were evaluated in BC₂F_4. On the basis of kernel measurements, the grain type was deduced. According to standard classification, the Kernel length and length/breadth ratio were plotted to decide the grain type. The female parent VDG 1 had a short bold grain type and the male parent, ADT 55 had a long slender grain type. According to mean observations recorded, all the seven F₄ breeding lines were scored as short bold i.e., they are like the preferred parent grain type VGD 1. Amylose content was estimated to be intermediate in VGD 1 (22.5 %) and for ADT 55, it was high (25.5). Six (F₄-11, F₄-12, F₄-56, F₄-94, F₄-112, and F₄-123) out of seven F₄ breeding lines were grouped to have intermediate amylose content, values ranging from 21.4 to 24.9, similar to VGD 1. The remaining breeding line F₄-1 is with high amylose content (25.2 %).

‘Rice is life’, since the daily dietary needs of more than half of the world’s population are met by rice. Numerous studies have found that in order to meet the rising demand for rice, the world’s rice supply must be doubled by 2050 (Ray et al., 2013). Many popular varieties are being improved according to farmer’s and people’s choices, who prefer fine-grain rice cultivars with strong market prospects. One such variety was VGD 1 from Tamil Nadu, a popular variety with good market values having a consumer-preferred characteristics. It has short grain having a good linear elongation ratio (LER) of 2.1 and with intermediate amylose of 21.9%. The cooked rice is tender and non-sticky, which is happily preferred for biryani and kushka making (Mohan et al., 2021). However, this cultivar is susceptible to bacterial light (BB), which is the major disease that results in significant losses in yield. Numerous rice varieties exhibiting broad spectrum defence against Xanthomonas oryzae pv oryzae (Xoo) isolates were created using MAS. Recently, more than 70 cultivars of rice or parental lines are improved, by cloned genes xa5, xa13 and Xa21, either alone for BB resistance or in combination with genes/QTLs providing tolerance to other stresses (Fiyaz et al., 2022). Embracing these steps, the current study envisaged to enhance bacterial blight resistance in VGD 1 through the introgression of BB resistance genes (xa5, xa13, and Xa21) by MAS without compromising grain quality traits. Even though multiple resistance sources are available for BB, used ADT 55 as a donor as it exhibits potential resistance to 11 races of Xoo, possessing xa5, xa13 and Xa21 BB resistance genes. A cross was made between VGD 1 and ADT 55, progenies were developed and true F₁’s were advanced to follow both marker assisted backcross breeding and marker assisted pedigree breeding. Foreground selection was done using linked/ functional markers xa5-1, xa13 prom, and pTA248 for xa5, xa13 and Xa21 respectively.

Backcross breeding’s main goal is to transfer single or more genes associated with desired traits into a foundational variety from a donor parent. This procedure normally requires 6 to 8 backcrosses to regain the phenotype of the recurrent parent (Hasan et al., 2015). But in the MAS strategy, 3 to 4 backcrossing generations are typically sufficient to obtain more than 99% of the RPG (Jiang 2013b). According to theory, BC₁, BC₂, BC₃ and BC₄ had theoretical % RPG of 75%, 87.5%, 93.8%, and 96.9%, respectively. Additionally, employing marker assisted selection will enhance the % RPG (Suh et al., 2013, Rajpurohit et al., 2011). In our study, initially, SSR markers were employed to find out polymorphism between parents and thereafter to utilize them for background selection. Such parental polymorphism survey between VGD 1 and ADT 55 provided 58 polymorphic markers out of 166 SSR markers with a parental polymorphism percentage of 35%. Closer to our study, a parental polymorphism survey was done using 463 SSR markers for two different parental cross combinations with recurrent parents ADT43 and ASD16 during gene pyramiding for multiple stress tolerance in rice, where 69 and 68 markers were found polymorphic respectively (Ramalingam et al., 2020; Rajarathinam et al., 2023). Utilizing the polymorphic markers (58) background selection was conducted, where the recurrent parent genome (RPG) recovery rates in BC₁F₁ and BC₂F₁ were 78.44% and 86.20% respectively. A closer background recovery rate was recorded when compared to the theoretical percent recovery rate by MAS, and the outcomes were comparable with those of previous investigations. In BC₁F₁ plants, Basavaraj et al., (2010)recovered RPG in a range of 71 to 79 percent. In the BC₁ and BC₂ generations, respectively, 73.4% and 84.8% were recovered. Thereafter, positive plant (Plant-12) (BC₂F₁) with the highest RPG was advanced to BC₂F_2, where we found homozygous resistance for BB genes in fifteen plants, in which eleven plants with similar grain trait of VGD 1 were advanced to BC₂F₃.Seven BC₂F₄ recombinant breeding lines possessing all three (xa5, xa13 and Xa21) resistance genes in homozygous conditions were evaluated for bacterial blight disease, agronomic traits and grain quality traits.

Bioassay results of BC₂F₄ breeding lines with three BB-resistance (xa5, xa13 and Xa21) genes were more resistant than those with one (Xa21) gene or two (xa13 and Xa21) genes. It also unfolded that the line with two genes (xa13 and Xa21) performed well then the line with one gene (Xa21). Gene interaction or quantitative complementarity between the R genes will results in a potential level of resistance (Yoshimura et al., 1995). These results agreed with those of earlier findings (Pradhan et al., 2015, Ramalingam et al., 2020). Major agronomic traits evaluation for BC₂F₄ breeding lines revealed that all of them were superior to donor parent ADT 55 and are more or less similar to VGD 1. Grain quality trait analysis revealed that all the BC₂F₄ lines possess grain qualities (grain type – short bold) closer to the recurrent parent (VGD 1). A similar evaluation of grain traits was done by Jamaloddin et al., (2020)in the ICF₂ population in the study of marker assisted gene pyramiding for bacterial blight and blast resistance in the rice variety ‘Tellahamsa’.

Seven genotypes harbouring BB resistance genes (xa5, xa13 and Xa21) with superior yield attributes and preferred grain trait were identified. These homozygous lines can be further multiplied and tested over environments for yield, resistance and other quality attributes. Studies examining the introduction of abiotic and biotic stress resistance have demonstrated the efficacy of marker–aided foreground selection in conjunction with phenotypic selection resembling our workflow (Joseph et al., 2004, Ellur, Khanna, Yadav, et al., 2016). Thus, the way of integrating genotyping and phenotypic selection techniques resulted in the evaluation of a better level of resistance to BB disease in the selected lines without compromising on desirable grain traits. Numerous studies have found that in order to meet the rising demand for rice, the world’s rice supply must be doubled by 2050 (Ray et al., 2013).

Bioassay results of BC₂F₄ breeding lines showed minimum mean lesion lengths of 0.76 ± 0.18 to 2.26 ± 0.04 as expected. Results also indicated that lines with three BB-resistance (xa5, xa13 and Xa21) genes were more resistant than those with one (Xa21) gene or two (xa13 and Xa21) genes. It also unfolded that the line with two genes (xa13 and Xa21) performed well then the line with one gene (Xa21). Gene interaction or quantitative complementarity between the R genes will results in a potential level of resistance (Yoshimura et al., 1995). These results agreed with those of earlier findings (Pradhan et al., 2015, Ramalingam et al., 2020). Major agronomic traits evaluation for F₄ breeding lines revealed that all of them were superior to donor parent ADT 55 and are more or less similar to VGD 1. Grain quality trait analysis revealed that all the BC₂F₄ lines possess grain qualities (grain type – short bold) closer to the recurrent parent (VGD 1). A similar evaluation of grain traits was done by Jamaloddin et al., (2020)in the ICF₂ population in the study of marker assisted gene pyramiding for bacterial blight and blast resistance in the rice variety ‘Tellahamsa’.

Overall reported that eleven genotypes in BC₂F₂ as well as the recombinant inbreed line BC₂F₄-56 harbouring BB resistance genes (xa5, xa13 and Xa21) with superior yield attributes and preferred grain trait were identified. These homozygous lines can be further multiplied and tested over environments for yield, resistance and other quality attributes. Studies examining the introduction of abiotic and biotic stress resistance have demonstrated the efficacy of marker–aided foreground selection in conjunction with phenotypic selection resembling our workflow (Joseph et al., 2004, Ellur, Khanna, Yadav, et al., 2016). Thus, the way of integrating genotyping and phenotypic selection techniques resulted in the evaluation of a better level of resistance to BB disease in the selected lines without compromising on desirable grain traits.

Development of genotype with multi-genic combination along with grain yield and grain quality is important. The market value of grain is prime importance to fetch high returns. In the current study, fine grain rice variety was improved for BB resistance without compromising its agronomic performance. The superfine variety, VGD 1 was enhanced with xa5, xa13 and Xa21 genes for bacterial blight resistance. Background selection performed for the heterozygous plants viz., Plant 10 and Plant 12 in BC₂F₁ generation exhibited RPG recovery of 82.75% and 86.20% respectively. Plant-12 showed the highest recovery of the genome from recurrent parent i.e., 86.20% and that was selfed to generate BC₂F₂ progenies. Genotypic screening of seven BC₂F₄ breeding lines for bacterial blight R genes (xa5, xa13 and Xa21) confirmed the homozygous condition for all three R genes. Seven pyramided lines were identified with high resistant to BB and grain quality similar to VGD1 via back cross based molecular breeding programme. The line 56 is promising with all desired agronomic and quality traits with improved bacterial blight resistance and higher yield than VGD1 was identified.

Acknowledgement

We thank the field technicians for their help in screen house screening work

Author Contribution:

SR, SRK, PR and SK carried out the back crosses and field trials. GC performed the screening work. SR and RJ wrote the manuscript and supervised the study. All authors approved the final manuscript

Funding

This work was funded by the Department of Biotechnology, Government of India (DBT/ATGC/127/ SP32889/ 2019).

Data Availability:

All data are given in the manuscript. Additional data were provided in the supplementary materials

Declarations:

The authors declare no competing interests.

Ethics Approval and Consent to Participate Not applicable.

Consent for Publication All authors have approved this manuscript for

publication.

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Table 1. Details of functional/linked markers employed in foreground selection

Gene/QTL	Marker	Chr. No.	Sequence (5’-3’)	Resistant band size (bp)	Susceptible band size (bp)	At(°c)	Enzyme	Reference
xa5	xa5-1	5	F-CTCTACCGGAGGTCCACCATTG R-AGGAACAGCAACATTGCAAC	299	100 and 199	56	BsrI	Iyer-Pascuzzi and McCouch (2007)
xa13	xa13-prom	8	F-GAGCTCCAGCTCTCCAAATG R-GGCCATGGCTCAGTGTTTAT	500	280	58	-	Chu et al.,(2006)
Xa21	pTA248	11	F-AGACGCGGAAGGGTGGTTTCCCGGA R-AGACGCGGTAATCGAAAGATGAAA	1000	650	58	-	Ronald et al.,(1992)

Table 2. List of number of polymorphic markers between VGD 1 and ADT 55

Chromosome number	Total No. of markers analysed	Number of polymorphic markers	Number of monomorphic markers	Polymorphism percentage (%)
1	20	8	12	40
2	15	10	5	66
3	18	5	13	27
4	14	8	5	57
5	13	5	8	38
6	15	5	10	33
7	16	8	8	50
8	9	2	7	22
9	10	2	8	20
10	16	3	13	19
11	7	2	5	28
12	13	0	13	0
Total	166	58	107	35

Table 3. Screening of BC₂F₄ breeding lines for bacterial blight disease

Cultivars	Genotyping result			Lesion length (cm) ± Standard error	Disease score	Host response
Cultivars	xa5	xa13	Xa21	Lesion length (cm) ± Standard error	Disease score	Host response
F₄-4	R	R	R	0.76 ± 0.18	2	Resistant
F₄-11	R	R	R	1.03 ± 0.04	3	Resistant
F₄-12	R	R	R	1.15 ± 0.49	3	Resistant
F₄-56	R	R	R	1.4 ± 0.84	3	Resistant
F₄-94	R	R	R	1.25 ± 0.21	3	Resistant
F₄-112	R	R	R	2.05 ± 0.02	3	Resistant
F₄-123	R	R	R	2.26 ± 0.04	3	Resistant
F₄-35	R	R	S	9.7 ± 2.05	5	Moderately Resistant
F₄-61	R	S	S	11.5 ± 0.80	7	Moderately susceptible
VGD 1	S	S	S	17.5 ± 0.23	8	Susceptible
ADT 55	R	R	R	0.85 ± 0.35	2	Resistant

Table 4. Estimation of recurrent parent genome of cross combination VGD 1 and ADT 55

Generation / Plant number	Number of Polymorphic markers (n)	Number of homozygous markers (x)	Number of Heterozygous markers (y)	Recovery of recurrent parent genome (RPG%)
BC₁F₁ Plant-7	58	30	28	75.86
BC₁F₁ Plant-18	58	33	25	78.44
BC₁F₁ Plant-36	58	26	32	72.41
BC₁F₁ Plant-58	58	28	30	74.13
BC₂F₁ Plant-10	58	38	20	82.75
BC₂F₁ Plant-12	58	42	16	86.20

Table 5. Agronomic performance and grain quality attributes of selected pyramided lines in BC₂F₄ generation

Entries	Plant height (PH) (cm)	Number of productive tillers per plant (NPT)	Panicle length (PL) (cm)	Kernel length	Kernel breadth	Kernel length/ breadth ratio	Grain type	Amylose Content	Remark	1000-grain weight (g)	Single plant yield (SPY) (g)
F₄-4	85.8 ± 0.8	22.2 ± 1.5	24.6 ± 0.5	5.30	2.1	2.33	Short bold	25.2	High	12.3 ± 0.05	34.70 ± 0.34
F₄-11	88.8 ± 0.4	24.8 ± 0.7	25.2 ± 0.8	4.68	1.9	2.46	Short bold	21.4	Intermediate	9.92 ± 0.03	34.18 ± 0.38
F₄-12	82.6 ± 0.5	22.4 ± 0.5	23.1 ± 0.8	4.80	2.2	2.18	Short bold	23.8	Intermediate	11.4 ± 0.02	32.60 ± 0.65
F₄-56	87 ± 0.8	25.2 ± 0.8	23.8 ± 1.0	4.30	1.8	2.38	Short bold	22.8	Intermediate	9.50 ± 0.08	35.60 ± 0.40
F₄-94	84.2 ± 1.1	23.2 ± 1.3	24.6 ± 1.1	4.81	2.1	2.29	Short bold	23.5	Intermediate	11.5 ± 0.05	32.30 ± 0.80
F₄-112	89.4 ± 0.9	24.3 ± 1.2	23.6 ± 1.1	4.75	2.0	2.37	Short bold	24.9	Intermediate	11.2 ± 0.15	34.43 ± 0.92
F₄-123	83.6 ± 0.4	22.6 ± 1.1	23.8 ± 0.4	4.90	2.0	2.45	Short bold	23.4	Intermediate	10.1 ± 0.02	31.24 ± 0.31
VGD 1	86.6 ± 0.7	25 ± 0.4	24.6 ± 0.5	4.10	1.9	2.15	Short bold	22.5	Intermediate	9.20 ± 0.05	35.24 ± 0.35
ADT 55	78.6 ± 0.4	15.4 ± 0.2	23.2 ± 0.7	6.00	2.1	2.85	Medium Slender	25.5	High	18.3 ± 0.06	30.19 ± 0.60
SE (d)	0.05	0.05	0.01							0.04	0.13
CD (5%)	0.17	0.15	0.03							0.14	0.41

No competing interests reported.

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Marker assisted improvement of super fine grain rice variety, VGD 1 for bacterial blight resistance

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