Improvement of bacterial blight resistance of the popular variety, Nellore Mahsuri, NLR34449 through marker-assisted breeding

To combat the dreaded diseases in rice like bacterial blight and blast, host plant resistance has been advocated as a sustainable method. Through the present study, we have successfully incorporated three major bacterial blight (BB) resistance genes viz., Xa21, xa13 and xa5 into NLR3449, a high yielding, blast resistant, ne-grain type popular rice variety through marker-assisted backcross breeding. Foreground selection was carried out using PCR based, gene-specic markers viz., pTA248 (Xa21), xa13prom (xa13) and xa5FM (xa5) at each generation of backcrossing, while 127 polymorphic SSR markers spanning on 12 chromosomes were used for background selection and backcrossing was limited to two rounds. At BC 2 F 1 generation, a single plant (NLR-87-10) with 89.9% recovery and possessing all the three bacterial blight resistance genes was forwarded to BC 2 F 2 generation. A solitary BC 2 F 2 plant viz., NLR-87-10-106 possessing all the three resistance genes and > 90% genome recovery was identied and advanced through selng till BC 2 F 4 generation by adopting pedigree method. Three best lines at BC 2 F 4 lines, possessing high level of resistance against bacterial blight and blast and equivalent or superior to NLR 34449 in terms of yield, grain quality and agro-morphological traits have been identied and advanced for multi-location trials. nearly of NLR 34449 at BC 2 F 4 generation were transplanted in the main eld of experimental farm of ICAR-IIRR located to assess their resistance against bacterial blight during wet season 2019. IX0-20, a local virulent isolate (collected from Hyderabad, Telangana State, India) of Xanthomonas oryzae pv. oryzae was used for screening of bacterial blight resistance of the breeding lines. Inoculation was done at maximum tillering stage following leaf clip et 1973 by the leaf tip of with scissors dipped into inoculum. were measured at after based on for their resistance/susceptibility.


Introduction
Rice is an important food crop that serves as a major carbohydrate source for nearly half of the world's population (Nguyen and Ferrero 2006;Sundaram et al. 2008 andPradhan et al. 2015). It is grown throughout the year in a variety of agro-ecosystems in India, which include irrigated, rain-fed, deep-water and hills ). We need to produce at least 40% more rice by 2030 to feed a growing population and there are many challenges in meeting the food security requirements for the future (Khush 2005). This target has to be met amidst the scarcity of natural resources and adverse effects from a rapidly changing climate and with less use of chemicals and unde constant battle against new emerging pathogens and pests (Pradhan et al. 2015). Rice is affected by many diseases and among them, bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) (Ishiyama, 1922) is known to cause yield losses ranging from 74%-81% (Srinivasan and Gnanamanickam 2005), when the disease occurs in severe form. BB is particularly severe in the irrigated and rain-fed lowland ecosystems (Sundaram et al. 2008). The disease occurs in the host plant at seedling, vegetative and reproductive stages, but infection at the tillering stage causes severe blighting of leaves resulting in yield loss (Shivalingaiah and Umesha 2011). Chemical control for BB is cost intensive and not effective as successful bactericidal chemicals are not available (Devadath 1989). Thus, the exploitation of disease resistant cultivars is the most effective and eco-friendly approach for reducing yield losses due to BB (Sundaram et al. 2008 Kim et al. 2018) have been identi ed so far from the primary gene pool of rice and pyramiding two or more resistance genes has been advocated to enhance the durability of resistance (Sundaram et al. 2008).
The rice variety, NLR 34449, popularly known as Nellore Mahsuri, was released from Rice Research Station, Acharya N.G. Ranga Agricultural University, Nellore, Andhra Pradesh, India. It is popular among the rice farmers of South India, as it is high yielding, possesses highly desirable medium slender (MS) grain type, has excellent blast resistance, is of short duration (120 days), possesses non-lodging plant type and is highly amenable for machine harvesting. It is highly preferred rice by the farmers of Andhra Pradesh and other South Indian states (http://www.kvknellore-angrau.org/index.php). Despite its high yield and blast resistance, the variety is highly susceptible to bacterial blight disease, which limits its production and adoption signi cantly. Keeping these points in view, through the present study, we have transferred three major bacterial blight resistance conferring genes, viz., Xa21, xa13 and xa5 from elite, ne grain, BB resistant variety Improved Samba Mahsuri (ISM) into the genetic background of NLR 34449 through markerassisted backcross breeding (MABB).

Material And Methods:
Plant material: NLR 34449 (popularly known as Nellore Mahsuri), a high yielding, medium slender, blast resistant and short duration variety (120-125 days) developed from the cross IR 72/BPT 5204 in the year 2009 was used as the recurrent parent. Improved Samba Mahsuri (ISM), a high yielding, medium-slender grain type, bacterial blight (BB) resistant variety developed by ICAR-Indian Institute of Rice Research (ICAR-IIRR), Hyderabad, India and CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, India, possessing three major bacterial blight (BB) resistance genes, viz., Xa21, xa13 and xa5 (Sundaram et al. 2008) was used as the donor parent for bacterial blight resistance.
Marker-assisted backcross breeding strategy: NLR 34449 was crossed with Improved Samba Mahsuri during dry season 2016-17. F 1 s developed from the above mentioned cross were con rmed for their heterozygosity using target resistance gene speci c markers viz., pTA248 speci c for Xa21 (Ronald et al. 1992), xa13prom, speci c for xa13 ) and xa5FM speci c for xa5 . True F 1 s thus identi ed were then backcrossed with the recurrent parent, NLR 34449 to generate BC 1 F 1 s. They were subjected for foreground selection using genespeci c markers and positive plants (i.e. plants which are heterozygous for all the three target BB resistance genes) were then analyzed for the recurrent parent genome recovery through background selection using a set of 127 SSR parental polymorphic markers evenly distributed across the 12 rice chromosomes (listed in Supplementary Table 1B) to identify those plants with maximum recurrent parent genome recovery (RPG). A single BC 1 F 1 plant positive for all three target genes and with maximum recurrent parent genome recovery was selected and backcrossed with NLR 34449 to generate BC 2 F 1 s. Marker-assisted foreground and background selection was repeated among the BC 2 F 1 plants and a single BC 2 F 1 plant possessing the target resistance genes and also with maximum recurrent genome recovery were selfed to develop BC 2 F 2 s. Finally, a single BC 2 F 2 plant possessing Xa21, xa13 and xa5in homozygous condition along with maximum RPG and closely resembling NLR 34449 (based on morphological features) was identi ed and advanced through pedigree method of breeding till BC 2 F 4 ( Figure 1). The extent of donor parent segment introgression among the selected backcross derived plants at BC 2 F 4 generation was assessed utilizing the software tool, Graphical Genotype V 2.0 (Van Berloo, 1999) and the BC 2 F 4:5 lines were also further evaluated for bacterial blight resistance and key agro-morphological traits.
Phenotypic screening for BB and blast resistance: Bacterial blight screening: Thirty-day old seedlings of the improved breeding lines of NLR 34449 at BC 2 F 4 generation were transplanted in the main eld of experimental farm of ICAR-IIRR located at Rajendranagar, Hyderabad, India to assess their resistance against bacterial blight during wet season 2019. IX0-20, a local virulent isolate (collected from Hyderabad, Telangana State, India) of Xanthomonas oryzae pv. oryzae was used for screening of bacterial blight resistance of the breeding lines. Inoculation was done at maximum tillering stage following leaf clip method of Kauffman et al. 1973 by clipping the leaf tip (about 1 to 2cm) of the uppermost leaf with scissors dipped into the inoculum. Symptoms were measured at 15 days after inoculation based on the IRRI SES (IRRI, 2013) for their resistance/susceptibility.

Screening for blast resistance:
During dry season 2019-20, gene pyramided lines possessing Xa21, xa13 and xa5at BC 2 F 5 generation were analysed for their resistance against blast in uniform blast nursery along with the susceptible (HR12) and resistant (C101A51 and Tetep) checks using a local isolate, SPI-40 of the blast pathogen, Magnaporthae oryzae as per the protocol described in Mohan et al. (2011). After 15days of inoculation, the lines were scored for their resistance/ susceptibility to the disease based on IRRI-SES scale, 1996 (IRRI, 1996).

Screening of the improved lines for agro-morphological characters:
During dry season 2020, thirty-day old seedlings of the recurrent parent NLR 34449 and donor parent ISM along with ve best backcross derived lines at BC 2 F 5 generation, possessing bacterial blight resistant genes in the genetic background of NLR 34449 were transplanted in the main eld of experimental farm of ICAR-IIRR located at Rajendranagar, Hyderabad, India at a spacing of 15 X 20cm in 2 m 2 plots in three replications and the eld was applied with the recommended dose of NPK fertilizers (@ 220:70:80kg/ha). Phenotypic data was collected for the selected plants for key agro-morphological traits, viz., plant height (cm), days to 50% owering (DFF), panicle length (cm), number of productive tillers per plant, panicle exertion, grain yield per 33 plants (i.e. per m 2 ; in g), number of grains per panicle, grain type, L/B ratio, 1000-grain weight (g) among 33 plants for each replication (n = 3) as explained in . The data was statistically analysed as per the procedure described in Freeman et al. (1978). Least Signi cance Difference (LSD) values at 5 to 7 percent level of signi cance and Coe cient of variation (CV) were calculated using standard errors of mean (S. Em. ±) using Microsoft Excel package. The software package, R studio (R Core, 2016) was used for analysis of variance (ANOVA) to determine signi cant variation among the improved breeding lines of NLR 34449.

Results:
Marker-assisted introgression of Xa21, xa13 and xa5 into the genetic background of NLR 34449 Ninety-ve F 1 plants, which were derived from the cross NLR 34449 X ISM were con rmed for their hybridity with respect to the three target BB resistance genes i.e., Xa21, xa13 and xa5 with the help of gene-speci c markers viz., pTA248, xa13prom and xa5FM, respectively during wet season2017. A total of 80 F 1 plants were found to be heterozygous for all the three target BB resistant genes.
These plants (i.e. Xa21xa21 xa13Xa13 xa5Xa5) were used as pollen donors to develop BC 1 F 1 s . Among 175 BC 1 F 1s, 21 plants were found to be heterozygous for all the three target BB resistant genes (i.e. Xa21, xa13 and xa5). These 21 plants were analyzed to assess the recovery of the recurrent parent genome using a set of 127 parental polymorphic primers (Supplementary table 1A, 1B) and the analysis revealed that a single BC 1 F 1 plant (NLR-87) had the highest recurrent parent genome (RPG) recovery of 78.5%. The plant, NLR-87 was then used as a pollen donor to produce BC 2 F 1 s . A total of 105 BC 2 F 1 plants were raised and evaluated through foreground selection with gene-speci c markers and thirteen were found to be positive for all the three target BB resistant genes and they were subjected for background genome recovery analysis with parental polymorphic SSR markers and a single plant (NLR-87-10) with maximum recurrent parent genome re recovery (89.9%) was identi ed. This plant was selfed to produce BC 2 F 2 plants. A total of 425 BC 2 F 2 plants, which were generated by sel ng the selected BC 2 F 1 plant (NLR-87-10), were grown in the eld. All of them were subjected to phenotypic screening with a local isolate of Xoo, IX0-20 at maximum tillering stage to screen for their resistance against BB. A total of 359 plants were found to be resistant.
When the phenotypically resistant plants were subjected for foreground selection with the help gene-speci c markers, a total of ve plants were identi ed to be homozygous for all the three target resistant genes ( Figure 2). A solitary plant (NLR-87-10-106) which was homozygous resistant to all the three target resistant genes and possessed with maximum recovery of NLR34449 genome (94.6%) was identi ed and was advanced further through pedigree method of breeding to BC 2 F 4 generation. The details of number of plants screened and selected in each generation of backcrossing were given in Table 1. A set of ve promising BC 2 F 4 lines, viz., (NLR-87-10-106-40-50, NLR-87-10-106-41-51, NLR-87-10-106-42-52, NLR-87-10-106-43-53, NLR-87-10-106-44-54) identi ed to be identical to recurrent parent NLR 34449 with respect to morphological traits and these lines were subjected for analysis of their resistance against bacterial blight, blast, yield and agro-morphological traits. The ve selected BC 2 F 4 lines were further analysed to assess the extent of linkage drag around the target bacterial blight resistant genes i.e., Xa21, xa13 and xa5. This analysis revealed that for Xa21 gene located on Chr. 11L, a segment of 0.3 Mb was introgressed at both proximal and distal ends from the donor parent genome in the best BC 2 F 4 plant (NLR-87-10-106-40-50); thus, in total, a segment of 0.6Mb was introgressed from the donor parent with respect to the genomic region in the vicinity of Xa21 ( Figure 3A). With respect to xa13 gene located on Chr. 8L, a segment of 0.5 Mb was introgressed at proximal end while another segment of 0.8 Mb was introgressed at distal end from the donor parent genome in the best BC 2 F 4 plant (NLR-87-10-106-40-50); thus, in total, a segment of 1.3Mb was introgressed from the donor parent with respect to the genomic region in the vicinity of xa13 ( Figure 3B). With respect to xa5 gene located on Chr. 5S, a segment of 0.2 Mb was introgressed at proximal end, while 0.3 Mb introgressed at distal end from the donor parent genome in the best BC 2 F 4 plant (NLR-87-10-106-40-50); thus, in total, a segment of 0.5Mb was introgressed from the donor parent with respect to the genomic region in the vicinity of xa5 ( Figure 3C). All the ve selected introgressed BC 2 F 4 lines were subjected to screening for bacterial blight and blast resistance and also evaluated for their agro-morphological traits.
Assessment of BB and Blast resistance in the improved lines of NLR 34449: The donor parent Improved Samba Mahsuri (ISM), RPBio-Patho-1 and RPBio-Patho-2 were observed to be highly resistant to the disease with a lesion length ranging from 0.0 to 1.7 ± 0.3 (Score 1). The recurrent parent, NLR 34449 was observed to be highly susceptible to the disease with a lesion length of 8.7 ± 0.0 (Score 9) when screened with IXO-20. All the ve selected BC 2 F 4 were observed to be resistant to BB disease, showing a lesion length of 0-1 cm, which was similar to the donor parent (i.e. ISM) (Fig 4A, Table 2). With respect to blast disease, the susceptible check, HR12 and the parent, donor ISM were highly susceptible to blast, with a score of 9, while the resistant check, Tetep and the recurrent parent, NLR 34449 were found to be resistant to the disease with a score of 1 and 3, respectively. The selected BC 2 F 5 lines were observed to show resistant reaction against blast disease with a disease score Table 2) Among the improved lines, BC 2 F 4 plant, NLR-87-10-106-44-54 displayed better attributes with respect to most of the agromorphological traits when compared to the recurrent parent, Nellore mahsuri. All the introgressed lines were slightly taller than the recurrent parent, Nellore Mahsuri and shorter than donor parent, Improved Samba Mahsuri, except a single line NLR-87-10-106-42-52 (75.5 ± 0.9 cm) which was observed to be slightly shorter than recurrent parent ( Table 3). The introgressed lines were found to perform equivalent or better than both the recurrent as well as donor parents in terms of panicle length, number of grains per panicle, thousand grain weight and grain yield per plant with good panicle exertion ( Figure 5, Table 3). Two improved lines, NLR-87-10-106-40-50 and NLR-87-10-106-42-52 recorded grain yield per plant (24.1 ± 0.6 g) equivalent to the recurrent parent. The analysis of variance for ten agro-morphological traits among the selected BC 2 F 4 lines and the recurrent parent, Nellore Mahsuri revealed that the mean sum of squares due to treatments (<0.01) were highly signi cant for all the traits under study, indicating signi cant variability among the lines as compared to Nellore Mahsuri (Table 3) (Table 3).

Discussion:
Rice yield and productivity is drastically reduced due to two major diseases, viz., bacterial blight (BB) and blast (Ou 1985 Agricultural University (ANGRAU), Nellore, Andhra Pradesh, India. This variety is popular among the farmers, as it is high yielding, semi-dwarf in plant stature, blast resistant, early maturing (120 days) and is non-lodging type (suitable for machine harvesting). It is highly preferred by rice farmers in the states of Andhra Pradesh, Telangana and other South Indian states (http://www.kvknelloreangrau.org/index.php). Further, it possesses medium-slender grain type, which is highly desirable among the rice consumers in South India and varieties with medium-slender grain type like Samba Mahsuri, Sona Mahsuri etc. are getting increasingly popular across India. As NLR 34449 is highly susceptible to bacterial blight disease, we attempted to improve NLR 34449 for durable bacterial blight resistance by using marker assisted introgression of three bacterial blight resistance genes, Xa21, xa13 and xa5.
In breeding programmes, molecular markers that are tightly linked to target genes/QTLs are very useful to overcome the limitations of conventional phenotype based breeding and improve the selection e ciency of target genes or traits in backcross breeding programmes (Jena and Mackill, 2008). This is especially through 'foreground selection' where markers are used alone or in combination with phenotypic screening in selection for the target trait(s) (Hospital and Charcosset, 1997). In the present study, the codominant markers, pTA248 (Ronald et al. 1992), xa13prom and xa5FM ) were used to screen for the presence of three bacterial blight resistance genes, viz., Xa21, xa13 and xa5 respectively and all the three markers are reported to be functional markers or very closely linked for the respective genes, with no chance or very little chance for recombination ).
These markers were also used by Ramalingam  The donor parent and recurrent parent used in the present study are having the same background (i.e. Samba Mahsuri); hence backcrossing was restricted to two rounds. The recurrent parent genome (RPG) recovery was 78.5% (plant # NLR-87) in the best BC 1 F 1 plant, while the value was 88.5% (plant # NLR-87-10) in the best BC 2 F 1 plant. In BC 2 F 2 generation, the RPG was observed to be 94.6% Phenotypic screening of the improved versions of the NLR 34449 against bacterial blight disease revealed that all the lines are resistant to the disease with a score of 1. Further, they were also resistant to blast with a score of 2-4 (Fig. 4, Table 2). The results are in agreement with earlier reports, wherein the bacterial blight resistance genes, Xa21, xa13 and xa5 were deployed either singly or in  Sundaram et al. 2008;Yoshimura et al. 1995) and the derived lines showed resistance against the disease. The objective of the present study was to develop pyramided lines of NLR3449 with durable resistance against BB. In earlier studies, introgressed lines possessing two or more genes against bacterial blight were observed to possess higher level of resistance, possibly due to quantitative complementation (Sanchez et al. 2000;Sundaram et al. 2008). Cultivation of breeding lines of NLR34449 possessing the three resistance genes in bacterial blight endemic areas may be bene cial to farmers and also the resistance is expected to be more durable, as three resistance genes with distinct functions ) have been deployed. There have been various reports, wherein a single major blast resistance gene likes Pi1 or Pi2 have shown the desired level of resistance against the disease (Fu et al. 2012). Similar to the recurrent parent, NLR 34449, which is highly resistant to blast disease, all the improved lines showed resistance against blast disease. Our preliminary analysis has indicated the possibility of presence of the major blast resistance gene, Pi1 in NLR34449, based on marker analysis (data not shown) and resistance in the breeding lines could be due to the presence of Pi1.
The results observed in the present study with respect to resistance against BB and blast are in correspondence with the results Most of the improved lines were observed to be equivalent to NLR 34449 in terms of grain yield and other agromorphological characters and some of them were found to be superior to NLR 34449 with respect to yield (Table 3) and all the lines were showing good panicle exsertion with plant height equivalent to NLR 34449 and hence are non-lodging (Fig. 5). Further all the selected improved lines, possessing bacterial blight and blast resistance were observed to be retaining the medium slender grain type and ne-grain quality attributes of the NLR 34449 (Fig. 5). It can be expected that the bacterial blight and blast resistant lines of NLR 34449 developed through present study may useful for the farmers to get the same price in the market, similar to NLR 34449 as they are identical in terms of grain quality and yield, plus added advantage of resistance against two major diseases. One of the signi cant achievement of this study is the complete recovery of the yield and yield related traits along with grain quality features in the improved lines of NLR 34449 possessing bacterial blight and blast resistance. Combining phenotype-based selection with MAS in the present study was helpful in not only recovering good features of the recurrent parent, but also helpful in selection of superior lines (with better yield and panicle exsertion) as compared to the recurrent parent, NLR34449.

Conclusion:
The improved versions of Nellore mahsuri possessing BB and blast resistance, developed in the present study may offer a distinct advantage to farmers of NLR34449, whose elds are affected by both bacterial blight and blast. Further, Cultivation of such improved backcross derived lines of NLR34449 possessing resistance against bacterial blight and blast could help to improve rice production in the disease endemic areas in many states of India, wherein ne-grain type varieties like NLR34449, Samba Mahsuri, HMT Sona etc.
are preferred. Further, the improved lines of NLR 34449 developed in this study can also be used as donors to transfer BB and blast resistance into other genetic backgrounds as they possess high yield and medium-slender grain type. Among the improved lines of NLR 34449, the backcross derived line # NLR-87-10-106-44-54 possessing Xa21, xa13 and xa5 showed very good phenotype and better panicle exsertion along with high yield and diseases resistance and hence it has been identi ed as one of promising lines ( Availability of data and materials: The data and information generated from the study are available in our laboratory as hard and soft copies and can be shared based on request.
Competing interests: The authors declare that they have no competing interests. Funding: Aleena is grateful to Bayer crop science for providing the nancial assistance during my research Outline of the crossing scheme. Nellore Mahsuri (NLR 34449) was crossed with Improved Samba Mahsuri (ISM) possessing bacterial blight resistant genes Xa21, xa13, xa5 in order to build bacterial blight resistance through step-wise, marker assisted backcross breeding strategy