Pyramiding of bacterial blight resistance genes into promising restorer BRRI31R line through marker-assisted backcross breeding and evaluation of agro-morphological and physiochemical characteristics of developed resistant restorer lines

BRRI31R is one of the Bangladesh’s most promising restorer lines due to its abundant pollen producing capacity, strong restoring ability, good combining ability, high outcrossing rate and genetically diverse from cytoplasmic male sterile (CMS) line. But the drawback of this line is that it is highly susceptible to bacterial blight (BB) disease of rice caused by Xanthomonas oryzae pv. oryzae. The present study highlighted the pyramiding of effective BB resistance genes (xa5, xa13 and Xa21) into the background of BRRI31R, through marker–assisted backcrossing (MABC). Backcross progenies were confirmed and advanced based on the foreground selection of target genes. Pyramided lines were used for pathogenicity test against five Bangladeshi Xanthomonas oryzae (BXo) races (BXo93, BXo220, BXo822, BXo826, BXo887) and confirmed the dominant fertility restore genes, Rf3 and Rf4 and further validated against SNP markers for more confirmation of target resistance genes. All pyramided restorer lines consisted of Xa4 (in built), xa5, xa13, Xa21, and Chalk5 with two fertility restorer genes, Rf3, Rf4. and these restorer lines showed intermediate amylose content (<25%). Restorer lines BRRI31R-MASP3 and BRRI31R-MASP4 showed high levels of resistance against five virulent BXo races and SNP genotyping revealed that these lines also contained a blast resistance gene Pita races. Gene pyramided restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 can directly be used as a male parent for the development of new BB resistant hybrid rice variety or could be used as a replacement of restorer line of BRRI hybrid dhan5 and 7 to enhance the quality of hybrid seeds as well as rice production in Bangladesh.

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Introduction
Rice (Oryza sativa L.) is the world's most influential and economically significant crop, with more than half of the world's population relying on rice to meet their daily calorie requirements [1,2].Bangladesh has secured the third position for total rice production in the world and food security depends on rice security [1,2].It has been assumed that, in 2050, the population of Bangladesh will be 215.4 million, when 44.6 MT of clean rice will be required [3].Climate change and the emergence of different biotic and abiotic stresses are threatening rice production in the world including Bangladesh [1,4,5].To ensure the basic food demand of the burgeoning population of Bangladesh, hybrid rice could be a viable solution to increase rice production with a 15-20% yield advantage compared to inbred varieties [6,7].
In Bangladesh, till now, 32 rice diseases have been reported and based on severity and incidence, blast [8], sheath blight [9], BB [10,11], tungro [12] and false smut [13] considered as major disease across the country.At present, BB is one of the major threats to both inbred and hybrid rice cultivation in the world specifically in South-east Asia including Bangladesh [14][15][16][17][18].In South and Southeast Asian nations, BB caused by Xanthomonas oryzae pv.oryzae (Xoo) causes 10-50% yield loss, but it can exceed 80%, depending on variety, crop stage, location, and favorable environment [19][20][21].The input requirement of hybrid is more compared to inbred varieties and these phenomena favor the epidemics of diverse diseases and pests.Highyielding varieties require high dose of nitrogen which favors the occurrence of BB in different countries of the world including Bangladesh [22].Ansari et al. [22] found that due to BB around 5.8-30.4% yield loss occurred in field conditions of Bangladesh.However, the damage caused by bacterial blight disease depends on many factors including location, virulence of the race, crop stage, environmental condition and cultivar [23,24].But till now, no effective chemical control measure is suggested to combat the disease.So far, 237 hybrid rice varieties have been released in Bangladesh but no variety has any resistance characteristics because all hybrid parental lines are susceptible to available races of Xoo [25].
Therefore, stacking resistance genes into hybrid parental lines is a durable strategy to manage bacterial blight disease [25].Moreover, by adopting conventional breeding it is tough to introgress more than one gene into the single lines and sometimes dominance and epistasis effects of genes make it more difficult to identify the effective gene [26,27].However, the development of DNA markers linked to resistance genes makes it easy to detect two or more genes in the introgressed lines [8,[28][29][30].Trait-specific gene transformation through markerassisted selection (MAS) is a very popular technique to develop inbred and hybrid lines with desired characters [8,31].To date, more than 46 resistance genes have been identified as effective against bacterial blight pathogens [32].Among them, Xa4, xa5, Xa7, xa13, Xa21, and Xa23 gens have been extensively deployed and incorporated into rice cultivars for the development of BB resistant cultivars [33].Resistance genes i.e., xa5, xa13 and Xa21 were found effective for the bacterial blight pathogens of Bangladesh [23,25,34,35].Moreover, molecular markers of these resistance genes are widely used in breeding programs to develop bacterial blight resistant rice varieties [36,37].Moreover, it is crucial to characterize genotypes using trait-specific SNPs in order to examine the frequency of advantageous alleles for various traits of interest associated with biotic and abiotic stressors as well as grain quality characteristics.The high throughput SNP platform has made it possible to choose genotypes for elite parents with exceptional traits utilizing contemporary breeding techniques like markerassisted forward breeding and genomic selection.

Plant materials
BRRI31R is a restorer background with medium long slender, medium duration, strong restorers but susceptible to bacterial blight disease.IRBB60, a pyramided line carrying four resistance genes (Xa4 + xa5 + xa13+ Xa21) was used as the donor for introgression of BB resistance genes into the most promising restorer line BRRI31R.We considered this line as promising because this parental line was also used in the background of two popular hybrid rice varieties (BRRI hybrid dhan5 and BRRI hybrid dhan7) of Bangladesh.Before hybridization, the selected parents were used for morphological screening against five BXo races and validation of markers against BB resistance genes, Xa4, xa5, xa13 and Xa21 for foreground selection.

Marker-assisted breeding strategy for restorer line development
A cross was made between BRRI31R and IRBB60.The F1 plants were backcrossed thrice with recurrent parent (BRRI31R) to produce BC3F1 plants, which were then selfed to get up to BC3F5 progenies.Whole backcross was confirmed and advanced from BC3F2 to BC3F5, based on gene-linked molecular markers.F1 to BC3F1 were followed for background recovery of recurrent parent and foreground selection was started from BC1F1 generation and continued till BC3F5 (Fig. 1) where pure homozygous lines for all three resistance genes were detected.
The selected and confirmed pyramided restorer lines were used for pathogenicity test against five BXo races.

DNA extraction and PCR amplification
Leaves of twenty-one days old seedlings were obtained from the field to extract DNA.

Validation of pyramided restorer lines by SNP markers
Finally, selected pyramided restorer lines were further validated by SNP markers against 20 QTL traits which are shown the Supplementary Table 2.

Molecular Study for fertility status of pyramided restorer lines
Selected five pyramided restorer lines with one fertility restorer and non-fertility restorer lines were used as a check to confirm fertility restorer genes, Rf3 and Rf4 with restorer allele through molecular markers (Table 1) during Transplanted Aman season, 2020 (wet season).

Constitute of source nursery
A total of five pyramided restorer lines along with two wild abortive (WA) CMS lines, IR75608A and IR79156A were constituted in the source nursery during the Boro season (dry season), 2020.Each line was grown in three (3) sets with different planting times for proper synchronization of flowering to make a test cross for the validation of fertility status.

Pollen fertility and spikelet fertility status of testcross f1s
The pollen grains were put on a glass slide and dyed with one drop of 1% Iodine Potassium Iodide (IKI) solution.The anthers were gently smashed with a needle to release pollen grains, and pollen fertility was determined using a compound microscope at 10X magnification.Spikelet fertility percentage (seed sett %) was counted manually.When the dark black cell was seen in I-KI solution under a microscope, it meant that male parents were confirmed to be suspected restorers.The fertility of pollen and spikelet was measured by the following formula: Pollen fertility (%) = (No. of fertile pollen/No. of total pollen)*100; Spikelet fertility (%) = (Filled spikelets per panicle/Total spikeletsper panicle)*100.

Screening for BB Resistance
The parent and selected pyramided restorer lines were inoculated with five bacterial blight races (BXo93, BXo220, BXo826, BXo822 and BXo887) suspension following the adapted clipping procedure of Rashid et al., [23] modified from Kauffman et al., [39].At the maximum tillering stage, ten hills per entry were selected for BXo race inoculation and 8-10 leaves were trimmed from the apex.Before plunging into the fresh isolate mixture, the scissors were rinsed three times with sterilized water and then sterilized in 70% ethanol for 30 minutes.Following 14 days of inoculation, eight (8) infected leaves per hill were collected from the field for lesion length measurement.The disease response was graded using the modified scale of Akter et al., [25].Briefly, disease data was categorized into six scales (0 to 9) i.e., 0-Highly resistant (HR, lesion length <0.

Agro-morphological traits and grain quality test of pyramided restorer lines
Five pyramided restorer lines and their recurrent parent were seeded in the Transplanted Aman season (wet season) of 2020.Twenty-one days old seedling was transplanted with a spacing of 20 cm × 15 cm and other agronomic practices.Data was noted on five plants from each pyramided restorer line in BC3F5 generation for agronomic traits like plant height (cm), days to 50% flowering (day), days to maturity (day), number of effective tiller/hill, panicle length (cm), number of spikelet/panicle, spikelet fertility (%), 1000 grain weight (g), Grain yield/ hills (g).On the other hand, grain quality parameters such as milling outturn, head rice recovery, milled rice length, milled rice breadth, length breadth ratio, amylose content, protein content, cooking time, elongation ratio and imbibition ratio were measured in the laboratory of grain quality and nutrition department of Bangladesh Rice Research Institute, Gazipur, during 2020.

Validation of markers for foreground selection of parents
The repeatability and polymorphism of gene-based/linked markers were checked between the recurrent parent, BRRI31R and donor parent IRBB60 (Table 2).Among the parents, the restorer line BRRI31R showed monomorphic nature with a resistant allele (150bp) of the Xa4 gene but showed susceptible alleles (198bp, 1322bp and 650bp) for xa5, xa13 and Xa21 genes with polymorphic nature respectively.Gel photographs of the markers for the four bacterial blight resistance genes in the parents are presented in Fig. 2a.

Pathogenicity test of parents against bacterial blight pathogen
After the morphological screening, the recipient parent, BRRI31R showed (23.5 cm) similar mean lesion length compared to susceptible check IR24 (23.8 cm) while the resistant check showed mean lesion length 0.9 cm (Table 2).The disease reaction of parents along with susceptible check against bacterial blight pathogen is shown in Fig. 2b.3).The selected five homozygous BC3F2 plants were forwarded to BC3F3 and continued from BC3F3 to BC3F5 progenies.BRRI31R had by default Xa4 gene (Fig. 2a) and for this reason, further confirm the four BB resistance genes (Xa4, xa5, xa13 and Xa21) through gene-based/linked markers in the cross combination, BRRI31R × IRBB60.Five pyramided restorer lines with four (4) BB resistance genes were confirmed through molecular markers (Figs. 3a-d) and these lines were used in the evaluation nursery for pathological screening against five BXo races and the confirmation of fertility status.Validation of BB resistance genes with additional traits using 20 SNP panels by molecular approach and also agro-morphological traits with grain quality test were evaluated.

Selected pyramid lines screening against bacterial blight pathogen
The BB resistance of pyramided restorers with the parents and susceptible check IR24 were evaluated by five BXo races.The mean lesion length of pyramided restorers (Xa4, xa5, xa13 and Xa21) was very less (<3 cm) compared to recurrent parents and susceptible check IR24 (Fig. 4).Out of five, two pyramid restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 were showed highly resistant reaction against all the races (BXo93, BXo220, BXo826, BXo822 and BXo887) with mean lesion length <0.5 cm which was similar to the mean lesion of the donor parent IRBB60.This result indicates that the introgression of xa5, xa13 and Xa21 genes significantly enhanced the resistance of the improved pyramided restorer lines to BB.The mean lesion length (cm) of all pyramided restorers lines (resistant to highly resistant) with parents and susceptible checks are shown in Table 4.

Evaluation of fertility status of pyramided restorer lines Fertility status observed by gene-based/linked markers
Five pyramid restorer lines were subjected to PCR analysis using gene-based or linked markers against fertility restore genes Rf3 & Rf4.All pyramid restorer lines showed restorer alleles, Rf3 and Rf4 where band size was 115bp and 800bp, respectively (Figs. 3e-f).So, these lines had the ability to restore fertility.

SL Pyramided restorers
Important traits present

Agro-morphological and grain quality test of pyramided restorer lines
No significant differences were found between the pyramided restorer lines and the respective recurrent parent.All the selected pyramided restorer lines had more or alike agromorphological and grain-quality characters as recipient parent (Table 7, Table 8 and Fig. 5).Out of the five two pyramided restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 produced higher yields than the recipient parent.

Discussion
BB disease is a key limitation for hybrid rice farming in Bangladesh since most hybrid rice parental lines are sensitive to BB. Introgression of bacterial blight resistance genes into parental lines of hybrid rice using MABC is the best way to develop durable bacterial blight resistance lines.Because through MABC, the desired genes can be easily transferred into the elite lines and can quickly recover the recurrent parent characteristics with a minimum number of backcrosses.We developed five pyramided lines having Xa4, xa5, xa13 and Xa21 genes with fertility restorer genes.Many reviewers previously studied and introgressed BB resistance genes into promising to restore lines for the development of BB resistant pyramid hybrid rice [41][42][43][44].
MAS offers a simpler and more efficient method to improve rice cultivars or lines and markerassisted introgression of major genes/QTLs has helped to develop resistant varieties against major diseases such as BB, blast, tungro etc. [8,30,[45][46][47].But conventional backcross cannot precisely transfer more than one gene into the cultivars and also the process needs a significant amount of time [48][49][50].In this study, selected parents, BRRI31R and IRBB60 were validated against four genes-based or genes-linked markers for foreground selection.It was observed that the restorer line, BRRI31R showed monomorphic nature with a resistant allele against the Xa4 gene but showed a susceptible allele against the xa5, xa13 and Xa21 genes.
In this study, BC3F5 pyramid lines have been developed by backcrossing and selfing, and at each stage, BB resistant plants were selected based on molecular screening by MAS, pathogenicity test and phenotype comparison to their respective recurrent parent.The similar approach followed by earlier researchers and successfully introgressed two or three or four BB resistance genes into the rice cultivars or lines [29,[51][52][53].Our restorer lines were selected with target BB resistance genes using molecular markers and so there is a chance to carry linkage drag with the target gene.Backcross breeding is helpful to remove the linkage drug [54,55] and to eliminate linkage drag, backcross progenies were selected in each backcross and segregating generation through foreground and phenotypic selection.Therefore, it was proceeded by three backcrosses (BC3) and advanced BC3F1 to BC3F5 generation and foreground selection was performed up to BC3F5 generations to ensure the presence of the desired genes and phenotypically compared to respective recurrent parents.
In the present study, the segregated populations were analyzed in BC3F2 progenies against the xa5, xa13 and Xa21 genes by chi-square (χ2) test.Based on gene combinations of cross combination BRRI31R  IRBB60, it was observed that the segregated genotypic ratio of 1:2:1 which fitted expected segregation ratio for single gene model.The inheritance pattern of BB resistance controlled by single gene and segregation analysis on xa5, xa13 and Xa21 genes revealed that the chi-square (χ2) value is lower than the tabulated value at 5% (5.99) and 1% (9.21) levels which prove that these genes individually followed the first law of Gregor Johan Mendel [56][57][58].
Five homozygous pyramided restorer lines were selected from the cross combination, BRRI31R  IRBB60, with three BB resistance genes, xa5 + xa13 + Xa21 in BC3F5 progenies which showed highly resistant reaction against BB pathogens.Combination of more than one resistance gene is effective to defend against BB pathogen and gene combination of xa5 +xa13 + Xa21 are effective against the wide range of BB isolates of India [53,[59][60][61].It was noted that BRRI31R had the Xa4 gene by default and for this reason, finally, five homozygous pyramided restore lines were selected with four BB resistance genes, Xa4 + xa5 + xa13 + Xa21 from the cross combination, BRRI31R  IRBB60 in BC3F5 progenies.Though Xa4 is not effective against the BB pathogen of Bangladesh but mbination of this gene might enhance the function of other resistance gene and a combination of these four genes are also very effective against the diverse isolates of BB pathogens [23,25,34,59,62].These gene combinations also gives long-lasting and broad-spectrum resistance in the R8012 restorer line for Chinese CMS Zhong-9A restoration [63].
All selected pyramided restorer lines with respected recurrent parent were screened against five BXo races.It was observed that all pyramided lines showed resistance to highly resistance to all five races of Xoo and mean lesion length below 3 cm and ranged from 0.2 to 1.3 cm.More than one gene, specifically a combination of xa5+xa13+Xa21 is effective against more than one BB virulent isolates of Bangladesh [25,34].The data indicates that pyramided BB resistance genes in parental lines of rice hybrid could improve resistance to Xoo.Four gene combinations (Xa4 + xa5 + xa13 + Xa21) showed highly resistant and comparatively showed a higher degree of resistance than parent and susceptible check and findings are consistent with previous investigations [23,30,33,53,59].
The discovery of restorers is critical for the commercialization of heterosis breeding programs in hybrid rice utilizing the cytoplasmic male sterility (CMS) method [64].In this study, all five pyramided restorer lines viz., BRRI31R-MASP1, BRRI31R-MASP2, BRRI31R-MASP3, BRRI31R-MASP4 and BRRI31R-MASP5 showed fertility restorer alleles of Rf3 and Rf4.F1s pollens were found and of these F1s pollen were studied under compound microscopic and seed set (%) was observed under field conditions.Under both observations, all restorer lines displayed full fertility.So, all pyramided restorer lines possessed fertility restoring ability and it proves that the resistance genes of the study had not any negative effect on the restoring ability [44,65].These pyramided restorer lines will be used in line × tester analysis for finding the suitable combiner with the best heterotic combination compared to standard checks by General combining ability (GCA) and specific combining ability (SCA) analysis.After molecular screening, selected 5 pyramided restorer lines were screened against 20 QTLs trait using SNP markers.In this study, out of 20, 5 QTLs were also confirmed.
The agronomic characters analysis revealed that BC3F5 restorer lines derived from the BRRI31R were more or less similar to the recurrent parent and advanced lines of the backcross population are similar to the recipient parent because of background recovery [33].However, out of five, two pyramided restorer lines produced higher grain yields per plant than the recurrent parent.In addition, all pyramided restorer lines were not significantly different in milling outturn in percent, head rice recovery, milled rice length, milled rice breadth, length breadth ratio, amylose content, protein content, cooking time, elongation ratio and imbibition ratio, indicating that BC3F5 pyramiding lines had grain quality consistent with BRRI31R.The findings revealed that there were no decreases in yield or grain quality as a result of the pyramiding of the four BB resistance genes (Xa4, xa5, xa13 and Xa21).

Conclusion
We have successfully introgressed BB resistance genes into a promising restorer line,

Competing Interest
Authors declare that they have no any competing interest.

Funding
Authors received funds from Strengthening Physical Infrastructure and Research Activities (SPIRA) Project, Bangladesh Rice Research Institute, through the Ministry of Agriculture, Bangladesh to conduct this experiment.
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As a result of the aforementioned facts, the current study project was initiated with the following goals in mind: (i) introgression of xa5, xa13 & Xa21 genes in the promising restorer lines, BRRI31R using MABC; (ii) assessment of introgressed pyramided restorer lines by molecular and conventional approach; (iii) confirmation of fertility restorer genes (iv) evaluation of morphological and grain quality characters of developed pyramided lines in comparison with respective recurrent parent.However, the development of pyramided BB resistant restorer line carrying four resistance genes (Xa4, xa5, xa13 and Xa21) has a promising future to develop a bacterial blight-resistant hybrid variety in Bangladesh.

Fig. 2 .
Fig. 2. (a) Gel photographs showing banding pattern of the markers for the four bacterial blight (BB) resistance genes such as Xa4, xa5, xa13 and Xa21 in the parents.(b) Phenotypic

Fig. 2 .
Fig. 2. (a) Gel photographs showing banding pattern of the markers for the four bacterial blight (BB) resistance genes such as Xa4, xa5, xa13 and Xa21 in the parents.(b) Phenotypic screening of selected parents along with resistant and susceptible check against BXo races of bacterial blight (BB) disease.

Fig. 4 .
Fig. 4. Phenotypic screening of selected pyramiding restorer lines along with respective recurrent parent, donor parent and susceptible check against BXo races of bacterial blight (BB) disease.
BRRI31R using MABC.The pyramided restorer lines, BRRI31R-MASP1, BRRI31R-MASP2 and BRRI31R-MASP5 consisted of Xa4, xa5, xa13, Xa21, Chalk5, Rf3 and Rf4 genes.Moreover, BRRI31R-MASP3 and BRRI31R-MASP4 consisted of blast resistance gene Pita with Xa4, xa5, xa13, Xa21, Chalk5, Rf3 and Rf4 genes and these two pyramided restorer lines showed high resistant reactions against BXo races.It also demonstrated the utility of foreground selection in discovering target genes for bacterial blight resistance.These studies, concluded that the developed pyramided restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 with fertility restore genes would be used as donor parent to transfer bacterial blight resistance genes from pyramided restorer lines into highly susceptible restorer lines of promising hybrid rice.These lines, BRRI31R-MASP3 and BRRI31R-MASP4 might be used for parental lines improvement program by (R x R) crossing based on desired traits and also be used as a male parent for the development of BB resistant heterotic hybrids.The newly developed resistant lines could be also a replacement of our released popular hybrid rice variety of BRRI hybrid dhan5 and BRRI hybrid dhan7.Bangladesh.The authors are highly grateful to Plant Pathology Division and Hybrid Rice Division for providing all necessary support.The authors are extremely thankful to the Plant Pathology Division and the Hybrid Rice Division for providing all required assistance.

Table 1 .
Molecular markers used for foreground selection.

Table 3 )
were evaluated in test cross nursery by two ways such as a microscopic study and phenotypic study for confirmation of fertility status at the flowering stage by following the Hybrid Rice Breeding Manual, IRRI, Replication means data of the yield contributing traits and physicochemical and cooking characters under the study were subjected to univariate analysis.Univariate analysis of the individual character was done by computer using Excel program 2010 software.Statistical

Table 2 .
Evaluation of parents for BB resistance genes Xa4, xa5, xa13 and Xa21 genes using MAS and artificial inoculation genes and selfed to produce BC3F2 progenies.Finally, two hundred BC3F2 progenies were tested using linked markers to identify homozygous plants for different R genes of BRRI31R × IRBB60.A gel photograph of the BC3F2 population is presented in.Plants with single, double and triple gene combinations with BB resistance were identified in this study.
heterozygous condition for xa5, xa13 and Xa21 genes and these plants were further backcrossed to produce BC3F1 populations.From BC3F1 populations, 24 out of 65 plants were selected with three BB resistance

Table 3 .
Marker analysis in segregating BC3F2 populations for xa5, xa13 and Xa21 gene RR: plants with a banding pattern a like to the resistant parent alleles; Rr: heterozygous plants; rr: plants with a banding pattern a like to the susceptible parent alleles;  2 : actual value of the chi-square test for resistant/susceptible ratio.

Table 5 .
Restoration confirmation of pyramided restorer lines Sl

Table 7 .
Agro-morphological traits of pyramided restorer lines and recurrent parent

Table 8 .
Mean performance of grain quality parameters of pyramided restorer lines and parent