Pyramiding of Four Broad Spectrum Bacterial Blight Resistance Genes in Cross Breeds of Basmati Rice

Pyramiding of major resistance (R) genes through marker-assisted selection (MAS) is a useful way to attain durable and broad-spectrum resistance against Xanthomonas oryzae pv. oryzae pathogen, the causal agent of bacterial blight (BB) disease in rice (Oryza sativa L.). The present study was designed to pyramid four broad spectrum BB-R genes (Xa4, xa5, xa13 and Xa21) in the background of Basmati-385, an indica rice cultivar with much sought-after qualitative and quantitative grain traits. The cultivar, however, is susceptible to BB and was therefore, crossed with IRBB59 which possesses R genes xa5, xa13 and Xa21, to attain broad and durable resistance. A total of 19 F1 plants were obtained, some of which were backcrossed with Basmati-385 and large number of BC1F1 plants were obtained. In BC1F2 generation, 31 phenotypically superior genotypes having morphological features of Basmati-385, were selected and advanced up to BC1F6 population. Sequence-tagged site (STS)-based MAS was carried out and phenotypic selection was made in each successive generation. In BC1F6 population, potentially homozygous recombinant inbred lines (RILs) from each line were selected and evaluated on the bases of STS evaluation and resistance to local Xanthomonas oryzae pv. oryzae (Xoo) isolates. Line 23 was found pyramided with all four BB-R genes i.e., Xa4, xa5, xa13 and Xa21. Five genotypes including line 8, line 16, line 21, line 27 and line 28 were identified as pyramided with three R genes, Xa4, xa5 and xa13. Pathological study showed that rice lines pyramided with quadruplet or triplet R genes showed the highest level of resistance compared to doublet or singlet R genes. Thus, line 23 with quadruplet, and lines 8, 16, 21, 27, and 28 with triplet R genes, are recommended for replicated yield and resistance trials before release as new rice varieties. Further, traditional breeding coupled with MAS, is a solid way to attain highly effective BB-resistant rice lines with no yield cost.


Introduction
Rice (Oryza sativa L.) as one of the most important cereal crops and major source of nutrition is feeding about 2.5 billion people around the world. Asian countries are the main producers and consumers of rice, with China and India contributing to more than half of Table 1. PCR analysis of RILs for bacterial blight R genes.

Identification of Pyramided Lines in BC1F6 Population
Line-23 was identified to possess all the four BB-R genes (Xa4, xa5, xa13 and Xa21) while line-8, line-16, line-21, line-27 and line-28 possessed three R genes among the advanced BC1F6 population developed in the present study ( Figure 5).

Responses of RILs to BB Isolates
Three local isolates of BB were used to evaluate the responses of selected genotypes. Analysis of variance showed significant differences among the genotypes for mean lesion length developed by isolates Xoo1, Xoo2, and Xoo3. Lesion size of RILs on inoculation with

Identification of Pyramided Lines in BC 1 F 6 Population
Line-23 was identified to possess all the four BB-R genes (Xa4, xa5, xa13 and Xa21) while line-8, line-16, line-21, line-27 and line-28 possessed three R genes among the advanced BC 1 F 6 population developed in the present study ( Figure 5).

Identification of Pyramided Lines in BC1F6 Population
Line-23 was identified to possess all the four BB-R genes (Xa4, xa5, xa13 and Xa21) while line-8, line-16, line-21, line-27 and line-28 possessed three R genes among the advanced BC1F6 population developed in the present study ( Figure 5).

Responses of RILs to BB Isolates
Three local isolates of BB were used to evaluate the responses of selected genotypes. Analysis of variance showed significant differences among the genotypes for mean lesion length developed by isolates Xoo1, Xoo2, and Xoo3. Lesion size of RILs on inoculation with

Responses of RILs to BB Isolates
Three local isolates of BB were used to evaluate the responses of selected genotypes. Analysis of variance showed significant differences among the genotypes for mean lesion length developed by isolates Xoo1, Xoo2, and Xoo3. Lesion size of RILs on inoculation with Xoo1 ranged from 1.3 to 60.1% with mean value of 24.1%, followed by 1-58% with mean  Out of 31 RILs, 18 genotypes showed resistant reactions to Xoo1, while 15 genotypes showed susceptible reactions. Among the former, lines 1, 16, 23 and 27 were highly resistant (HR), lines 5, 6, 8, 17, 20, 21 and 28 were resistant (R) and lines 2, 3, 4, 9, 11 and 14 were moderately resistant (MR). Among the latter, lines 7, 10, 12, 13, 18, 19, 22, 24, 25, 26, 30, 31 and  A phylogenetic tree was constructed based on percent diseases incidence. The tree was divided into three main clades. Clade 1 was further subdivided into two sub clades, C1A and C1B. Clade C1A consisted of 9 genotypes, including lines 6, 16, 21, 1, 8, 27, 28, 23 and IRBB-59 and was the most important clade consisting of all resistant genotypes. Both clades 2 and 3 comprised of 8 genotypes, each of which were further subdivided into two sub clades. The genotypes present in these clades were moderately susceptible to Xoo isolates (Table 2; Figure 9).      A phylogenetic tree was constructed based on percent diseases incidence. The tree was divided into three main clades. Clade 1 was further subdivided into two sub clades, C1A and C1B. Clade C1A consisted of 9 genotypes, including lines 6, 16, 21, 1, 8, 27, 28, 23 and IRBB-59 and was the most important clade consisting of all resistant genotypes. Both clades 2 and 3 comprised of 8 genotypes, each of which were further subdivided into two sub clades. The genotypes present in these clades were moderately susceptible to Xoo isolates (Table 2; Figure 9).

Discussion
Basmati rice is praised all over the world for its traits of unique taste, aroma and grain length. Its characteristic morpho-physiological ideotypes, however, lacks resistance against the BB disease in almost all tropical rice growing regions of the world [47], con- Figure 9. Phylogenetic tree constructed on the basis of percent diseases incidence.

Discussion
Basmati rice is praised all over the world for its traits of unique taste, aroma and grain length. Its characteristic morpho-physiological ideotypes, however, lacks resistance against the BB disease in almost all tropical rice growing regions of the world [47], considerably slashing the net rice productivity. The approach of 'defect elimination' of crop ideotype was used in this study to introgress BB resistance genes from IRBB59 to the susceptible Basmati-385 variety [48]. Introgression of R genes in Basmati varieties aided by molecular markers has been well reviewed in BB management programs [49]. Out of 45 BB resistance (R) genes identified [22,23], R genes Xa4, xa5, Xa7, xa13 and Xa21, have been individually incorporated into rice cultivars. However, the single gene-associated resistance is prone to break down and has been overcome by new strains of the pathogen. MAS-based pyramiding of major R genes into a single genotype seems to be a resource-feasible approach to attain durable and broad-spectrum resistance [20]. The approach was adopted in the current study to pyramid quadruplet BB-R genes in Basmati-385 rice background.
The quadruplet R genes have been exclusively and successfully pyramided in different rice varieties or their backgrounds. Resistance was achieved in rice variety Tapaswini for lowland ecology by introgressing Xa4, xa5, xa13, and Xa21 genes [50,51]. The disease incidence of Putra-1 was significantly lowered to 6.35% from 53% in the introgressed F 1 lines when pyramided with quadruplet Xa4, xa5, xa13, and Xa21 from the donor IRBB60 [15]. Similarly, the Indian susceptible varieties Mahsuri, PRR78, and KMR3 were bred for resistance by pyramiding Xa4, xa5, xa13 and Xa21 R genes, resulting into 1-3 cm infection in the resistant lines compared to lesion lengths of 22.6 cm, 18.8 cm and 18.2 cm in the susceptible parents [52]. The resistance response of the CNYBB4R03 line pyramided with Xa4, xa5, xa13 and Xa21 genes was 0.43 cm compared to 6.75-12.56 cm in susceptible varieties, TNG82, TCS10, TN1 and IR24 [38]. In the current study, RIL 23 pyramided with Xa4, xa5, xa13 and Xa21 genes showed highest level of resistance with just 1.4-2.4% disease lesion length compared to Basmati-385 in response to local Himalayan Xoo strains.
Similar to quadruplets, combination of triplet R genes also showed significant BBresistance in different susceptible rice cultivars [45,53]. The triplet set of R genes, Xa4, xa5 and Xa21, has been exclusively used in different rice breeding for BB resistance programs. The triplet-genes-transformed CSR-30 rice cultivar, for example, exhibited an incompatible mean lesion length of 0.4 cm, near to the mean lesion length of 0.5 cm of donor IRBB-60 against Xoo. Similarly, the high-yielding deep-water rice variety, Jalmagna and PKBB 15-116 lines showed reduced lesions of 1.4-2.9 cm and 4% compared to 9.4-12.8 cm and 60% in the respective controls when pyramided with Xa4, xa5, and Xa21 triplet genes [41,46]. Pyramided with Xa4, xa5, and Xa21 from donor line IRBB57, the Korean elite japonica variety Mangeumbyeo, showed a drastic regression of <0.3 cm lesion length, near to the <0.5 cm lesion length of donor IRBB-57, compared to 9-18 cm lesion length when challenged with 18 Xoo isolates. The NILs introgressed with either of individual R genes, Xa4, xa5 or Xa21 were, however, exhibited S, MR and R reactions, respectively, to the 18 isolates in question [54]. In the current results, 6 RILs having three genes Xa4, xa5 and xa13, showed high resistance reaction compared to RILs with single or double genes, suggesting the additive nature of the resistance.
The mean lesion length on the doublet R genes combinations: Xa21 and xa13, Xa21 and xa5, and xa5 and xa13 was 3.1-3.9 cm, 3.5-4.8 cm, and 4.9-5.7, respectively [41]. Xa21 and xa5, individually, showed resistance with the mean lesion lengths of 1.2 cm and 1.1 cm, respectively, though xa13-harboring genotype was moderately resistant with of 4.8 cm lesion length when challenged with Xoo [53]. Rashid et al., [55] studied the response of different lines to 118 local Xoo isolates and found the lines with single R genes did not show much resistance. There were, however, pyramided lines with xa5 and xa13 or xa13 and Xa21 genes combinations, showing the highest amount of resistance frequency (100%). Similarly, Yugander et al. [56] observed IRBB lines with single R genes were susceptible to 73.2-97.2% of the isolates. Current study showed that the quadruplet-and triplet-genes-introgressed lines were the best lines which showed resistance against the selected Himalayan Xoo strains, corroborating the cumulative impact of multiple R genes.
The epistatic effect of the dominant R genes on plant morphology and physiology, and quantitatively indistinguishable impact between different R genes on two or more races of Xoo, make the identification of and differentiation between different R genes a tough task. Molecular markers tightly linked with each of the R genes make the identification of plants with two or more R genes possible [45]. Several genes for resistance to BB have been tagged with molecular markers [57]. These include restriction fragment length polymorphism, randomly amplified polymorphic DNA markers and STS markers to validate the presence of introgressed R genes in lines of interest [58]. It is possible to develop closely linked molecular markers for each of the R genes within a plant [45,59]. The potential of STS markers was, therefore, assessed to identify rice lines with multiple BB-R genes in the current study. These RILs developed were resistant to the selected strains collected from the foothills of Himalayas in Mansehra district, which otherwise, were virulent to the parent Basmati 385. The local farmers of the district can grow Basmati-like RILs in the foothills of Himalaya, unlike the parent Basmati-385 which is generally grown in the Punjab province of Pakistan, without any significant BB disease incidence. Further, such RILs can be tested in the mainstream rice growing regions of Pakistani Punjab and its adjoining Indian Punjab, to evaluate their qualitative and quantitative traits before their adoption in these mainstream regions.

Pyramiding BB-R Genes into Basmati-385
Basmati-385 was crossed with IRBB-59, in which the former contributes single BB-R gene Xa4, while the later contributes triple BB-R genes, Xa21, xa5 and xa13 ( Figure 10). The breeding program was started in rice growing season 2014. A large number of immature spikelets of Basmati-385 were emasculated early in the morning and shed with sufficient pollens of IRBB59 in the afternoon. The pollinated panicles were covered with crossing bags. The hybrid seeds were harvested 30 days after pollination. A total of 19 hybrid seeds were obtained and germinated in sterile petri plates to grow F1 hybrids. Some of the F1 hybrids were back crossed with Basmati-385 and thus BC1F1 population The breeding program was started in rice growing season 2014. A large number of immature spikelets of Basmati-385 were emasculated early in the morning and shed with sufficient pollens of IRBB59 in the afternoon. The pollinated panicles were covered with crossing bags. The hybrid seeds were harvested 30 days after pollination. A total of 19 hybrid seeds were obtained and germinated in sterile petri plates to grow F 1 hybrids. Some of the F 1 hybrids were back crossed with Basmati-385 and thus BC 1 F 1 population was obtained and selfed to produce a large number of BC 1 F 2 population. Phenotypically, superior plants were advanced up to BC 1 F 6 population. Finally, 31 potentially homozygous recombinant inbred lines (RILs) were selected and evaluated on the bases of molecular markers and their resistance to local Xoo isolates.

Extraction of Genomic DNA
The genomic (g) DNA was extracted using CTAB method [20]. Fresh leaves at early seedling stages were collected in Eppendorf tubes and put immediately in liquid nitrogen. Crushed with a glass rod and 700 µL of heated (60 • C) 2× CTAB buffer (50 mM Tris-HCl, pH 8.0, 25 mM EDTA, 300 mM NaCl and 2% CTAB) was added to each sample. The samples were then incubated at 56 • C overnight and mixed again with the help of a glass rod. Followed by addition of 700 µL Chloroform: Isoamyl alcohol (24:1) solution and kept at room temperature for 30 min. The samples were then centrifuged at 9000 rpm for 20 min and a clear supernatant (500 µL) was transferred to a newly labeled Eppendorf tubes. Then 500 µL ice cold isopropanol and 40 µL sodium acetate were added to it and incubated at −20 • C for at least 1 h or overnight. The samples were centrifuged at 9000 rpm for 20 min to make DNA pellet. The supernatant was discarded, and the pellet was washed with 70% ethanol and dried at room temperature. Then, 40 µL TE buffer was added to each sample. For RNA degradation 1µL RNAse was added to each tube and incubated at 37 • C for one hour. The quality and quantity of DNA was checked on 1% agarose gel stained with ethidium bromide. The concentration of DNA was adjusted from 20 to 50 ng/µL by using double distilled water and stored at 4 • C for further use.

PCR Amplification of Bacterial Blight R Genes
PCR reactions were carried out in 50 µL reaction volumes having 1-2 µL genomic DNA, 1 µL each of forward and reverse STS primers. Amplification was carried out in DNA thermal Cycler (Applied Bio System), at 94 • C for 6 min as initial denaturation, 36 cycles at 94 • C for 1 min, 55 • C for 1 min, and 72 • C for 2 min. Final extension was carried out at 72 • C for 7 min. PCR products were run on 2% agarose gel in TAE buffer. For identification of xa13 gene, initially 5µL PCR product was used in gel electrophoresis. The remaining PCR product was used for restriction digests. The reaction mixture for restriction of PCR amplicon of xa13 gene consisted of 3.2 µL sterile distilled water, 1.5 µL restriction buffer (tango buffer), 0.3 µL restriction enzyme Hinf1(10 U/µL) and 15 µL of PCR product. Incubation of the reaction mixture varied from 4 h to overnight at 37 • C. The resultant fragments were separated by gel electrophoresis on 1.5% agarose gel. The gel was stained with ethidium bromide (10 ug/mL) and observed under UV light (Table 3).

Xanthomonas oryzae pv. oryzae (Xoo) Strain Isolation
Rice leaves showing clear BB symptoms were collected from various rice fields at district Mansehra and kept in refrigerator. For isolation of bacterial strains, the samples were washed with sterilized distilled water and air dried in laminar flow hood. Four Eppendorf tubes were filled with sterilized water, while one Eppendorf tube with 70% ethanol. Leaves were cut at points of fresh disease symptoms into 2-4 cm fragments and washed with 70% ethanol, followed by rinsing with sterile distil water 2-3 times and crushed in 1 mL sterilized water using sterilized blue tip in a fresh Eppendorf tube. The crushed material was incubated at 30 • C for one hour so that bacteria may ooze out into the water. After dipping the sterilized wire loop into the crushed material, bacteria were streaked on the petri dishes having nutrient agar media, and kept in incubator for 4 days at 28 • C. Pure cultures were derived from single round, smooth, golden yellow and mucous colonies and streaked in new petri dishes having nutrient agar media and incubated for 2-3 days at 28 • C.

Preparation of Inoculum from Pure Cultures
For preparation of inoculum 10-15 mL distilled water was taken in 30 mL tubes and mixed with a 2-days pure cultures of Xoo. Selected genotypes were inoculated at seedling stages using clip method as reported by [63]. Disease symptoms were observed on a daily basis till the 16th day and compared with Intentional Rice Research Institute (IRRI) standard scale as reference for diseases scoring (Table 4).

Data Analyses
Analysis of variance (ANOVA) was performed and Tukey test was used for comparison of means using statistical software Statistix 8.1. Cluster analysis was performed using Past software.

Conclusions
The present study conclusively proves RIL 23 as the only quadruplet-genes-introgressed line showing the best resistance against the selected Himalayan Xoo strains, followed by 5 triplet-genes-introgressed RILs consisting of 8, 16, 21, 27 and 28. Though having the high possibility of adoption in Northern Pakistan, country-wide multi-location trials for the RILs are needed to evaluate their resistance against extant of Xoo strains and yield performance before incorporating them in national rice breeding programs.