Allele Profiling of Bacterial Blight Resistance Genes Xa4 , Xa38 , and Xa21 in Selected Sri Lankan Rice Germplasm

In rice, resistance to bacterial blight (BB) is conveyed by several Xa genes, among which Xa4 , Xa21 and Xa38 convey durable resistance. Most Sri Lankan rice germplasm is uncharacterized for the alleles carried at Xa genes. Knowledge on the allele profile of major Xa genes of rice accessions/varieties is essential for making informed decisions in rice breeding programs. In the current study, the allele profiles of 42 Sri Lankan rice accessions/varieties were developed targeting three Xa genes ( Xa4 , Xa21 , and Xa38 ) using intragenic/linked markers amplifying known resistance/susceptible alleles. According to allele profiles, the rice accessions/varieties were grouped into 11 allele-haplotypes. The varieties, Bg 250, Bg 251 and At 354 carried resistance alleles at Xa4 , Xa21 , and Xa38 (H1 and H3). Most other accessions/varieties reported either one ( Xa4 - H8 and Xa38 - H10) or two resistance alleles ( Xa4 and Xa21-H2; Xa4 and Xa38 - H9; Xa21 and Xa38 - H11). Three allele-haplotypes were reported with a novel allele at Xa4 (H4, H5, and H6). Ma wee and Kuru wee reported susceptible alleles at all three Xa genes (H7). A significant association ( p ≤0.05) between the Xa allele-haplotypes and the BB disease response was not observed. To achieve durable BB disease resistance in rice, it is recommended to introgress resistance alleles of the major Xa genes when releasing rice varieties, for which the


ABSTRACT
In rice, resistance to bacterial blight (BB) is conveyed by several Xa genes, among which Xa4, Xa21 and Xa38 convey durable resistance.Most Sri Lankan rice germplasm is uncharacterized for the alleles carried at Xa genes.Knowledge on the allele profile of major Xa genes of rice accessions/varieties is essential for making informed decisions in rice breeding programs.In the current study, the allele profiles of 42 Sri Lankan rice accessions/varieties were developed targeting three Xa genes (Xa4, Xa21, and Xa38) using intragenic/linked markers amplifying known resistance/susceptible alleles.According to allele profiles, the rice accessions/varieties were grouped into 11 allelehaplotypes.The varieties, Bg 250, Bg 251 and At 354 carried resistance alleles at Xa4, Xa21, and Xa38 (H1 and H3).Most other accessions/varieties reported either one (Xa4-H8 and Xa38-H10) or two resistance alleles (Xa4 and Xa21-H2; Xa4 and Xa38-H9; Xa21 and Xa38-H11).Three allele-haplotypes were reported with a novel allele at Xa4 (H4, H5, and H6).Ma wee and Kuru wee reported susceptible alleles at all three Xa genes (H7).A significant association (p≤0.05) between the Xa allele-haplotypes and the BB disease response was not observed.To achieve durable BB disease resistance in rice, it is recommended to introgress resistance alleles of the major Xa genes when releasing rice varieties, for which the reported allele profile of Xa4, Xa21 and Xa38 genes in the selected panel of Sri Lankan rice varieties/accessions will be of great importance.

INTRODUCTION
Rice is a major cereal crop known to provide the main caloric requirement of approximately 2/3 rd of the world's population.It is predicted that the world population will reach 9.5 billion by the year 2050 (FAO, 2009), hence, feeding the increasing number of heads will be a main challenge in the rice sector.The final yield and quality of rice depends on the genetic potential, plant vigor, environment, and pre-and post-harvest practices.To meet the demand, improving both yield and quality attributes, and production of a climate resilient rice crop tolerant to major abiotic stresses, and resistant to pests become a key target in rice breeding programs globally.
Biotic and abiotic factors are major causes of yield and quality losses in rice.Rice is susceptible to many diseases; among them bacterial blight (BB) is one of the devastating diseases which cause severe damages in rice fields all over the world.In an epidemic, BB has been reported to cause significant economic losses in both yield and quality, leading to yield losses up to 80% in some regions (Wonni et al., 2016;Dilla-ermita et al., 2017).
The gram-negative bacteria Xanthomonas oryzae pv.oryzae (Xoo) is the causal agent of BB and under high humidity and mild temperatures the pathogen is known to develop and spread faster in rice fields.Controlling BB in rice is one of the major challenges faced by rice farmers all over the world.There is no specific regime of chemicals that can be effectively used to control BB (Song et al., 2016;Kumar et al., 2020).Though antibiotics have been reported as a control strategy, it is considered neither as a feasible, nor cost effective disease management option.As a long-term, stable disease management strategy plant breeders try to incorporate gene-bound resistance against BB into newly released rice varieties (Lang et al., 2010;Perumalsamy et al., 2010;Lua et al., 2014;Kim et al., 2015).
Nearly 45 Xa genes have been reported so far and their combined action is known to deliver resistance against BB (Fiyaz et al., 2022).The relative contribution of resistance from each Xa gene for mitigation of disease development of BB is different; some exert major effects, some minor effects, some specific effects for a few Xoo strains and some work against a broader range of Xoo strains (Akhtar et al., 2008;Fred et al., 2016).The Xa4 (Os11g0691280; Chr11: 27668321..27675334), Xa21 (Os11g0569733; Chr11: 21273533..21277443), and Xa38 (Os04g0621500; Chr4: 31576517..31583927) are a few of the known major BB resistance genes which deliver high, durable and stable resistance (Huang et al., 1997;Sun et al., 2003;Sabar et al., 2016;Zhang et al., 2017;Xu et al., 2019).Molecular marker-assisted selection (MAS) can be used to introgress the resistance alleles of these Xa genes into elite rice varieties.In previous studies, intragenic markers Xa4_F/R and ABUOP0001_F/R were used to track BB resistance alleles of genes Xa4 and Xa21, respectively (Hajira et al., 2016;Yap et al., 2016;Nanayakkara et al., 2020).For the gene Xa38, the linked marker Os04g53050-1_F/R was recommended (Bhasin et al., 2012).
Rice germplasm around the world is known to be diverse in its genetic potential with respect to BB response.Sri Lanka has a diverse collection of rice germplasm, accounting to approximately 1,500 rice accessions (Withanawasam., 2017).However, the allelic diversity of the Xa genes in Sri Lankan rice germplasm remains mostly unknown.Identification of existing alleles will enable the rice breeders to make informative decisions during parental selection for varietal development and other pre-breeding activities.The current study was conducted to profile the known resistance/susceptible alleles carried at Xa4, Xa21, and Xa38 BB resistance gene loci as an allele-haplotype in a core rice panel of 42 rice accessions/varieties comprising of Sri Lankan traditional rice accessions (SLTA) and Sri Lankan newly improved rice varieties (SLNIV).

METHODOLOGY Bacterial blight disease responses of the core rice panel
The BB responses of 42 Sri Lankan rice accessions/varieties (16 SLTAs and 26 SLNIVs) from the Yala season of 2017 was previously reported in Nanayakkara et al. (2020).In the current study, the estimated median score (EMS) reported for these 42 Sri

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Lankan rice accessions/ varieties were used for further analysis under the Creative Commons Attribution License (Nanayakkara et al., 2020;Supplementary Table 1-Column G).

Identification of alleles carried at BB resistance genes Xa4, Xa21 and Xa38 in the core rice panel
The primer pairs Xa4_F/R (Xa4; Yap et al., 2016) and Os04g53050-1_F/R (Xa38; Bhasin et al., 2012) were used for PCR amplification of the previously reported resistant/susceptible alleles carried by the 42 Sri Lankan rice accessions/varieties.The PCR amplification was done using a CT1000 thermal cycler (Bio-Rad Laboratories, Inc., California, USA) with a 15 µL final PCR reaction volume that included 150 ng of template DNA, 1× GoTaq ® Green master mix (Promega Corporation, Maddison, USA), 0.125 μM primer concentration and 1 mg/mL bovine serum albumin (Promega Corporation, Maddison, USA).In the notemplate control, the template DNA was replaced with nuclease-free water (Promega Corporation, Maddison, USA).The template DNA was initially denatured at 94 °C for 5 min, followed by 35 cycles of PCR amplification: 94 °C denaturation for 30 s, annealing at a primer-specific annealing temperature (58 °C for Xa4_F/R and 56 °C for Os04g53050-1_F/R) for 30 s and a primer extension at 72 °C for 30 s.The PCR cycle was completed with an additional primer extension step of 72 °C for 5 min.The amplified PCR products were resolved on a 3% agarose gel (Sigma-Aldrich ® , Merck KGaA, Darmstadt, Germany) prestained with 5% ethidium bromide and was visualized using a gel documentation system (UVCI-1100, Major Science, USA).The alleles were called resistant and susceptible by comparison to previously reported fragment sizes (Table 01).The newly reported alleles in the core panel were noted separately.For the Xa21 gene the allele calls for marker ABUOP0001 previously reported for the 42 rice accessions were extracted from Nanayakkara et al. (2020)

RESULTS AND DISCUSSION
Bacterial blight is a major rice disease prevailing in Sri Lanka.Therefore, introgression of genetic resistance against BB to elite varieties destined for wider cultivation is an effective and eco-friendly control strategy.For most Sri Lankan rice accessions the alleles carried at major BB resistance genes are not available, except for those reported for Xa21 in Nanayakkara et al. (2020).Knowledge on the alleles carried at major BB resistance genes is important for rice breeders as well as for pre-breeders working on BB resistance.Here we report, the resistance/susceptible alleles carried at the major BB resistance genes Xa4 and Xa38 for a panel of 42 Sri Lankan rice accessions/varieties and the association of BB disease responses to allele-haplotype of three major BB resistance genes Xa4, Xa38 and Xa21 (extracted from Nanayakkara et al. (2020).
In the current study, the disease categorization reported in Acharya and Sujiata, (2021) was modified further to categorize the rice accessions/varieties according to the BB disease response expressed.Accordingly, the 42 Sri Lankan rice accessions/varieties were categorized as resistant (EMS of 1 and 2), moderately resistant (EMS of 3 and 4), moderately susceptible (EMS of 5 and 6), susceptible (EMS of 7 and 8) and highly susceptible (EMS of 9).Compared to Koch, (1989) adopted in Nanayakkara et al. (2020), the current categorization is reflective of a broader disease response spectrum and hence, is more appropriate for deducing the association of disease responses to the allele-haplotypes.The BB disease responses are driven by the cumulative effect of the resistance/susceptible alleles of Xa genes in a particular rice accession/variety.In the current study, the alleles of two major Xa genes, namely Xa4 and Xa38 were identified for 42 Sri Lankan accessions/varieties (Supplementary Table 1) and the alleles of Xa21 for the same rice panel were extracted from Nanayakkara et al. (2020).Based on the alleles reported for these three BB disease resistance genes Xa4, Xa21 and Xa38, 11 allelehaplotypes (H1-H11) were deduced in the selected core panel of 42 Sri Lankan rice accessions/varieties (Figure 1).
With respect to the three Xa genes Xa4, Xa21 and Xa38, only SLNIVs At 354, Bg 250, and Bg 251 (in a heterozygous state at the Xa38 locus) carried resistance alleles at all three loci (Figure 1 -H1 and H3; Supplementary Table 1).None of the studied SLTAs reported resistant alleles at all three of these Xa genes.According to Supplementary Table 1, varieties Bg 250, Bg 251 and At 354 reported a resistant disease response.However, it is noteworthy to mention that according to the varietal descriptors, Bg 250 and Bg 251 are reported as moderately resistant and the variety At 354 was reported as susceptible (https://doa.gov.lk/rrdi_rice_varities/).Given the fact that BB is a quantitative trait which is highly dependent on the environment (Burt et al., 2014;Han et al., 2014) and due to the strain-specific resistance delivered by Xa genes, such variations could be expected when comparing responses from different seasons and experimental locations.
In the core panel, SLTAs Kuru wee and Murungakayan carried the susceptible alleles at all three genes Xa4, Xa21 and Xa38 (Figure 1 -H7, Supplementary Table 1).However, Kuru wee and Murungakayan reported a resistant and moderately resistant BB disease response, respectively (Supplementary Table 1).Being traditional Sri Lankan accessions, | 147 these accessions could be carrying unique genetic make-up, with potentially novel QTL/genes that are responsible for resistant BB disease responses, hence, it is recommended to carry out QTL mapping studies to deduce the Xa genes responsible for the observed BB resistant/moderately resistant disease response, despite carrying a susceptible allele-haplotype at the major Xa genes Xa4, Xa21 and Xa38 With respect to other Xa gene combinations, SLNIVs Bg 305, Bg 352, Bg 358, Bg 360, Bg 366, Bg 369, At 362, At 373, Bw 367, Bw 14-509 and Ld 365, and SLTA Yakada wee reported carrying resistance alleles at the two Xa genes, Xa4 and Xa38 (Figure 1 -H9, Supplementary Table 1).These varieties exerted a disease response of resistant (Bg 352, Bg 360 and Bw 367), moderately resistant (Bg 305, Bg 366, Bg 369, At 362, At 373 and Bw 14-509) and susceptible (Bg 358, Ld 365 and Yakada wee) (Supplementary Table 1).Furthermore, SLNIV Ld 371 was the only variety to carry the resistance allele combination of genes Xa21 and Xa38 (Figure 1 -H11, Supplementary Table 1) and reported a moderately resistant BB disease response (Supplementary Table 1).The SLTA Mahakuru wee reported a resistant allele combination for Xa4 and Xa21 (Figure 1 -H2, Supplementary Table 1) and a resistant disease response (Supplementary Table 1).The SLTAs Pokkali, Dik wee, Herath Banda, Kattaran, Wannidahanala, Podihatatha, Pachchaperumal, Gonabaru, and SLNIV Bg 357 reported only the known resistance allele at Xa4 (Figure 1 -H8, Supplementary Table 1).Furthermore, Mada el, Batapola el, Suwandel and At 307 reported a new allele for Xa4.In previous research, only the resistance allele 217 bp and susceptible allele 198 bp was reported for Xa4 (Yap et al., 2016).The novel allele detected has a fragment size which is in between the previously reported alleles (approximately 225 bp) on a 3% agarose gel.Therefore, it is recommended to size it through a capillary gel electrophoresis approach and validate its association to the BB disease response.Nevertheless, the detection of such novel alleles indicates the existence of a unique genetic diversity among the Sri Lankan rice germplasm.
Based on Kruskal-Wallis test no single allelehaplotype (H1 -H11) was significantly (p ≤ 0.05) associated with a BB disease response expressed as EMS.The inability to find a clear association between the allele haplotypes of these Xa genes and the BB disease response could well be because of the additive effect of other known Xa genes in play and novel Xa genes responsible for resistance/susceptibility which are yet to be identified in this unique germplasm.Therefore, it is worthy to further expand the screening of alleles to cover other known major Xa genes and to conduct QTL mapping to identify novel genomic regions associated with BB resistance/susceptibility. Given the severity of the impact of BB in rice production in countries such as Sri Lanka where rice is the main staple food of majority of the people, identification of the accessions that carry resistance alleles gives an important perspective when selecting rice accessions in breeding programs.In this regard, the findings of the current study are very much important for the Sri Lankan rice breeders to make informative decisions with respect to development of rice varieties deploying genetic resistance to mitigate BB in rice cultivation.Further, the study reveals opportunities for pre-breeders to explore the genomes of the mostly uncharacterized Sri Lankan rice germplasm to discover novel QTL related to BB disease resistance.It is a known fact that a single Xa gene cannot deliver the expected durable resistance against BB given the existence of several pathogen strains and dependance on the environmental conditions in the cultivation areas.Based on our observations, it is recommended to introgress resistant allele-haplotypes (considering the major Xa genes such as Xa4, Xa21, Xa38 and other) through gene pyramiding using MAS when releasing modern varieties.

CONCLUSIONS
The current study reports 11 allelehaplotypes (H1 -H11) in a core panel of 42 Sri Lankan rice accessions/varieties considering three major Xa genes Xa4, Xa21 and Xa38 delivering resistance against BB in rice.Only three SLNIVs (Bg 250, Bg 251 and At 354) carried the resistance alleles for all the three Xa genes under consideration.While most accessions/varieties reported one or more combinations of resistance alleles for these major Xa genes, accessions/varieties Murungakayan and Kattaran reported susceptible alleles for all three Xa genes.None of the identified allele-haplotypes showed a significant association (p ≤ 0.05) to any of the BB disease response categories (EMS 1 -8), indicating additional genes at play. the International Development Research Centre (IDRC), Ottawa, Canada.The views expressed herein do not necessarily represent those of UNESCO, IDRC or its Board of Governors or the University of Peradeniya, Sri Lanka.The authors also wish to acknowledge Plant Genetic Resource Centre, Gannoruwa, Sri Lanka and Regional Rice Research and Development Centre, Bombuwala, Sri Lanka for providing seeds to carry out the experiment.

Figure 1 :
Figure 1: Allele-haplotypes identified for bacterial blight disease resistance genes Xa4, Xa21 and Xa38 in a core panel of 42 Sri Lankan rice accessions/varieties

Table 1 . Details of the DNA markers used to amplify the alleles of Xa4 and Xa38 bacterial blight resistance genes in the core rice panel
(Supplementary Table -1; Column E) under the Creative Commons Attribution License.