Exploring of bacterial blight resistance in landraces, mining of resistant gene(s) and genetic divergence of resistant germplasm through molecular markers and quantitative traits

Bacterial blight, one of the oldest and severe diseases of rice poses a major threat towards global rice production and food security. Thereafter, sustainable management of this disease has given paramount importance globally. In the current study, we explored 792 landraces to evaluate their disease reaction status against three highly virulent strains (BXo69, BXo87 and BXo93) of Xanthomonas oryzae pv. oryzae (Xoo). Subsequently, we intended to identify the possible candidate resistant (R) genes responsible for the resistant reaction using six STS markers correspond to Xa4, xa5, Xa7, xa13, Xa21 and Xa23 genes and finally, we evaluated morphological and molecular diversity of the potential bacterial blight resistant germplasm using quantitative traits and ISSR markers. Based on pathogenicity test, a single germplasm was found as highly resistant while, 33 germplasm were resistant and 40 were moderately resistant. Further molecular study on these 74 germplasm divulged that 41 germplasm carried Xa4 gene, 15 carried xa5 gene, 62 carried Xa7 gene, 33 carried xa13 gene, and 19 carried Xa23 gene. Only a single germplasm consisted of Xa21 gene. Interestingly, we found a wide range of gene combinations ranged from 2 to 4 genes among the germplasm resistant to bacterial blight and G3 genotype (Acc. No. 4216; highly resistant) having Xa4, Xa7, xa13, Xa21 and G43 genotype (Acc. No. 1523; resistant) having Xa4, xa5, xa13 and Xa23 gene combination being the most effective against all the Xoo strains. Nonetheless, UPGMA dendrogram and heatmap analysis based on quantitative traits identified two important clusters viz. cluster-III and cluster-VIII with multiple desired traits However, genetic similarity based on ISSR marker data pointed out 3 germplasm, namely, G20 (Acc. No. 4004; contained Xa4, Xa7, xa13 genes), G17 (Acc. No. 3981; contained Xa7, xa13 genes) and G6 (Acc. No. 991; contained Xa4, Xa7, xa13, Xa23 genes) having comparatively lower genetic similarity (0.30, 0.37 and 0.38, respectively) with elite variety BRRI dhan28. Notably, Mantle test of molecular and morphological data indicated that there was a positive correlation (r = 0.113) between them. The outcome of this study would enrich and diversify the rice gene pool and would be useful for the development of durable bacterial blight resistant varieties.

Nonetheless, epistasis and masking of gene effect rendered the conventional approach ineffective 75 in identifying cultivars carrying multiple disease resistant genes [18]. Alternatively, molecular 76 approach is widely accepted and utilized as a highly reliable and efficient method in identifying 77 resistant genes [12,19,20]. Interestingly, in a recent pathotype profiling study conducted on 118 78 Xoo isolates of Bangladesh, we identified xa5, xa8, xa13, Xa21 and Xa23 genes as the most 79 effective R genes against diverse Xoo strains [21]. In line with our previous finding, we aimed to 80 identify five cloned R genes viz. Xa4, xa5, xa13, Xa21, Xa23 and one un-cloned but widely 81 utilized R gene, Xa7, from a local germplasm collection of Bangladesh using STS markers.

82
On the other hand, knowledge of genetic diversity of germplasm collection helps to attain 83 durable selection gain while, plays a major role in parent identification and thus increases the 84 likelihood of crop improvement [22][23][24]. Then again, molecular markers play a leading role in based on quantitative traits. So, by combining molecular and morphological approach will be a 93 more comprehensive strategy to assess genetic divergence and to ensure a precise genotype 94 documentation along with parental line selection for breeding programs.

95
In the current study, we explored 792 landraces of Bangladesh through molecular and 96 morphological approaches with an objective to identify bacterial blight resistant germplasm and 97 evaluate genetic diversity among them to enrich and diversify the rice gene pool for future 98 breeding programs. The outcome of this study will be useful for research on rice and rice 99 improvement programs.

101
Collection of germplasm and experimental design 102 A total of 792 germplasm (S1 Table) (Table 1). On the contrary, genetic diversity of the selected germplasm were 142 determined using ISSR marker. A total of 22 ISSR markers were taken for polymorphism study 143 and finally 17 markers were selected based on their unambiguous reproducible polymorphic 144 band generation capability for genetic diversity analysis. PCR reaction mixture was formulated 145 using 5 μl GoTaq G2 green PCR master-mix (Promega, USA), 1 μl primer (forward and revers,    and 40 germplasm were found moderately resistant (score 3) against three virulent strains ( Fig   188   1). Rest of the germplasm showed moderately susceptible to highly susceptible disease reaction 189 (S1 Table). IRBB60 showed highly resistant score (0) while BRRI dhan28 showed highly 190 susceptible reaction (score 9).      Here, G1 to G74 represents 74 potential germplasm identified based on the disease reaction 272 against 3 Xoo strains while G75 represents BRRI dhan28 and G76 represents IRBB60.

314
According to the PCA analysis, 94.049% variability was observed due to first three component  Here, G1 to G74 represents 74 potential germplasm identified based on the disease reaction 324 against three Xoo strains while G75 represents BRRI dhan28 and G76 represents IRBB60.

326
The clustered germplasm from UPGMA dendrogram showed a variable mean performance in 327 respect to quantitative traits. However, none of the clusters was found to have all the desired 328 traits in respect to mean values (Fig 11). Notably, germplasm from Cluster-I had the highest Mantel test was performed to find correlation between molecular and morphological data matrix 345 (Fig 12). Our findings suggested that a positive but non-significant correlation (Matrix 346 correlation r= 0.113) existed between molecular and morphological variability data.

366
The STS markers utilized in the study were found highly efficient to identify the resistant genes.