Frequency distribution of ShB disease in the F2 and F2:3 populations
The resistant parent O. nivara acc. IRGC81941A and highly susceptible recipient parents PR121 showed contrasting responses to R. solani during the five years of screening. The O. nivara acc. IRGC81941A showed a consistent disease score of 3 on a 0-9 scale with a mean RLH of 19-24 % against a highly virulent strain of R. solani over the period from 2017 to 2021. The recipient parent PR121 and PR114 consistently display a score of 9 (highly susceptible) with mean RLH 62-75 % over five years of screening against R. solani. We have developed a mapping population and backcross population by crossing resistant parent O. nivara acc. IRGC81941A and highly susceptible recipient parents PR121 for the identification of ShB resistance QTL (Fig. 1). Phenotypic evaluation for the three ShB-related traits viz. LH, RLH, and PH were performed on 479 F2 individuals with their parental lines in the year 2017. The phenotypic data in the year 2017 varied from 21 to 52 for LH, from 0.15 to 0.70 for RLH, and from 65 to 185 for PH. Continuous variations with transgressive segregation were observed in the F2 populations for LH, RLH, and PH (Fig. 2A). The Shapiro–Wilk (w) normality test indicates that the phenotypic data in the F2 population was normally distributed (Table 1). A total of 241 F2:3 families (10 plants each) were evaluated in the year 2018 against R. solani (Supp. Table 1) The phenotypic data for three traits was slightly skewed towards the recipient parent (Fig. 2B). The obvious reason for the skewness is selection biases in the F2:3 populations and practically, the F2:3 may not always correlate to the F2. The phenotypic data on ShB was also recorded on F3:4 progenies in the year 2019. Besides, the average of F2 and F2:3 was more stabilized and exhibited normal distribution (Fig. 2C). Therefore, the mapping was conducted in mean data as well as F2 and F2:3 populations.
The different disease variables measured in the years 2017, and 2018 and their means were significantly (P < 0.05) correlated (Supp. Fig. 1). A strong and positive correlation was observed between LH and RLH in the years 2017 (0.74), and 2018 (0.54) which indicates that these two traits were highly associated with each other under different environmental conditions. However, PH showed a significantly negative correlation with LH and RLH in the years 2017, and 2018 and their means. It suggested that PH and ShB resistance are controlled by a different genetic mechanism. RLH was negatively correlated to PH with a correlation coefficient of -0.75 in 2017, and -0.77 in 2018, whereas LH displayed a negative correlation with PH in 2017 (-0.15), and poor correlation in 2018 (0.08). It indicates that PH had little effect on the LH and RLH across the two environments and their means.
Identification of promising lines in the BC2F2 and BC2F3 populations
Significant variations were observed for three traits PH, LH, and RLH in the BC2F2 and BC2F3 populations. Approximately 300 BC2F2 and their corresponding BC2F3 were phenotyped against highly virulent strains of R. solani. In the year 2019, approximately 300 BC2F2 were screened. Among them, three BC2F2 progenies (6645-6, 6645-12, 6646-13, and 6646-16) were selected which showed good resistance ranging from 3-5 score with RLH <28% having desirable agronomic traits. These progenies were self-pollinated as BC2F2 progenies (1547, 1554, and 1556) and evaluated in the year 2020. The frequency distribution of RLH for three progenies of BC2F2 and their corresponding BC2F3 populations were represented in Fig. 3. The distribution of phenotype in the BC2F2 was nearly symmetrical and bell-shaped whereas populations in the BC2F3 for RLH were skewed towards the resistant parent except for population 1556 which showed symmetrical distribution for the trait. The BC2F3 progeny 1547 was developed in the background of highly susceptible parent PR121 whereas 1554 and 1556 were developed in the genetic background of PR114. A total of 13 and 30 individuals from the progenies 1554 and 1556, respectively were selected which showed 3.0 disease score. Whereas, 6 individuals were showing resistance response to R. solani with superior agronomic features were identified from the BC2F3 progeny 1547. These promising resistant BC2F3 lines exhibit substantial variations for major agronomic traits such as plant height, small awn, early maturity, etc without any significant reduction in the yield (Table 2). Transgressive segregations reported for plant height, earliness, short awns, and grain number indicate the contribution of beneficial and yield-enhancing alleles from wild species. However, some lines showing big awn, late maturity, and tall stature were discarded. The superior Shb resistant lines showing desirable agronomic traits could be excellent pre-breeding germplasm resources for broadening the genetic base of the cultivated gene pool.
Genotyping of the F2 population using ddRAD-seq
In total 3,32,663 SNP markers were obtained with ddRAD sequencing (Table 1). The SNP markers with indel or heterozygote SNP in either of the parents were removed, and only SNPs in the homozygous state (AA, CC, TT, and GG) were used for scoring the polymorphism between O. sativa cv. PR121 and O. nivara accession IRGC81941A. With this approach, we were left with only 51,320 polymorphic SNPs between the parents for further linkage analysis. After filtering the significantly distorted markers (ꭓ2 ≥ 10) and missing values ≥10, a total of 1971 markers were retained for genetic map construction. The SNPs markers with distorted segregation were analyzed using the Chi-square test. A total of 1971 markers were thus used for the construction of the linkage map using RECORD (Supp Table 2).
Linkage and quantitative trait locus mapping
Based on an SNP-based linkage map with 1971 SNP markers and the phenotypic data, we conducted QTL analysis using composite interval mapping in QTL cartographer software. QTLs were detected above 2.5 LOD as the threshold and 1000 permutations were considered significant intervals. A QTL detected above 3 LOD with > 10 phenotypic variances were considered a major QTL. A total of 29 QTLs were detected for sheath blight resistance and component traits using composite interval mapping. These QTLs were located on all the rice chromosomes except 5, 9, 10, and 12, and the total phenotypic variance explained (PVE) ranged from 4.70 to 48.05% (Table 3). Favorable alleles from 13 QTLs were inherited from the susceptible cultivars PR121 while 16 QTLs were inherited from partially resistant parent O. nivara acc.81941A. Among them, 11 QTLs were detected on chromosome 1. Five loci on chromosome 1 and four loci on chromosome 3 were mapped to the nearly same location and stable across the years 2017, 2018, and the mean data whereas other loci were mapped at a different position or undetected under different environmental conditions. A total of eight QTLs were identified for PH, including four in the year 2017, two each in the year 2018, and the mean. They were located on chromosomes 1, 2, 4, 6, and 8 and explained 0.5 to 37.69 % phenotypic variance. Among them, six QTLs were major explaining >10 % phenotypic variance while two were minor QTLs. A major QTL located on chromosome 1 was consistently detected in all the environments and the mean exhibiting phenotypic variance ranged between 17.3 to 37.69 %. This QTL was co-localized near the QTL controlling RLH in all the environments and the mean. Eight QTLs were identified for LH, including five in the year 2017, one in 2018, and two in the mean with R2 ranging from 4.82 to 23.08%. Thirteen QTLs were detected for RLH, four each in the years 2017 and 2018, and five in the mean data, and R2 ranged from 4.70-48.05%.
Validation of the ShB resistance QTL
It was observed that locus mapped on chromosomes 1, 3, and 11 were consistent and detected over multiple generations. To validate the locus on chromosome 1, 94 F4:5 populations were used. 40 SSR markers were employed. Out of which, 3 were polymorphic between two parents and the rest were monomorphic. Only one marker RM212 from chromosome 1 showed normal Mendelian segregation whereas the other two showed distorted segregation in the F4:5 populations. SSR marker RM212 was closely linked to the four loci namely qRLH1.1, qRLH1.2, qRLH1.5, and qRLH1.8 (Fig. 4). We analyzed few recombinants with five SSR markers at this region. The chromosomal region carrying donor segments and heterozygotes comparatively showed lower RLH. The segment derived from recurrent parent exhibit susceptible response to R. solani with relatively higher RLH (Table 4). Further, the SSR marker RM212 was amplified in a panel of 81 rice cultivars including basmati, non-basmati and landraces. Most of the non-basmati rice cultivars showed amplification corresponding to susceptible variety PR114. However, traditional basmati and aromatic rice germplasm showed amplicons corresponding to the O. nivara allele (Supp. Fig 2). The basmati cultivars are not prone to attack with the sheath blight disease as they are shown late. The land races and aromatic germplasm are known to have resistance to the sheath blight disease indicating usefulness of the linked marker in marker-assisted breeding programs.
Identification of candidate genes underlying ShB resistance QTL
The rice reference genome was searched using the coordinates of linked SNP markers associated with the QTLs. Stable QTLs qRLH1.1 spanned at 202 kb genomic region, qRLH1.2 in 0.096 kb, qRLH1.5 in 76.84 kb, and qRLH1.8 in 420.1 kb region harboring 35, 1, 10, and 67 candidate genes, respectively. The detailed list of candidate genes predicted in the QTL intervals is presented in Supplementary Table 3. The qRLH1.1 consists of some important genes such as pectin acetyl esterase (LOC_Os01g66830, LOC_Os01g66840, and (LOC_Os01g66850), serine/threonine protein kinase (LOC_Os01g66860), BTBZ1-Bric-a-Brac, Tram track (LOC_Os01g66890), Ser/Thr protein phosphatase (LOC_Os01g66920), zinc finger (LOC_Os01g66970), auxin-responsive protein (LOC_Os01g67030), few expressed protein and transposon proteins. The qRLH1.2 consists of LTPL39 - Protease inhibitor/seed storage/LTP family protein (LOC_Os01g68589). The qRLH1.5 constitutes NAM protein (no apical meristem protein, LOC_Os01g66490), phosphoribosyl-formyl-glycinamidine synthase (LOC_Os01g66500), MLO domain-containing protein (LOC_Os01g66510), serine/threonine-protein kinase RIO-like (LOC_Os01g66520), C2H2 zinc finger protein (LOC_Os01g66570), and DUF260 domain-containing protein (LOC_Os01g66590). The QTL qRLH1.8 contains disease resistance-responsive family protein (LOC_01g62030), serine/threonine-protein kinase (LOC_01g62080), thaumatin protein (LOC_01g62260), laccase precursor protein (LOC_01g62480, LOC_01g62490 and LOC_01g62600), aspartic proteinase nepenthesin precursor (LOC_01g62630), and avr9/Cf-9 rapidly elicited protein (LOC_01g62670). A concise list of important disease resistance candidate genes is given in the table 5.