Mol Biol Rep DOI 10.1007/s11033-016-4066-z

ORIGINAL ARTICLE

Phenotyping at hot spots and tagging of QTLs conferring spot blotch resistance in bread wheat Virender Singh1,3 • Gyanendra Singh1 • A. Chaudhury3 • Ashish Ojha1 B. S. Tyagi1 • A. K. Chowdhary2 • Sonia Sheoran1

•

Received: 21 November 2015 / Accepted: 19 August 2016 Ó Springer Science+Business Media Dordrecht 2016

Abstract Spot blotch is a major foliar disease of wheat caused by Bipolaris sorokiniana in warm and humid environments of the world including South Asian countries. In India, it has a larger impact in Indo-Gangetic plains of the country. Therefore, the present study was undertaken to phenotype a mapping population at different hot spots of India and to detect quantitative trait loci (QTL) for resistance to spot blotch in wheat. For this study, 209 single seed descent (SSD) derived F8, F9, F10 recombinant inbred lines (RILs) of the cross ‘Sonalika’ (an Indian susceptible cultivar)/‘BH 1146’ (a Brazilian resistant cultivar) were assessed for spot blotch resistance at two hot spot locations (Coochbehar and Kalyani) for three years and for two years under controlled conditions in the polyhouse (Karnal). The population showed large variation in spot blotch reaction for disease severity in all the environments indicating polygenic nature of the disease. Microsatellite markers were used to create the linkage maps. Joint and/or individual year analysis by composite interval mapping (CIM) and likelihood of odds ratio (LOD) [2.1, detected two consistent QTLs mapped on chromosome 7BL and 7DL and these explained phenotypic variation of 11.4 percent and 9.5 percent over the years and locations, respectively. The resistance at these loci was contributed by the parent ‘BH 1146’ and shown to be independent of plant height & Sonia Sheoran [email protected] 1

Indian Institute of Wheat and Barley Research, Karnal, India

2

Uttar Banga Krishi Viswavidyalaya, Pundibari, Coochbehar, India

3

Department of Bio and Nano Technology, Bio and Nano Technology Centre, Guru Jambheshwar University of Science and Technology, Hisar, India

and earliness. Besides, association of some agro-morphological traits has also been observed with percent disease severity. These identified genomic regions may be used in future wheat breeding programs through marker assisted selection for developing spot blotch resistant cultivars. Keywords Bipolaris sorokiniana  Phenotyping  QTL mapping  Spot blotch  Wheat

Introduction Bread wheat (Triticum aestivum L.) is the principal cereal crop next to rice in India and is well recognized as a major food source of South Asian region countries (Bangladesh, India, Nepal and Pakistan). It plays an important role in ensuring food security in this densely occupied region of the world [1, 2]. Spot blotch, caused by the hemibiotrophic pathogen Bipolaris sorokiniana (Sacc.) Shoem syn. Drechslera sorokiniana (Sacc.) Subrm and Jain (syn. Helminthosporium sativum, telemorph Cochliobolus sativus) [3], has surfaced as a key production constraint in the North Eastern part of India and South Asia’s intensive cropping system [2, 4]. Typical symptoms of B. sorokiniana occur in warm and humid environments, but, this disease has been expanding towards non-traditional cooler regions of wheat producing states in Northern India [2] and European countries due to global warming. It has been reported that the annual yield losses due to spot blotch in the Indian subcontinent ranges from 18 to 50 % [5–7]. The disease appears initially as brown eye shaped necrotic spots surrounded by yellow halo on leaves. Later such spots coalesce to make a larger area of leaf blighted and thus adversely affect photosynthesis. The disease intensity increases in the grain filling duration and considerable

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yield loss in addition to deterioration of grain quality was observed in susceptible cultivars [5]. The symptoms induced by B. sorokiniana are usually visible on leaf, sheath and stem [8]. Although, during intense conditions the disease also infects spikelets and results in shriveled grains [9] with black point at the embryo end of kernels [3]. An integrated approach, with host resistance as a major component, is therefore considered the best way to control the disease [10]. Some morphological traits such as leaf angle [11], stay green trait [12] and leaf tip necrosis (Ltn) having pleiotropic effect of Lr34 gene [13, 14] showed positive association with the disease resistance. The correlations of some morphological and physiological traits with spot blotch resistance have been analyzed [15, 16]. The genetics of spot blotch resistance behaves like a quantitative trait in wheat [17]. Using bulked segregant analysis, Sharma et al. [18] first reported some simple sequence repeat (SSR) markers linked with resistance to spot blotch in G162 (CIMMYT CID 93926), a source of resistance. Thereafter, QTL for spot blotch resistance have been mapped in the resistant sources ‘Yangmai 6’ [19], ‘Ning 8201’ and ‘Chirya 3’ [20]. Most recently two more sources of resistance ‘Saar’ [14] and ‘SYN1’ a CIMMYT synthetic-derived bread wheat [21] have been explored to map QTLs for resistance to spot blotch. Association mapping studies has also been carried out to detect markers/ QTLs linked to spot blotch blotch resistance using DArT [22] and genome-wide SNP markers [23] in diverse spring wheat germplasm. The above studies with genetic markers have further confirmed that many genes are accountable for resistance to this disease. The increasing threat of spot blotch calls for more concerted efforts to understand the genetics of resistance. Therefore, the objective of this study was to map the genomic regions responsible for spot blotch resistance in a large size population comprising of 209 RILs resulting from the cross between ‘Sonalika’ and ‘BH 1146’, phenotyped under controlled and natural field conditions at two hot spot locations in India. Here in this study, we used a larger mapping population than in previous studies, which increases the likelihood of detecting smalleffect QTL. The multilocation phenotyping for disease will further validate the authenticity of the QTLs observed against different pathotypes and environments.

Materials and methods Plant materials A total of 209 SSD derived recombinant inbred lines (RILs-F8, F9, F10) of the cross ‘Sonalika/BH 1146’ were evaluated in polyhouse and field trials for resistance to spot blotch. ‘Sonalika’ [II54-368/An/3/Yt54/N10B//LR64] was

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once a popular cultivar of Northeastern India and is highly vulnerable to spot blotch. Whereas, ‘BH 1146’ [Fronteira/ Mentana//Ponta Grossa1] is a Brazilian cultivar carrying resistance to spot blotch. Evaluation of disease severity The 209 RILs along with two parents were assessed in polyhouse and natural field conditions during three consecutive years 2011–12, 2012–13 and 2013–14 at two hot spot locations of North Eastern India, namely Agricultural Research Farm UBKV (Uttar Banga Krishi Viswavidyalaya), Coochbehar (altitude 138ft, 25°570 N, 89°540 E, Temp. range 3.9-39.9 °C), and BCKV (Bidhan Chandra Krishi Viswavidyalaya) Agricultural Research farms, Kalayani (altitude 36 ft, 22°590 N, 88°48E, Temp. range 19–37 °C) and also under polyhouse condition at IIWBR (Indian Institute of Wheat and Barley Research), Karnal. The field layout followed a lattice design with three replications and in all the replications each RIL was planted in single rows of 1 m on the irrigated land. Plant-to-plant and row-to-row distance was 5 and 25 cm, respectively with 0.5 m distance between the blocks. To promote disease build up and spread, one row of the spreader consisting of a blend of highly susceptible cultivars RAJ 4015, A-9-30-1, Kanchan and Sonalika was planted around each block providing an equal chance of infection in test entries. Sowing was done in the first week of December, to allow post-anthesis stage to coincide with a warm and humid environment conducive to the disease that occurs in March [24]. For controlled conditions RILs were sown in polyhouse in two replications and in hill planting method with plant-to-plant and row-to-row spacing of 15 and 10 cm, respectively. By taking into account mean of all environments, the RILs were categorized into six groups based on the reactions to spot blotch as highly resistant, resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible in the corresponding 0–10, 11–20, 21–40, 41–60, 61–80, 81–100 % disease severity ranges, respectively (Fig. 1).

Epiphytotic conditions in polyhouse Epiphytotic conditions in the polyhouse were created by artificial inoculation to minimize the environmental effects. Spot blotch inoculum as a mixture of virulent isolates of B. sorokiniana collected from different hot spot locations of India (namely, Faizabad, Pantnagar, Coochbehar and Kalyani) was multiplied on autoclaved sorghum grains. Spore suspension adjusted to roughly 104 spores/ml of water was evenly sprayed at three different Zadoks growth stages [25], viz., tillering (GS-20), flag leaf emergence (GS-37) and anthesis (GS-65) in the evening hours. The

No. of RILs

Mol Biol Rep 80 70 60 50 40 30 20 10 0

and making an angle of 0–60°) and drooping (half or more than half the length of flag leaf, from tip to base, was drooping). This was done at growth stage corresponding to the 50–55 stage [25]. Microsarellite analysis 0-10 HR

11-20 R

21-40 MR

41-60 MS

61-80 S

81-100 HS

Percent disease severity range and infection response Fig. 1 Categorization of the RILs on the basis of infection response to spot blotch as highly resistant (HR), resistant (R), moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS)

inoculation was followed by misting to retain a high level of moisture.

Disease assessment Spot blotch severity in each plot was visually scored at early dough stage (GS-83) on a Zadoks scale [25]. To assess spot blotch disease a double-digit scale (00–99) as a modification of Saari and Prescott’s severity scale [26] was used. The upper most leaves, the flag leaf (digit D1) and the penultimate leaf (digit D2) were considered for scoring the disease. For each evaluation, percent disease severity was estimated based on the following formula: Disease severity (%Þ ¼ ðD1=9Þ  ðD2=9Þ  100: This disease severity (%) calculated from the double digit scale data, recorded at early dough stage (GS-83) was used in QTL analysis.

Assessment of phenological and yield traits Plant height (PH) and days to maturity (DTM) were assessed for all lines. PH was measured at the hard dough stage (Zadoks growth stage 87) [25], while DTM were counted as the number of days from sowing until the grains were completely hard and possessed moisture levels less than 12 % (GS-92). Days to heading (DTH) were counted as the number of days from sowing until 50 % of the ear emerges in each line (GS-50 to GS-55). Thousand grain weight was assessed after the harvest. Canopy colour was visually scored at GS-41 and divided the lines in three groups as dark green, green and pale green. Leaf angle was measured at Inflorescence emergence [27] in all the lines, dividing the lines into three groups: erect (flag leaf nondrooping and making an angle of 60–90° with respect to the horizontal plane); semi-erect (flag leaf non-drooping

Total genomic DNA of the RILs (F8) along with parental genotypes was extracted from 2 week old leaves following modified CTAB method [28]. PCR was performed with SSR markers with each reaction mixture containing 50–100 ng template DNA, 0.25 lM of each primer, 200 lM of each dNTP, 19 PCR buffer and 1 U of Taq DNA polymerase. The PCR programme consisted of initial denaturation at 94 °C for 3 min, followed by 45 cycles of 94 °C for 1 min, annealing temperature (50–60 °C) for 1 min depending on the primer choice, 72 °C for 2 min with a final extension of 10 min at 72 °C [29, 30]. PCR products were resolved on 3 % agarose gels (Himedia, USA) and 6 % polyacrylamide gels in EtBr staining highthroughput vertical gel electrophoresis system (C-DASG400-50, CBS Scientific). Map construction and QTL mapping Total 700 SSR markers, by selecting at least twenty evenly distributed markers of each chromosome from the reference Somers wheat consensus map, were screened for polymorphism between parental genotypes ‘Sonalika’ and ‘BH 1146’ [30]. A subset of 46 RILs (23 highly resistant and resistant lines and 23 susceptible and highly susceptible lines) out of 209 was then screened with identified polymorphic markers. At first, single marker regression analysis was performed to test each marker for the significance of disease severity using software QTL Cartographer version 2.5 [31]. Then, 110 SSR markers which showed significance at P \ 0.001 were used to screen remaining RILs and linkage groups were generated using computer software JoinMap 4 [32] for QTL analysis. After that, more markers were added for further enrichment of the obtained linkage groups on chromosomes 7B and 7D. The Likelihood of odds ratio (LOD) of [3 was used to create the linkage map. QTL analysis was done following composite interval mapping (CIM) using software QTL Cartographer version 2.5 [31]. The parameters setting for CIM were similar to that of Kumar et al. [19]. QTLs were confirmed by LOD values compared to empirical genomewide significant threshold calculated from 1000 permutations for P \ 0.01 to minimize Type-I error. LOD values and coefficients of determination were estimated by CIM for individual QTL. QTLs were considered to have significant effect when LOD statistics exceeded a threshold of 2.1. Adjusted mean for percent disease severity values of

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each year were calculated before pooling the statistics from all the years. The QTL names were given according to the International Rules of Genetic Nomenclature (http:// wheat.pw.usda.gov/ggpages/wgc/98/Intro.htm): QSb.iiwbr as a QTL for spot blotch resistance identified at Indian Institute of Wheat and Barley Research, Karnal, India. Statistical analysis Analysis of variance was done with the PROC GLM procedure in SAS (SAS Institute Inc., v. 9.1.). The heritability of percent disease severity in the population was calculated from the variance components derived from PROC MIXED with all effects [genotype, year, replication (year), genotype 9 year] that were considered random: H2 ¼ r2 g=r2 g þ ðr2 ge=eÞ þ ðr2 =reÞ where, H2 represents the broad sense heritability, r2g is genetic variance, r2ge is genotype 9 environment variance and r2 is error variances, r is the number of replications and e is the number of environments. Pearson correlation coefficients among traits were estimated by the PROC CORR procedure in SAS. Multiple regression was done using Fit model of JMP version 9.0 (SAS Institute Inc., Cary, NC, USA) taking disease severity as dependent variable while days to heading and plant height as independent variables was used to eliminate the effect of these traits. Residuals thus obtained were used to map true QTL for disease resistance.

Results and discussion The distribution pattern of 209 RILs for spot blotch percent disease severity (Fig. 2) indicated that resistance to spot blotch is controlled by multiple genes and not by a single gene in the ‘Sonalika/BH 1146’ cross. The genetics of spot blotch resistance in this mapping population was not explored previously. Polygenic control of the inheritance of spot blotch resistance had also been reported by earlier researchers [17]. In the present study, the parental genotypes ‘Sonalika’ and ‘BH 1146’ showed similar susceptible and resistant reactions to spot blotch isolates, respectively, in all experiments over years and locations. Phenotypic variation The spot blotch epiphytoties were created well for all the eight trials at three locations. Mean percent disease severity of the susceptible (Sonalika) and the resistant (BH 1146) parents recorded at GS-83 [25] ranged from 54 to 89 % and 16 to 23 %, respectively, during three crop seasons (2011–14) under natural field conditions at two hot spot

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locations and also over 2 years (2012–14) under controlled conditions (Table 1). The percent disease severity of RILs ranged from minimum zero (immune lines) to a maximum 96 % (highly susceptible lines) during the same period and location (Table 1). The population showed large variation in spot blotch reaction among the 209 RILs for percent disease severity in all the environments (Fig. 2). The analysis of variance (ANOVA) for disease severity (%) showed a significant variation for the genotypes, year, genotype-by-year, genotype-by-location and genotype-byyear-by-location interaction (Table 2). However, a nonsignificant variation for years was observed in the disease under polyhouse (Table 3). A highly significant variation due to genotypes was observed in each year and location. As a result, the data from different years and locations were used individually for QTL analysis. Based on the percent disease severity values of 3 years broad sense heritability was estimated to be 0.89 and 0.87 for Coochbehar and Kalyani, respectively. Whereas, under polyhouse broad sense heritability was estimated to be 0.92 for 2 years. High estimates of heritability for spot blotch had also been recorded previously [33]. The range and mean values of different agro-morphological traits for parental lines and the RILs are presented in Table 4. Trait correlations Significant positive correlation coefficients were found among percent disease severity over 3 years at two hot spots and in polyhouse during 2 years, ranging from 0.55 to 0.86 at P \ 0.001 (Table 5). Disease severity between 2 years in polyhouse was highly correlated (0.86, P \ 0.001) and significant correlation was observed between 2nd year data of polyhouse and field data of two locations over 3 years than the 1st year disease data of polyhouse (Table 5). Coochbehar 2nd year disease data was highly correlated with all the other field and polyhouse trials (Table 5). Similar observations were reported by Kumar et al. [20]. The correlation coefficient between days to heading (DTH) and disease severity (DS) was found significant and showed negative correlation when calculated over the 2 years and across two locations that ranged from -0.50 to -0.64 at P \ 0.001 (Table 6). This finding demonstrates that early heading lines showed increased frequency of DS in comparison to lines with late heading dates which is consistent with previous reports [15, 34, 35]. Negative correlations between foliar disease and heading dates are common, but it is not known whether this is due to pleiotropy, linkage or escape [36]. Plant height among the plant type traits, is one of the key trait to wheat breeders. The result showed negative correlations between disease severity and plant height for each of the environments

Mol Biol Rep

Coochbehar 2012

Coochbehar 2013

Coochbehar 2014

Kalyani 2012

Kalyani 2013

Kalyani 2014

Polyhouse 2013

Polyhouse 2014

Mean of all environments

Percent Disease Severity Fig. 2 Histograms showing frequency distribution pattern of spot blotch disease severity (%) values in total eight environments along with the average of all the environments for 209 RILs of the cross Table 1 Ranges and mean scores of spot blotch percent disease severity for the parents and RILs of ‘Sonalika/BH 1146’population

Genotype

between ‘Sonalika/BH 1146’. Sonalika and BH 1146 are denoted by the letters ‘S’ and ‘B’, respectively

Coochbehar 2012

2013

Kalyani 2014

2012

Polyhouse 2013

2014

2013

2014

BH 1146

23 ± 3.6

16 ± 3.0

16 ± 2.3

17 ± 2.3

23 ± 3.6

17 ± 2.3

20 ± 5.2

17 ± 2.3

Sonalika

89 ± 6.3

73 ± 5.3

54 ± 5.3

68 ± 5.4

68 ± 5.4

72 ± 3.6

68 ± 5.4

69 ± 5.7

RILs mean

36 ± 1.6

38 ± 1.8

34 ± 1.5

38 ± 1.5

35 ± 1.6

44 ± 1.8

34 ± 1.4

35 ± 1.4

RILs range

0–96

0–96

0–93

0–85

0–89

0–96

0–89

0–94

SE standard error

(-0.24 to -0.45, P \ 0.001), were found consistent with previous reports [15, 21, 37]. Increased plant height may have reduced the rate of movement of disease up the canopy due to splashing of spores. The disease also showed negative and significant correlation (-0.40 and -0.59, P \ 0.001) with days to maturity (DTM), indicating that late maturing genotypes are less prone to disease infection and similar results were observed by Singh et al. [35]. In a

previous study, spot blotch has been reported to be independent of PH and DTM [38]. However, no significant correlation of disease with spike length was observed in this population similar to the findings of Rosyara et al. [15]. Thousand kernel weight (TKW) showed very low positive correlation with disease (0.15, P \ 0.05) in only single environment (Kalyani, 2012–13) and was non-significant during crop season 2011–12 both at Kalyani and

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Mol Biol Rep Table 2 Analysis of variance for percent disease severity of spot blotch under natural field conditions at two hot spot locations and over 3 years in the ‘Sonalika/BH 1146’ RIL population Source

DF

Type III SS

GENO (G)

208

1608633.555

YEAR (Y)

2

4479.547

1 416

8630.155 233142.451

LOC (L) G9Y

F value

Pr [ F

7733.815

102.62

\0.0001

2239.773

29.72

\0.0001

8630.155 560.439

114.52 7.44

\0.0001 \0.0001

MS

Y9L

2

26029.993

13014.996

172.70

\0.0001

G9L

208

130484.355

627.329

8.32

\0.0001

G9Y9L

416

153435.942

368.836

4.89

\0.0001

Table 3 Analysis of variance for percent disease severity of spot blotch under controlled conditions in polyhouse in the ‘Sonalika/BH 1146’ RIL population Source

DF

Type III SS

MS

F value

Pr [ F

GENO (G)

208

319339.4141

1535.2856

20.52

\0.0001

YEAR (Y) G9Y

1 208

31.7426 24716.4936

31.7426 118.8293

0.42 1.59

0.5152 \0.0001

Coochbehar locations. Rosyara et al. [15] also observed low phenotypic correlation for hundred kernel weight (HKW) with spot blotch AUDPC. Previously a negative and significant correlation of spot blotch disease with grain yield had been reported [33], which is indicative of the significance of the disease. A very low positive and significant correlation (0.14–0.19, P \ 0.05) was noticed

between leaf angle and disease severity. Joshi and Chand [11] found a moderately positive phenotypic correlation between leaf angle and spot blotch. These results showed that the erect leaves might have contributed positively to reduce spot blotch disease severity. The erect leaves generally hold less free water, which is essential for pathogen spores to germinate. Spot blotch disease developed best when wheat plants were continuously exposed to conditions of 25 °C and 100 % relative humidity (RH) for 24 h [39]. The canopy color also showed significantly low negative correlation (-0.15 to -0.24) with disease severity. In general, RILs with dark green canopy colour showed reduced disease severity as compared to pale green leaves. Correlation coefficients between disease severity and different agro-morphological traits have been presented in Table 6. Significant correlation was observed for days to heading, days to maturity and plant height with spot blotch although to establish any definitive link between these phenological traits and disease reaction, further studies need to be done in wider prospective. Large size mapping populations with only single contrasting phenological traits at a time could be used to confirm whether pleiotropy or linkage is involved, or the genes controlling the spot blotch are independent of the genes of plant height and earliness. Marker polymorphism and linkage groups For polymorphism between the parents ‘Sonalika’ and ‘BH 1146’ a total 700 SSR markers covering the wheat genome were used. Out of these, 167 (24 %) SSRs were found polymorphic and used for screening a subset of 46 RILs. The percentage of polymorphic SSR markers found was

Table 4 Range and mean values of agro-morphological traits for the parents and RILs of the ‘Sonalika/BH 1146’ cross Traits

Coochbehar Sonalika

Kalyani BH 1146

RILs Mean

RILs range

Sonalika

BH 1146

RILs mean

RILs range

2011–12 DTH

64 ± 0.5

70 ± 0.5

75 ± 0.5

63–95

56 ± 0.5

65 ± 0.7

68 ± 0.5

53–89

98 ± 2.5

105 ± 1.5

107 ± 0.3

98–119

67–136

78 ± 4.0

112 ± 2.3

93.3 ± 0.9

63–126

34 ± 2.0

40 ± 1.5

39.7 ± 0.2

30–43

DTM PH

94 ± 4.6

139 ± 3.4

100.79 ± 1.1

TKW

35 ± 0.8

41 ± 1.8

38.5 ± 0.5

19–56

SL

10 ± 0.6

9 ± 0.2

9.71 ± 0.1

7.3–13.1

64 ± 0.3

69 ± 1.2

75.62 ± 0.5

63–91

2012–13 DTH DTM PH

104 ± 2.0

128 ± 6.6

99.31 ± 1.0

70–130

TKW SL

10 ± 0.05

8 ± 0.3

9.75 ± 0.1

59 ± 1.0

65 ± 0.7

70 ± 0.5

56–88

97 ± 1.5

103 ± 0.6

108 ± 0.4

96–118

101 ± 3.0

143 ± 15

100 ± 1.0

63–131

36 ± 3.0

40 ± 0.8

39 ± 0.3

31–49

6.8–12.4

DTH days to heading, DTM days to maturity, PH plant height, TKW thousand kernel weight, SL spike length ± standard error

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Mol Biol Rep Table 5 Correlation coefficients between the spot blotch disease severity (%) over the years and across locations Traits

CB 1st year

CB 2nd year

CB 2nd year

0.76***

CB 3rd year

0.69***

0.79***

KLY 1st year

0.67***

0.70***

CB 3rd year

KLY 1st year

KLY 2nd year

KLY 3rd year

POL 1st year

0.68***

KLY 2nd year

0.68***

0.84***

0.67***

0.71***

KLY 3rd year

0.62***

0.74***

0.70***

0.75***

POL 1st year

0.56***

0.67***

0.55***

0.59***

0.60***

0.59***

POL 2nd year

0.59***

0.72***

0.62***

0.66***

0.68***

0.67***

0.71*** 0.86***

CB Coochbehar, KLY Kalyani, POL Polyhouse *** Significant at P \ 0.001

Table 6 Pearson correlation coefficients between percent disease severity and different agro-morphological traits in the RIL polulation of the cross ‘Sonalika/BH 1146’ during crop season 2011–12, 2012–13 at two hot spots

orientation of the mapped SSR markers in this study were in conformity with those in the map of the ITMI population [41].

Traits

QTL identification

Coochbehar DS 2012

DS 2013

-0.49909

***

-0.29035

***

-0.11496

NS

SL

-0.12191

NS

CC

-0.15124*

LA

0.14068*

DTH

Kalyani DS 2012

-0.64243

***

-0.44909

***

DTM PH TKW

-0.60686

-0.61045***

-0.39617***

-0.58687***

***

-0.36804***

-0.23527 0.0383

-0.06505

DS 2013 ***

NS

0.14876*

NS

-0.16687* 0.19069**

-0.23778***

-0.19641**

0.17148*

0.15926*

DTH days to heading, DTM days to maturity, PH plant height, TKW thousand kernel weight, SL spike length, CC canopy colour, LA leaf angle * Significant at P \ 0.05; ** Significant at P \ 0.01; *** Significant at P \ 0.001; NS Non-significant

similar to earlier reports [19, 20]. Maximum frequency of marker polymorphism was observed in the B genome (42.5 %) followed by A genome (33.0 %) and D genome (24.5), which are in concurrence with the results of Kumar et al. [19]. Eight chromosomes (1B, 2A, 2B, 2D, 5B, 7A, 7B, 7D) out of twenty-one exhibited comparatively higher polymorphism and most marker loci with few exceptions segregated in the expected ratio of 1:1 (P \ 0.05). All the 209 RILs were genotyped with significant polymorphic markers emphasizing more on the identified eight chromosomes. McDanie et al. [40] reported that linkage maps were not hindered by segregation distortion. Therefore, the distorted markers were also used in the analysis. The linkage groups constructed on chromosomes 7B with 11 marker loci and 7D with 9 markers covered genetic distances of 126.4 and 188.3 cM, respectively. The order and

Two consistent QTLs were identified for spot blotch percent disease severity. The LOD scores ranged from 2.9 to 6.4 and the corresponding phenotypic variation (R2) ranging from 5.6 to 18.3 percent in the individual years and locations when calculated using composite interval mapping (Tables 7, 8). These two consistent QTLs mapped on the long arm of chromosome 7B and the long arm of chromosome 7D (Fig. 3), were identified in all the environments. The QTL on 7BL explained phenotypic variation ranging from 7.7 to 12.3 percent and 11.4 % with mean of disease severity over years and locations (Table 7). The QTL detected on 7D explained 5.6–18.3 % of phenotypic variation and 9.5 % with mean over years and locations (Table 8). The effect of significantly correlated traits (plant height and days to heading) with disease severity was eliminated to map the true QTL. Residuals were calculated using multiple regression analysis by taking 2 years mean of disease severity (2011–12, 2012–13) for individual locations. The QTLs obtained with the residuals were mapped in the same genomic region of chromosome 7B (Table 7) and 7D (Table 8) showing the true resistant QTLs and not merely the pleiotropic effect of earliness or plant height genes. The previous studies on QTL mapping for spot blotch resistance showed more phenotypic variance, which may be due to their small population size. Small population size often resulted in that only QTLs with large phenotypic effects could be detected [42–44]. So far, QTLs for resistance to spot blotch have been reported on 10 wheat chromosomes (1B, 2A, 2B, 2D, 3B, 5A, 5B, 6D, 7B, and 7D) by different researchers [19–21] through linkage mapping. Apart from these QTL reports, three SSR

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Mol Biol Rep Table 7 Effect of QTLs on 7B chromosome for reduced spot blotch disease severity detected by composite interval mapping (CIM) in the ‘Sonalika/BH 1146’ RIL population

Environment

Marker interval

LOD score

R2 (%)

Position interval (cM)

Additive

CB2012

wmc758-wmc335

4.8

9.0

8.6

-7.2

CB2013

wmc758-wmc335

6.4

12.3

8.6

-9.4

CB2014

wmc758-wmc335

4.1

8.4

8.6

-8.2

KLY2012

wmc758-wmc335

4.2

7.7

8.6

-6.3

KLY2013

wmc758-wmc335

5.2

10.4

8.6

-10.1

KLY2014

wmc758-wmc335

4.5

9.0

8.6

-7.8

POL2013

wmc758-wmc335

4.1

8.6

8.6

-6.0

POL2014

wmc758-wmc335

5.0

10.3

8.6

-6.6

Mean

wmc758-wmc335

6.3

11.4

8.6

-6.7

Res-CB

gwm758-wmc335

3.4

7.2

8.6

-4.6

Res-KLY

wmc696-wmc758

5.4

10.2

3.2

-5.7

CB Coochbehar, KLY Kalyani, POL Polyhouse, Res-CB Coochbehar Residual mean (2011–12, 2012–13)

Table 8 Effect of QTLs on 7D chromosome for reduced spot blotch disease severity detected by composite interval mapping (CIM) in the ‘Sonalika/BH 1146’ RIL population

Environment

Marker interval

LOD score

R2 (%)

Position interval (cM)

Additive

CB2012

wmc653-cfa2040

4.1

10.6

47.7

-7.7

CB2013

wmc653-barc121

3.9

11.4

26.6

-8.7

CB2014

wmc653-cfa2040

3.8

9.7

47.7

-7.2

KLY2012

wmc653-barc121

5.4

18.3

26.6

-9.6

KLY2013

wmc653-barc121

4.2

14.6

26.6

-8.8

KLY2014

wmc653-cfa2040

3.2

9.4

47.7

-8.0

POL2013

wmc653-cfa2040

2.9

5.6

47.7

-4.8

POL2014

wmc653-cfa2040

3.4

6.4

47.7

-5.2

Mean Res-CB

wmc653-cfa2040 wmc653-cfa2040

4.6 4.2

9.5 8.0

47.7 47.7

-6.2 -4.8

Res-KLY

wmc653-cfa2040

4.0

7.7

47.7

-3.9

CB Coochbehar, KLY Kalyani, POL Polyhouse, Res-KLY Kalyani Residual mean (2011–12, 2012–13)

markers linked to spot blotch resistance in the resistant parent G162 (CIMMYT CID 93926) were reported using bulked segregant analysis [18]. These SSR markers, gwm067, gwm570 and gwm469 are placed on the chromosome 5BL, 6AL and 6DS, respectively. QTL, QSb.iiwbr-7B was located on the long arm of chromosome 7B in the marker interval wmc758-wmc335 (8.6 cM). The second QTL, QSb.iiwbr-7D observed on chromosome 7D was located in the marker interval wmc653-barc121 (26.6 cM) in Coochbehar during 2012–13 and also in Kalyani during 2011–12, 2012–13 and between markers wmc653 and cfa2040 (47.7 cM) with barc121 as a peak marker in the rest of the environments. For the two QTLs identified in the mapping population ‘Sonalika/BH 1146’, the alleles were received from the donor parent ‘BH 1146’ for reduced disease severity. The mean disease score of the RILs without any QTL observed was 51.2, whereas for the QTL allele on chromosome 7B (wmc758) and chromosome 7D (barc121) showed mean disease scores of 31.2 and 30.1, respectively. The

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resistance allele of both the QTLs together showed mean disease score of 25.8 indicating reduction in disease severity (Fig. 4). The QTL ‘QSb.iiwbr-7B’ observed in this study lies close to the chromosomal region of previously identified QTL on 7B in ‘Chirya 3’ between markers gwm263 and gwm255 [20]. The second QTL ‘QSb.iiwbr7D’ on the long arm of 7D chromosome is mapped *100 cM away from Sb1 gene reported by Lillemo et al. [14] on the short arm of 7D chromosome showing it as a distinct QTL for spot blotch resistance. A minor QTL was also reported on 7D in a sub-population of ‘Saar’ line having susceptibility allele of Lr34 between marker wPt7654 and gdm88 [14]. In addition to this Kumar et al. [20] identified a major QTL on chromosome 7D in ‘Ning 8201’ and ‘Chirya 3’. The importance of the genomic regions on chromosome 7B and 7D for conferring resistance to spot blotch is also evidenced by association mapping studies [22, 23]. Gurung et al. [23] reported significant association of the SNP marker IWA4145 with spot blotch resistance in spring wheat germplasm using association mapping

Mol Biol Rep Fig. 3 LOD curves for two QTLs detected on chromosome 7B and 7D by composite interval mapping (CIM) for spot blotch resistance in the RIL population of the cross between ‘Sonalika/BH 1146’. QTLs are shown for individual environments, mean of all the eight environments and residual mean for individual locations (Coochbehar and Kalyani). Genetic positions are shown in cM on the horizontal axis. The vertical line indicates the threshold LOD value (2.1) determining significant QTLs

Chromosome 7B

Chromosome 7D

Mean disease score of Lines

60 50 40 30 20 10 0 None QTL

7B QTL

7D QTL

7B and 7D QTL

Fig. 4 Graph showing the mean disease scores of lines without any of the two QTL, only 7B, only 7D and with the resistance allele of both QTLs. Error bars show the standard error of the mean (SE)

approach. The marker IWA4145 is 4.92 and 1.34 cM distal to wmc758 and wmc335, respectively, based on the integrated map recently developed by Maccaferri et al. [45]. Therefore, it is very likely that the 7B QTL identified in this study corresponds to the previously reported 7BL QTL [23]. The confidence interval of the 7D QTL identified in

this study is flanked by wmc653 and cfa2040, with barc121 corresponding to the peak of the QTL. Adhikari et al. [22] reported association of resistance to spot blotch with the DArT marker wPt-66459, which is 4.84 cM proximal to cfa2040 and 82.88 cM distal to barc121 on the basis of the integrated map created by Maccaferri et al. [45]. The DArT marker wPt-66459 also co-localizes with wPt-664164, wPt-663943, and wPt-665516 [22]. Direct comparison of the locations of the QTLs identified in the present work with the previous studies [14, 19–21] may not be feasible due to different fungal isolates, source of resistance, and molecular marker systems used. Due to global warming and climate change, rains are shifting towards the later stages of crop growth, therefore, it has become more important to understand the molecular genetics of spot blotch resistance. In this study, it is highlighted that the QTL ‘QSb.iiwbr-7B’ and ‘QSb.iiwbr7D’ are responsible for conferring resistance to spot blotch in resistant parent BH 1146. These QTLs have equivalent effects in the reduction of spot blotch disease development in typically different spot blotch prone environments and even with different pathotypes of the spot blotch fungus.

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Mol Biol Rep

Integration of conventional breeding with molecular approach will help out in mitigating the problems of spot blotch disease in wheat through incorporation of useful QTLs in susceptible yet popular genotypes that are adapted to a particular region. Thus, the information generated here will allow improvement of spot blotch resistant wheats through marker assisted selection strategy in future wheat breeding programs. Even a marginal reduction in disease level may be of significance for wheat growing areas, especially those in developing countries. Acknowledgments The authors thankfully acknowledge the financial support from Department of Biotechnology (DBT), New Delhi, India for the present study. The first author is also grateful to University Grants Commission (UGC), New Delhi, India for providing financial support in form of UGC fellowship to carry out doctoral degree.

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