Euphytica

, Volume 203, Issue 1, pp 97–107 | Cite as

Introgression of multiple disease resistance into a maintainer of Basmati rice CMS line by marker assisted backcross breeding

  • Ashok K. Singh
  • Vikas K. Singh
  • Atul Singh
  • Ranjith K. Ellur
  • R. T. P. Pandian
  • S. Gopala Krishnan
  • U. D. Singh
  • M. Nagarajan
  • K. K. Vinod
  • K. V. Prabhu
Article

Abstract

Globally, blast incited by Magnaporthe oryzae and sheath blight (ShB) by Rhizoctonia solani Kuhn forms two major fungal diseases that cause significant yield loss in rice. Pusa 6B, the Basmati quality maintainer line of the popular superfine grain aromatic rice hybrid Pusa RH10, is highly susceptible to both the diseases. The rice cultivar Tetep was used as the donor for transferring a major blast resistant gene, Pi54 and three ShB resistant quantitative trait loci (QTLs) namely, qSBR11-1, qSBR11-2 and qSBR7-1 into Pusa 6B using a marker assisted backcross breeding scheme with restricted number of backcrosses. Plants heterozygous for the alleles of interest and phenotypic similarity to the recurrent parent were used for generating BC1F2 population by selfing selected BC1F1 plants. Selected BC1F2 plants homozygous for Pi54 were selfed to generate BC1F3 families that were subjected to a step-wise reductive screening for the three ShB resistant QTLs. Final selections were advanced to BC1F5 generation through selfing while subjecting to stringent phenotypic selection. The advanced selections carrying blast and ShB resistant genes (Pi54, qSBR11-1, qSBR11-2, qSBR7-1) in the background of Pusa 6B were resistant to highly virulent strains of rice blast as well as ShB isolates without compromising the grain and cooking quality of Pusa 6B. Marker assisted transfer of blast and ShB resistance into Pusa 6B will aid in developing CMS lines with inbuilt resistance to these diseases. When combined with restorer lines possessing resistant genes/QTLs for these diseases, the improved Pusa6A lines will aid in development of improved Pusa RH10 and other novel aromatic hybrids with resistance to blast and ShB diseases. The present study demonstrates successful use of a restricted backcrossing strategy for introgression of multiple loci conferring resistance to two important fungal diseases in rice.

Keywords

Basmati rice hybrid Maintainer line Marker assisted selection Rice blast Sheath blight Disease resistance 

Introduction

Hybrid rice based on cytoplasmic male sterility (CMS) system has been successfully utilized for improving rice yields by 30 % over the pureline rice varieties (Yuan 1998). Globally, rice productivity is seriously affected by several factors among which diseases and pests play a major role in limiting rice yields. Among the major diseases, two fungal diseases namely, rice blast caused by Magnaporthe oryzae and sheath blight (ShB) caused by Rhizoctonia solani Kühn are the most destructive. These diseases can cause yield losses above 50 % and affect grain quality to a great extent (Savary et al. 2006). Pusa Rice Hybrid 10 (PRH10), the world’s first superfine-grain aromatic rice hybrid is very popular among farmers of India for its excellent cooking and eating quality, early maturity and higher yield (Basavaraj et al. 2010). However, like most of the released hybrids in India, PRH10 also is highly susceptible to rice blast and ShB diseases (Singh et al. 2011).

Rice blast is associated with rice production environments worldwide, due to its co-existence in crop favorable environments. Breeding rice varieties with durable blast resistance is the most logical and environment-friendly solution. Till date, no rice cultivar is known to possess durable blast resistance owing to the highly adaptive nature of the pathogen (Sharma et al. 2002, 2010). Several strategies have been proposed to develop durable blast resistance in rice such as pyramiding of major and partial resistant genes either separately or in a combination of both (Kato et al. 2004). A major resistant gene, Pi54, mapped on the long arm of chromosome 11 from a Vietnamese indica rice variety Tetep (Sharma et al. 2010) has been shown to provide resistance to predominant rice blast races in India (Sharma et al. 2002). This gene, encoding for a nucleotide binding site–leucine-rich repeat (NBS–LRR) domain containing protein has been cloned (Sharma et al. 2005).

As that of rice blast, ShB is also widely prevalent in all rice production areas worldwide (Ou 1985), and causes an estimated yield loss of up to 54.3 % in India (Chahal et al. 2003). Breeding for resistance to ShB seems to be one of the viable solutions to manage ShB because several other techniques such as cultural, chemical and biological control have been found inadequate due to wide host range of the pathogen (Gangopadhyay and Chakrabarti 1982). So far, complete resistance has not been found against R. Solani, because ShB resistance in rice is of quantitative in nature (Pinson et al. 2005). In rice, a total of 33 QTLs associated with ShB resistance have been reported spanning over all 12 chromosomes (Srinivasachary et al. 2011), although the mechanisms of resistance of these QTLs are poorly understood. Among these, three major QTLs mapped in Tetep, qSBR11-1, qSBR11-2 and qSBR7-1 were found consistently associated with ShB resistance across location and years (Channamallikarjuna et al. 2010).

Marker assisted selection (MAS) has been proved to be an efficient selection tool for those traits that are difficult and expensive to evaluate in a shorter time period (Tanksley et al. 1989) such as rice blast and ShB resistance owing to its advantages of ease and precision in selection of target traits even before their expression. MAS coupled with stringent phenotypic selection can provide rich dividends in breeding success (Collard and Mackill 2008). Marker assisted backcross breeding (MABB) is widely demonstrated to be the most effective way of transferring specific gene(s)/QTLs into rice varieties/parental lines of hybrids such as resistance to bacterial blight (BB) (Joseph et al. 2004; Gopalakrishnan et al. 2008; Basavaraj et al. 2009), blast (Singh et al. 2012a, 2013), ShB (Singh et al. 2012b), submergence tolerance (Neeraja et al. 2007), salt tolerance (Islam and Gregorio 2013), phosphorus deficiency tolerance (Heuer et al. 2013) and yield under drought (Kumar et al. 2013).

Pusa 6B is a Basmati quality genotype that is characterised by its long grains and good cooking quality. Further, it is an excellent specific combiner and maintainer of WA CMS in rice. This has led to the exploitation of this line to develop its isogenic male sterile Pusa 6A that was successfully used in developing the popular aromatic rice hybrid, PRH10 by crossing with an aromatic restorer line PRR78 (Zaman et al. 2003). However, Pusa 6B is susceptible to major diseases of rice such as bacterial blight (BB), blast and ShB. In a previous study from our laboratory, Basavaraj et al. (2010) successfully incorporated two genes, xa13 and Xa21 conferring BB resistance into Pusa 6B, through MABB integrated with stringent phenotypic selection. In this study, to incorporate dual disease resistance into Pusa 6B against rice blast and ShB, a modified MABB approach was used for gene/QTL pyramiding in which backcross was restricted to one generation and MAS was done only at the early segregating generations such as F2 and F3. Rest of the generations were handled as in the case of pedigree approach (Fig. 1).
Fig. 1

Scheme of marker assisted backcross breeding (MABB). Marker assisted backcross breeding with restricted backcross coupled with MAS done at early segregating generations such as BC1F2 and BC1F3. For multiple QTLs a step-wise reductive selection was done. Rest of the generations was handled as in the case of pedigree approach

Materials and methods

Plant materials

Pusa 6B, the Basmati quality maintainer line of cytoplasmic male sterility (CMS) line Pusa 6A, which is the female parent of PRH10 was used as the recurrent parent in the present study. Tetep, a Vietnamese indica rice variety has been used as the donor for the blast resistance gene, Pi54 and ShB resistance QTLs, qSBR11-1, qSBR11-2 and qSBR7-1. Tetep was crossed with Pusa 6B (Fig. 2) and the single F1 plant was backcrossed with Pusa 6B, to generate the BC1F1. Marker assisted foreground selection among BC1F1 plants initially targeted the SSR marker RM206 linked for Pi54 which was coupled with selection for phenotypic similarity with the recurrent parent. Selected plants that were heterozygous for RM206 were selfed to generate the BC1F2 generation. After another round of selection and selfing, in the BC1F3 generation, the ShB resistance was screened and the resistant lines were subjected for SSR marker evaluation for QTLs, RM224 and RM7332 (qSBR11-1), RM209 (qSBR11-2), and RM336 (qSBR7-1) by reductive screening. The improved lines identified homozygous for Pi54 and the three ShB resistant QTLs were selfed to generate BC1F5 generation.
Fig. 2

Phenotypic evaluation of parents and backcross derived improved lines for ShB resistance. Resistance for sheath blight was evaluated in the prevalent field conditions using a virulent isolate at IARI, New Delhi. Recurrent parent (Pusa 6B) showed susceptible reaction with score of 7 and donor parent (Tetep) showed resistant reaction with score of 1. The backcross derived BC1F5 line (Pusa 1604-5-3-5) also showed resistant reaction with score of 1 similar to donor parent

Molecular marker analysis

Total genomic DNA was extracted from 15-day-old seedlings using standard protocol (Prabhu et al. 1998). The polymerase chain reaction was performed in 10 μl reaction as mentioned in our earlier studies (Singh et al. 2012a, b). Detailed description of the markers, their chromosomal and physical location, phenotypic variation explained (PVE), annealing temperature and fragment size of resistant and susceptible parents are presented in Table 1.
Table 1

Details of molecular markers used for marker-assisted backcross breeding for development of improved lines for blast and sheath blight resistance

Gene/QTL

R2 (%)

SSR marker

Physical position (Mb)

Type

Target trait

Distance

AT (°C)

Chr

Tetep (allele in bp)

Pusa6B (allele in bp)

Reference

Pi54

RM206

21.62

SSR

Blast

0.6 cM

55

11

160

150

Savary et al. (2006)

qSBR11-1

13.82

RM224

27.20

SSR

ShB

Peak marker

55

11

130

140

Channamallikarjuna et al. (2010)

qSBR11-2

7.81

RM209

17.80

SSR

ShB

Peak marker

55

11

150

140

Channamallikarjuna et al. (2010)

qSBR7-1

18.01

RM336

21.81

SSR

ShB

Peak marker

55

7

200

180

Channamallikarjuna et al. (2010)

R2 phenotypic variation explained (PVE) by the respective QTL, ShB sheath blight, AT annealing temperature of primers, Chr chromosome number

Screening for blast and sheath blight resistance

Twenty one-day-old seedlings grown in plastic trays were inoculated with four most virulent rice blast isolates, in two replications collected from Basmati grown locations of North India (Mo-nwi-007, Mo-nwi-012, Mo-nwi-018 and Mo-nwi-019). Details on the virulence spectrum of these four isolates can be found elsewhere (Singh et al. 2012a). The screening for blast resistance under artificial inoculation was carried out by standard screening procedure (Bonman et al. 1986) in which 50 ml of inoculum preparation with 5 × 104 conidia ml−1 was sprayed on the plants in each tray and kept for 24 h at 25 °C in a dew chamber and subsequently for 1 week at 25 °C in a mist chamber before scoring. Blast resistance was scored on a 0–5 scale using SES for rice (IRRI 2002).

For screening for ShB resistance, a virulent isolate of R. solani from Kapurthala in Punjab (Rs-K), was used to inoculate rice plants using an inserted inoculation method (Pan et al. 1997). Woody matchsticks infected with the pathogen were used as the inoculums carrier. Horizontal and vertical disease spread (relative lesion length) was recorded on three tillers in each of the two replications twice after 10 days and after 25 days of inoculation. The relative lesion height (RLH) were computed using formula, RLH = lesion height × 100/plant height. Based on RLH, ShB resistance was grouped into 4 categories (Ahn et al.1986).

Evaluation of agronomic performance and grain quality analysis

The improved lines in BC1F5 generation together with parental lines were evaluated for agronomic performance in a randomized complete block design with three replications with 20 × 15 cm spacing at the experimental farm of Division of Genetics, IARI, New Delhi during Kharif 2010. The data of five plants were recorded for various agronomic traits such as days to 50 % flowering (DF), plant height (PH), number of tillers (NT), panicle length (PL), filled grains per panicle (FG), spikelet fertility (SF), 1000-grain weight (GW) and yield (YD) using the standard evaluation system of rice (IRRI 2002). The grain quality was determined by grain size, kernel length before cooking (KLBC), kernel length after cooking (KLAC), kernel breath before cooking (KBBC), kernel breath after cooking (KBAC), length/breath ratio (LBR), elongation ratio (ER) alkali spreading value (ASV) and aroma (Gopalakrishnan et al. 2008).

Results

MABB for blast resistance

Pusa 6B was crossed as female parent with Tetep as donor parent to produce 21 F1 seeds and this cross combination were designated as Pusa 1604. To identify positive plants hybridity of F1 plants were tested using Pi54 linked maker RM206, which showed that all of F1 plants were true hybrids. Further, single F1 plant was selected to generate 119 BC1F1 seeds by backcrossing it with the recurrent parent Pusa 6B used as female. Out of the 119 plants, 26 plants were selected for molecular analysis on the basis of phenotypic similarity to recurrent parent. In the foreground selection using the marker RM206, 12 BC1F1 plants out of 26 were identified heterozygous for the gene of interest (Pi54). Further, out of 12 BC1F1 plants, four plants showing best resemblance to Pusa 6B in grain and cooking quality were selfed to generate four BC1F2 families comprising of 600 plants. On the basis of strict phenotypic selection for agronomic traits towards recurrent parent, 388 plants were selected, which represented all four F2 families for foreground selection. Subsequent analysis identified 86 plants homozygous for Pi54, indicated by the homozygosity of the gene linked marker, RM206. Stringent phenotypic selection for grain and cooking quality traits in comparison with Pusa 6B resulted in identification of 71 BC1F2 plants, which were selfed to generate 71 BC1F3 families, which were further analyzed for the presence of QTLs for ShB resistance.

MAS for QTLs governing sheath blight resistance

A stepwise reductive screening on target loci was performed on the 71 BC1F3 families each with 100 plants for identifying families with QTLs for ShB resistance. Pooled DNA from ten randomly selected plants from each family was screened for identification of families homozygous for ShB resistance QTL, qSBR11-1 using the peak marker, RM224. This resulted in identification of 33 families that were homozygous for qSBR11-1. Of these, 15 families were found homozygous for another ShB resistance QTL, qSBR11-2 using peak marker, RM209. These 15 families were subsequently screened for the third ShB resistance QTL, qSBR7-1 using its peak marker RM336, resulting in identification of seven plants homozygous for Pi54, qSBR11-1, qSBR11-2 and qSBR7-1 (Table 2). These seven selected plants which belonged to six different families, along with two additional plants possessing combinations such as Pi54 + qSBR11-1 and Pi54 + qSBR11-1 + qSBR11-2 were selfed up to BC1F5 through pedigree method of selection. Details about the selected lines, their genotype status and disease score are presented in Table 2.
Table 2

Marker genotypes and disease reaction score for blast and sheath blight isolates among Pusa1604 lines

Disease

Blast

Sheath blight (ShB)

Gene/QTL/Isolate

Pi54

Mo-ni-007

Mo-ni-012

Mo-ni-018

Mo-ni-019

qSBR11-1

qSBR11-2

qSBR7-2

Sh-K

Marker

RM206

RM224

RM209

RM336

Pusa 6B

S

S

S

S

7 (S)

Tetep

+

R

R

R

R

+

+

+

1 (R)

Pusa 1604-05-5-1

+

S

R

R

R

+

+

+

1 (R)

Pusa 1604-05-6-1

+

R

R

R

R

+

+

+

1 (R)

Pusa 1604-05-6-2

+

R

R

R

R

+

+

+

1 (R)

Pusa 1604-05-3-5

+

R

R

R

R

+

1 (R)

Pusa 1604-05-7-1

+

R

S

R

R

+

+

+

1 (R)

Pusa 1604-05-7-2

+

R

R

S

R

+

+

+

1 (R)

Pusa 1604-05-43-1

+

R

R

S

S

+

+

+

3 (MR)

Pusa 1604-05-46-5

+

R

R

S

S

+

+

+

3 (MR)

Pusa 1604-05-45-1

+

R

R

S

S

+

+

3 (MR)

Resistance to rice blast was evaluated on the basis of lesion size under artificial inoculation conditions and 0–5 scale of SES was adopted, wherein score 0–2 were considered as resistant (R), 3 as moderately resistance (MR) and 4–5 as susceptible (S). For evaluation of ShB resistance, a 0–9 score scale was used based on relative lesion height (RLH), wherein score 0 was considered as highly resistant (RLH = 0), 1 as resistant (RLH < 20), 3 as moderately resistant (RLH: 20–30), 5 as moderately susceptible (RLH: 31–45), 7 as susceptible (RLH: 45–65) and 9 (>65) as highly susceptible. + indicates presence of resistance allele while − shows presence of susceptibility allele

Evaluation for disease resistance of Pusa 1604 lines

Evaluation of Pusa 6B and Tetep along with the improved lines for blast and ShB resistance confirmed susceptible reaction of Pusa 6B to all four rice blast isolates used in the study, while Tetep showed resistance (Table 2). However, out of nine Pusa 1604 lines, only three lines such as Pusa 1604-05-06-1, Pusa 1604-05-6-2 and Pusa 1604-05-3-5 showed resistance similar to Tetep against all four isolates. Of the remaining, three lines showed resistance to three isolates and rest three showed only resistance to two isolates.

Similarly, while analyzing lines for ShB resistance using highly virulent isolate Rs-K under field conditions, Tetep showed resistance with a score of 1 and the recurrent parent Pusa 6B showed susceptible reaction with score of 7. Among the improved lines, only six lines showed resistance score of 1. The remaining three lines showed moderate resistance with a score of 3. Interestingly, Pusa1604-05-3-5 which showed presence of a single resistant QTL, qSBR11-1 exhibited resistance score of 1 similar to those lines possessing all three resistant QTLs (Fig. 2). On the contrary, Pusa 1604-05-45-1 possessing two and Pusa 1604-05-43-1 with three ShB resistance QTLs, showed moderate resistance (score of 3). Overall, two improved lines namely Pusa 1604-05-06-1 and Pusa 1604-05-6-2 showed high degree of resistance to all four virulent rice blast isolates and one ShB isolate.

Agronomic performance of Pusa 1604 lines

The agronomic performance of the backcross derived improved lines showed that most of the agronomic traits were similar to that of the recurrent parent Pusa 6B (Table 3). The DF of the improved lines ranged from 94 to 100 days with an average of 96 days similar to that of Pusa 6B. The average PL was 28.5 cm and with a range of 27.6–30.1 cm as against 28.7 cm in Pusa 6B. However, the maximum FG of 201 was observed in Pusa 1604-05-6-1 which was higher than Pusa 6B (189). Similarly, SF and GW of some of the improved lines (91.1 % SF in Pusa 1604-05-6-2 and GW of 26.8 g in Pusa 1604-05-6-1) were better that of Pusa 6B (SF 83.1 % and GW 23.6 g). The yield advantage of improved lines over Pusa 6B was ranged from −8.1 % (Pusa 1604-05-6-2) to 8.8 % (Pusa 1604-05-7-1). All of the selected lines had sturdy stem, dark green flag leaves unlike that of the donor parent Tetep.
Table 3

Agronomic performance of Pusa 1604 lines possessing blast and sheath blight resistant genes/QTLs

Designation

DF

PH

NT

PL

FG

SF

GW

YD

SUP

Pusa 6B

96

97.1

10.80

28.70

189

83.08

23.60

49.15

Pusa 1604-05-5-1

95

93.8

8.00

27.80

198

81.97

20.80

52.75

7.32

Pusa 1604-05-6-1

99

94.0

9.80

27.73

201

91.07

26.80

47.75

−2.62

Pusa 1604-05-6-2

100

103.6

12.60

27.63

177

91.10

22.10

45.30

−8.06

Pusa 1604-05-3-5

94

102.2

14.20

27.67

179

82.52

25.35

46.50

−5.85

Pusa 1604-05-7-1

94

96.8

10.00

30.10

185

80.30

20.70

53.25

8.82

Pusa 1604-05-7-2

97

114.2

11.60

29.07

184

87.82

24.70

53.50

8.17

Pusa 1604-05-43-1

96

112.8

13.20

29.37

165

84.75

23.50

50.50

2.52

Pusa 1604-05-46-5

98

97.5

11.04

28.56

167

82.65

23.35

48.15

−1.98

SD±

2.26

7.99

2.02

0.94

12.97

4.22

2.16

3.22

6.61

SE

0.75

2.66

0.67

0.31

4.32

1.41

0.72

1.07

2.20

DF days to 50 % flowering, PH plant height in cm, NT number of effective tillers, PL panicle length, FG filled grains per panicle, SF spikelet fertility in percentage, GW 1000 grain weight, YD yield in quintal ha−1, SUP percent yield superiority over Pusa 6B

Grain and cooking quality of Pusa 1604 lines

The grain quality of MAS derived lines (Table 4) showed that all of them were better in grain and cooking quality than that of the recipient parent, Pusa 6B (Fig. 3). The milled rice length (KLBC) ranged from 6.46 to 7.20 mm as against 6.33 mm of Pusa 6B, while the KLAC of the improved lines ranged from 12.00 (Pusa 1604-05-3-5) to 13.93 mm (Pusa 1604-05-5-1) for which Pusa 6B registered an average of 10.07 mm. Significant improvement was obtained in all the improved lines for ER which ranged between 1.71 and 2.03 as against 1.59 of the recipient parent. All of the selected lines was found to have an ASV score of 7 and aroma of 1–2 similar to that of Pusa 6B.
Table 4

Grain and cooking quality attributes of Pusa 1604 lines in comparison to the recurrent and donor parent of marker- assisted backcross breeding

Designation

GS

KLBC (mm)

KBBC (mm)

KLAC (mm)

KBAC (mm)

ER

ASV

Aroma

Pusa 6B

Medium

6.33

1.53

10.07

2.74

1.59

7

1

Tetep

Medium

5.58

1.93

8.40

2.87

1.50

3

0

Pusa 1604-05-5-1

Long

6.87

1.53

13.93

2.33

2.03

7

1

Pusa 1604-05-6-1

Long

6.46

1.67

12.67

2.33

1.96

7

2

Pusa 1604-05-6-2

Long

6.47

1.53

13.07

2.33

2.02

7

2

Pusa 1604-05-3-5

Long

6.47

1.53

12.00

2.33

1.85

7

2

Pusa 1604-05-7-1

Long

7.07

1.40

12.13

2.47

1.71

7

1

Pusa 1604-05-7-2

Long

6.60

1.47

12.47

2.33

1.89

7

2

Pusa 1604-05-43-1

Long

7.20

1.40

13.73

2.47

1.91

7

2

Pusa 1604-05-46-5

Long

6.80

1.47

13.53

2.33

1.99

7

1

SD±

 

0.47

0.16

1.89

0.18

0.21

1.33

0.73

SE

 

0.16

0.05

0.63

0.06

0.07

0.44

0.24

GS grain shape (Long: 6.61 mm to 7.50 mm, Medium: 5.51 mm to 6.60), KLBC kernel length before cooking; KBBC kernel breadth before cooking, KLAC kernel length after cooking, KBAC kernel breadth after cooking, ER elongation ratio, ASV alkali spreading value (1–2 high and 6–7 Low); Aroma (0 absent, 1 mild, 2 6 strong and 3 very strong)

Fig. 3

Grain and cooking quality of parents and backcross derived improved line. Comparison of grain and cooking quality of parental lines and backcross derived improved line, showing small seeded donor parent Tetep with poor kernel elongation after cooking as against longer kernels of the recurrent parent (Pusa 6B) and improved line, Pusa 1604-5-3-5. By adopting stringent phenotypic selection for grain and cooking quality in the initial generations we were able to recover some superior recombinants with good elongation after cooking and presence of high aroma better or on par with the recurrent parent

Discussion

The conventional breeding for disease resistance is cumbersome, time consuming and highly dependent on environmental conditions as compared to MAS procedures which is simpler, more efficient and accurate. For crop improvement for durable disease resistance, gene pyramiding is emphasized to integrate many complex biochemical pathways into plant (Datta et al. 2002). Different MAS approaches are now in practice, using background and foreground selection (Basavaraj et al. 2010; Singh et al. 2013), combined approach using foreground and stringent phenotypic selection (Joseph et al. 2004; Gopalakrishnan et al. 2008), and foreground coupled with stringent phenotypic selection and background analysis (Singh et al. 2013) those can aid accurate and efficient gene transfer including gene pyramiding into desired varietal backgrounds.

Among the two major diseases under focus in this study, currently rice blast resistance is being targeted genetically by using major R genes, while ShB is managed agronomically by the use of chemical agents (Jia et al. 2009). Development of ShB resistance cultivars remained elusive by conventional approaches because, identification of complete ShB resistance was difficult due to conditioning of the resistance by quantitative genes (Zuo et al. 2008). Recently, eight QTLs for ShB resistance were mapped on six chromosomes such as 1, 3, 7, 8, 9 and 11 in the genotype Tetep using QTL mapping strategy (Channamallikarjuna et al. 2010). In our study, after validation of all the eight QTLs using respective tightly linked peak markers we have chosen three QTLs (qSBR11-1, qSBR11-2 and qSBR7-1) for introgression. Among these QTLs, qSBR11-1 was mapped across two locations and seasons and qSBR7-1 was mapped under two locations, while qSBR11-2 was detected in one location (Channamallikarjuna et al. 2010).

The prevalence of rice blast and ShB diseases and the significant loss inflicted by them hamper widespread adoption of PRH10 due to its high susceptibility for both the diseases. Incorporation of multiple disease resistance including BB, rice blast and ShB can greatly improve further expansion of this hybrid. Being a three line hybrid, it is therefore imperative that all the three parents of PRH10 should be improved with different combination of resistant genes, such a way that combined resistance can be brought into the improved hybrid, without affecting the grain quality. Considerable progress in this direction has been achieved in our laboratory, by incorporating BB resistance genes xa13 and Xa21 into Pusa 6B (Basavaraj et al. 2009) and PRR78 (Basavaraj et al. 2010), as well as two blast resistance genes Piz5 and Pi54 into PRR78 (Singh et al. 2012a, 2013). In this paper, we report successful incorporation broad-spectrum blast resistance gene Pi54 and ShB resistant QTLs (qSBR11-1, qSBR11-2 and qSBR7-1) in the genetic background of Pusa 6B using a MABB approach with restricted backcrossing augmented with stringent phenotypic selection for agro-morphological and quality traits. The entire process of development of these improved lines took only 3 years, which could have otherwise taken many years using conventional approaches.

The marker assisted integration of genes and QTLs has improved the resistance to blast and ShB in all the improved lines while maintaining the agronomic traits and grain quality on par with Pusa 6B. Although most of the improved lines possessed all the target genes and QTLs, disease phenotyping revealed differential resistance reaction against the virulent isolates of both the diseases. This observation was similar to the resistance reaction reported against blast and ShB among the MAS improved lines of Pusa Basmati 1 (Singh et al. 2012a). One of the plausible reasons could be the presence of different proportion of donor parent genome in the improved lines. Similar observation has been made earlier with respect to MABB derived blast resistant Basmati rice genotypes (Singh et al. 2012b).

While transferring ShB resistance QTLs, notably, one line (Pusa 1604-05-3-5) possessing single QTL qSBR11-1 showed comparable disease reaction score as that of the donor parent, Tetep while Pusa1604-05-45-1 possessing two and Pusa1604-05-43-1 with three ShB resistance QTLs, showed moderate resistance only. This could be attributed to several reasons such as QTL-marker recombination (Moreau et al. 1998), QTL-background interactions (Wang et al. 2009; Chen et al. 2014), presence of un-known QTLs in the improved lines derived from donor (Tinker and Mather 1995) and/due to the competitive effectiveness of qSBR11-1 than the other two QTLs (Pandian et al. 2012). Further, disease resistance variations among the lines positive for all QTLs, can be attributed to epistatic effects and QTL-background interaction, a well-recognized component of natural genetic variation (Malmberg and Mauricio 2005).

We successfully performed a MABB coupled with restricted backcross to facilitate MAS for multiple loci from the donor in a single breeding programme. Started with a large BC1F2 population in order to select for combinations of several target loci into the final product, we used a step-wise reductive screening of target loci (Gopalakrishnan et al. 2008; Sreewongchai et al. 2009) that was very effective in systematically reducing the large population size in the advanced generations. By the successful isolation of improved lines having multiple disease resistance together with yield and grain quality similar to that of Pusa 6B, we have shown here that restricted backcrossing can help to target more number of traits in a stepwise fashion within a single breeding stream, leveraging the practical limitations of handling large population (Joseph et al. 2004). This demonstrates successful use of a restricted backcross strategy in practical breeding and the results here proves the successful introgression of broad-spectrum blast and ShB resistance from Tetep to Pusa 6B, for the first time into a rice CMS maintainer line. This will further facilitate development of the allocytoplasmic and isogenic CMS line containing the same set of introgressed genes, because CMS lines are always developed and maintained by backcross to their isogenic maintainers (Alam et al. 1999). Therefore, the Pusa 1604 lines and their corresponding male sterile lines can ultimately lead to the development of multiple disease resistant aromatic rice hybrids, including resynthesis of improved PRH10.

For Basmati rice, the physical, organoleptic and cooking qualities of grain are the most important breeding objectives and hence a stringent phenotypic selection has been maintained throughout the selection stream. We could recover phenotypically similar if not better improved lines than that of Pusa 6B, with all desired plant type and grain quality, in short span of selection time, with a single backcross and advancement to BC1F5 generation. Successful recovery of desired phenotypic quality with one backcross has already been demonstrated by Joseph et al. (2004), who could recover 80.4–86.7 % of the recurrent parent (Pusa Basmati 1) background by BC1F3 generation. Similar results are also reported by Gopalakrishnan et al. (2008) and Pandey et al. (2013).

Conclusion

The new stable improved lines of the Pusa 6B reported in the present study showed a high level of broad spectrum resistance for different isolates of blast and resistance for ShB coupled with comparable yield, cooking and eating quality to that of recurrent parent, Pusa 6B. These lines can be instantly employed either for the development of blast and ShB resistant CMS lines as well as be used as novel germplasm providing potential donors for multiple disease resistance for Basmati rice improvement.

Notes

Acknowledgments

The present study was supported under the Indian Council of Agriculture Research, New Delhi funded Network project on gene pyramiding in rice. The virulent isolates of Magnaporthae oryzae were collected under the ICAR-NAIP funded project entitled “Allele Mining and Expression Profiling of Resistance and Avirulence Genes in Rice-Blast Pathosystem for Development of Race Non-Specific Disease Resistance” (NAIP/C4/C1071).

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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Ashok K. Singh
    • 1
  • Vikas K. Singh
    • 1
    • 4
  • Atul Singh
    • 1
  • Ranjith K. Ellur
    • 1
  • R. T. P. Pandian
    • 2
  • S. Gopala Krishnan
    • 1
  • U. D. Singh
    • 2
  • M. Nagarajan
    • 3
  • K. K. Vinod
    • 3
  • K. V. Prabhu
    • 1
  1. 1.Division of GeneticsIndian Agricultural Research InstituteNew DelhiIndia
  2. 2.Division of Plant PathologyIndian Agricultural Research InstituteNew DelhiIndia
  3. 3.Rice Breeding and Genetics Research CentreIndian Agricultural Research InstituteAduthuraiIndia
  4. 4.International Crop Research Institute for Semi-Arid Tropics (ICRISAT)PatancheruIndia

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