Find the information such as human life, natural resource,agriculture,forestry, biotechnology, biodiversity, wood and non-wood materials.
Blog List
Monday, 12 December 2016
Marker-Assisted Pyramiding of Genes Conferring Resistance Against Bacterial Blight and Blast Diseases into Indian Rice Variety MTU1010
Published Date November 2016, Vol.23(6):306–316,doi:10.1016/j.rsci.2016.04.005 Open Access, Creative Commons license,Funding information Author
K. Arunakumari a
C.V. Durgarani a,,
V. Satturu a
K.R. Sarikonda b
P.D.R. Chittoor c
B. Vutukuri b
G.S. Laha d
A.P.K. Nelli a
S. Gattu a
M. Jamal a
A. Prasadbabu e
S. Hajira d
R.M. Sundaram d,,
aInstitute of Biotechnology, Professor Jayashankar Telangana State Agricultural University, Rajendranagar, Hyderabad 500030, India
bAndhra Pradesh Rice Research Institute, Acharya N.G. Ranga Agricultural University, Maruteru, West Godavari District 534122, India
cAgriculture Research Station, Acharya N.G. Ranga Agricultural University, Nellore 524004, India
dCrop Improvement Section, ICAR-Indian Institute of Rice Research, Rajendranagar, Hyderabad 500030, India
eUrmatt Ltd. Wang Chai, Chaingrai 57210, Thailand
Received 1 December 2015. Accepted 12 April 2016. Available online 16 November 2016. Managing Editor: Li Guan
Abstract
Two major bacterial blight (BB) resistance genes (Xa21andxa13) and a major gene for blast resistance (Pi54) were introgressed into an Indian rice variety MTU1010 through marker-assisted backcross breeding. Improved Samba Mahsuri (possessingXa21andxa13) and NLR145 (possessingPi54) were used as donor parents. Marker-assisted backcrossing was continued till BC2generation wherein PCR based functional markers specific for the resistance genes were used for foreground selection and a set of parental polymorphic microsatellite markers were used for background selection at each stage of backcrossing. Selected BC2F1plants from both crosses, having the highest recoveries of MTU1010 genome (90% and 92%, respectively), were intercrossed to obtain intercross F1(ICF1) plants, which were then selfed to generate 880 ICF2plants possessing different combinations of the BB and blast resistance genes. Among the ICF2plants, seven triple homozygous plants (xa13xa13Xa21Xa21Pi54Pi54) with recurrent parent genome recovery ranging from 82% to 92% were identified. All the seven ICF2plants showed high resistance against the bacterial blight disease with a lesion lengths of only 0.53–2.28 cm, 1%–5% disease leaf areas and disease scoring values of ‘1’ or ‘3’. The seven ICF2plants were selfed to generate ICF3, which were then screened for blast resistance, and all were observed to be highly resistant to the diseases. Several ICF3lines possessing high level of resistance against BB and blast, coupled with yield, grain quality and plant type on par with MTU1010 were identified and advanced for further selection and evaluation.
Keywords
gene pyramiding
bacterial blight resistance
blast resistance
rice
marker-assisted backcross breeding
Rice is the principal staple food crop of the world and rice production has so far kept pace with the growing population, principally due to cultivation of high-yielding, high-input demanding, and semi-dwarf varieties (Gnanamanickam, 2009). However, the introduction of semi-dwarf rice varieties and the large-scale use of inputs like fertilizers and insecticides have changed the dynamics of pests and diseases of rice, increasing their incidence significantly in the recent years. Bacterial blight (BB) and rice blast are the two most important diseases causing significant yield loss in rice (Zhang et al., 2015), and they are endemic to several rice growing states of India (Production Oriented Survey, DRR, 2008). In Andhra Pradesh of India (including the newly created state of Telangana), the yield loss is very severe due to BB and blast (Rajarajeswari and Muralidharan, 2006 and Sundaram et al., 2008). To minimize these problems, development of durable, broad-spectrum resistant varieties has been advocated (Jena and Mackill, 2008, Kumar et al., 2014 and Sundaram et al., 2014). At least 40 genes conferring BB resistance have been identified (Sundaram et al., 2014) and many of them have been fine-mapped and cloned (Natrajkumar et al., 2012). To date, 101 blast-resistant genes (Rajashekara et al., 2014) and 350 quantitative trait loci (QTLs) have been identified (Sharma et al., 2012), with many fine-mapped and a few cloned. Closely linked or functional markers are available for many BB and blast resistance genes (Sundaram et al., 2014).
MTU1010 (Cottondora Sannalu), a short duration rice variety released in 2000 derived from the cross Krishnaveni/IR64, is extremely popular with farmers and has been planted for many years on a minimum of one million hectares. This variety possesses brown planthopper tolerance with long slender grains. However, MTU1010 is highly susceptible to both BB and blast diseases, which limits its spread to areas where the two diseases are endemic. As the availability of several resistance genes to BB and blast, pyramiding multiple genes into MTU1010 is considered as an ideal strategy to improve its resistance to these major diseases. Breeding for host-plant resistance is considered as the most economical and eco-friendly strategy for management of pests and diseases of crop plants and achieving yield stability. Molecular markers can accelerate resistance breeding efforts, as segregating plants can be selected on the basis of molecular marker alleles instead of their phenotypes and introgression of multiple resistance genes or gene pyramiding can be tracked easily in a population (Sundaram et al., 2014).
Gene pyramiding through conventional phenotype-based screening is considered to be difficult and often impossible, due to the dominance and epistasis effects of genes governing disease resistance and also due to limitations related to screening against the two diseases across the year (Sundaram et al., 2009). The availability of molecular markers, closely linked to or located within the resistance genes (i.e. functional markers), makes the task of gene pyramiding easier (Singh et al., 2001, Sundaram et al., 2008, Shanti et al., 2010 and Zhao et al., 2014). Functional markers are developed from polymorphic sites within genes that casually affect target trait variation i.e. based on functional characterization of polymorphism. Hence, they are more reliable to be used in marker-assisted backcross breeding, circumventing the recombination issue there by getting rid of false positives. Among the BB resistance genes identified so far, the dominant gene, Xa21, originally discovered from an accession of the wild rice, Oryza longistaminata, confers broad spectrum resistance to many Xoo isolates in India and elsewhere. The gene has been cloned and fine-mapped on the long arm of rice chromosome 11, and a gene-specific functional marker, named pTA248 (Ronald et al., 1992), is available for marker-assisted breeding. BB resistance gene xa13 was first discovered in the rice variety BJ1, and mapped on the long arm of rice chromosome 8 (Ogawa et al., 1987, Zhang et al., 1996 and Sanchez et al., 1999) and very tightly linked markers are available for the gene (Sundaram et al., 2014). The BB resistance gene combination, Xa21 + xa13, is known to be very effective across India (Joseph et al., 2004 and Gopalakrishnan et al., 2008). Among the major blast resistance genes, Pi54 exhibits resistance to predominant isolates of the blast pathogen in India (Sharma et al., 2002 and Sharma et al., 2010) and is considered to be an ideal choice for introgression. In this study, we aimed to transfer two major BB resistance genes (Xa21 and xa13) and one major blast resistance gene (Pi54) into MTU1010 through marker-assisted backcross breeding.
MATERIALS AND METHODS
Rice materials
Improved Samba Mahsuri (ISM), with high-yielding, fine-grain type and BB resistant, released by ICAR-Indian Institute of Rice Research (ICAR-IIRR), Hyderabad, Indian, possessing xa5, xa13 and Xa21 (Sundaram et al., 2008), and NLR145 (Swarnamukhi), obtained from the parentage CICA4/IR625-23-3-1// Tetep, a popular long slender and long duration variety released from Agricultural Research Station, Nellore, Indian, possessing Pi54, were used as the donor parents for BB and blast resistance, respectively. MTU1010 was used as the recurrent parent.
Marker-assisted selection for BB and blast resistance
For targeted introgression of xa13, Xa21 and Pi54 into MTU1010, a simultaneous and stepwise marker-assisted backcross breeding strategy as illustrated in Supplemental Fig. 1 was adopted. Two separate backcrosses were carried out wherein the BB resistance genes Xa21 and xa13 from ISM, as well as the blast resistance gene Pi54from NLR145, were introgressed into MTU1010, respectively. The F1 plants derived were confirmed for their hybridity by MTU1010/ISM (i.e. heterozygosity) using the co-dominant markers, pTA248 (specific for Xa21; Ronald et al., 1992) and xa13-prom (specific for xa13; Sundaram et al., 2011), while Pi54 gene-specific co-dominant marker, Pi54-MAS (Ramkumar et al., 2011), was used for the hybridity by MTU1010/NLR145. The ‘true’ F1 plants were backcrossed with MTU1010. BC1F1plants (ISM/MTU1010//MTU1010) were screened with pTA248 and xa13-prom markers to identify plants heterozygous for Xa21 and xa13, respectively. Backcross plants of NLR145/MTU1010//MTU1010 were screened with the marker Pi54-MAS to identify plants heterozygous for Pi54. The primer sequence information is presented in Supplemental Table 1. The positive plants identified from the two BC1F1s were then screened with a set of parental polymorphic SSR markers (Supplemental Table 2) to identify the recovery of MTU1010 genome. Marker-assisted backcrossing was done till BC2 generation, after which the backcross plants (BC2F1) derived from the two crosses possessing the maximum recurrent parent genome recovery were intercrossed for pyramiding all the three resistance genes into MTU1010. The intercross F1s were confirmed for their heterozygosity as described earlier using pTA248, xa13-prom and Pi54-MAS, and ‘true’ intercross F1 with the maximum recurrent parent genome recovery were then selfed to generate intercross F2 (ICF2) plants. These were then screened with the three target-gene specific markers to identify ‘gene’ positive plants in homozygous condition, which were later screened using parental polymorphic SSR markers to identify the ‘best’ ICF2 plants. From ICF3generation onwards, pedigree-based selection was carried out to identify the best homozygous lines. For marker-assisted selection, DNA was isolated from the parents, backcross plants and intercrossed plants/lines according to Zheng et al (1995). PCR and gel electrophoresis protocols recommended by Sundaram et al (2008) and Ramkumar et al (2011) were adopted for marker-assisted selection of Xa21, xa13 and Pi54, respectively, while the background selection protocol recommended by Sundaram et al (2008) was adopted to identify backcross and intercross F2 plants possessing the maximum recurrent parent genome recovery by Graphical genotype version 2.0 (van Berloo, 2008).
Fig. 1. Foreground selection for xa13 (A), Xa21 (B) and Pi54 (C) among ICF2 plants.
R, Recurrent parent (MTU1010); D, Donor parent (ISM); M, 50 bp ladder molecular weight marker; Lanes 9–104 represent ICF2 plants.
Arrow indicates a triple gene homozygous plant (ICF2-16-59).
Table 1. Details of number of plants generated and confirmed to be resistance gene positive through marker analysis in each generation.
Cross combination
Particular of cross combination
No. of plants screened
No. of plants confirmed
Gene combination in the confirmed plants
MTU1010 × ISM (C1)
C1-F1
125
101
Xa13xa13Xa21xa21
MTU1010 × C1-F1
C1-BC1F1
293
8
Xa13xa13Xa21xa21
MTU1010 × C1-BC1F1
C1-BC2F1
534
11
Xa13xa13Xa21xa21
MTU1010 × NLR145 (C2)
C2-F1
110
74
Pi54pi54
MTU1010 × C2-F1
C2-BC1F1
80
35
Pi54pi54
MTU1010 × C2-BC1F1
C2-BC2F1
268
17
Pi54pi54
C1-BC2F1 × C2-BC2F1
ICF1
360
4
Xa13xa13Xa21xa21Pi54pi54
Selfed progeny of selected ICF1plant
ICF2
880
7
xa13xa13Xa21Xa21Pi54Pi54
Selfed progeny of ICF2
ICF3
–
7
xa13xa13Xa21Xa21Pi54Pi54
Table 2. Screening of three-gene positive ICF2 and ICF3 plants for resistance against bacterial blight (BB) disease and blast disease.
Plant identity
Allelic status of xa13, Xa21 and Pi54
Disease scoring scale for BB in ICF2
Disease scoring scale for rice blast in ICF3
Background genome recovery (%)
MTU1010
Xa13Xa13xa21xa21pi54pi54
9
7
–
Improved Samba Mahsuri
xa13xa13Xa21Xa21
1
–
–
NLR145
Pi54Pi54
–
3
–
ICF2:3-16-59
xa13xa13Xa21Xa21Pi54Pi54
1
1
92
ICF2:3-16-231
xa13xa13Xa21Xa21Pi54Pi54
1
2
85
ICF2:3-16-235
xa13xa13Xa21Xa21Pi54Pi54
1
2
82
ICF2:3-16-282
xa13xa13Xa21Xa21Pi54Pi54
1
2
83
ICF2:3-16-521
xa13xa13Xa21Xa21Pi54Pi54
1
1
82
ICF2:3-16-786
xa13xa13Xa21Xa21Pi54Pi54
3
2
83
ICF2:3-16-837
xa13xa13Xa21Xa21Pi54Pi54
1
2
88
Screening for BB resistance
A virulent isolate of the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae(Xoo), collected from Rajendranagar farm of Indian Institute of Rice Research, DRR Xanthomonas collection-022 (DX-022), was used to screen the donor and recurrent parents along with ICF2 plants for bacterial blight resistance under both glasshouse and field conditions. In the greenhouse, disease severity is assessed based on lesion length measurement or estimation of diseased leaf area. Due to the large amount of breeding lines assessed in the field, disease severity is usually measured in diseased leaf area. The Xoo strain was cultured and stored as described by Laha et al (2009). The rice plants were clip-inoculated with a bacterial suspension of 109 cfu/mL at the maximum tillering stage (50 d after transplanting) according to the method of Kauffman et al (1973). Approximately 10 leaves per plant were inoculated, and disease reaction was scored 14 d after inoculation. BB lesion length was measured and the disease score was calculated as per IRRI standard evaluation system (IRRI-SES) scale (IRRI, 1996).
Screening for blast resistance
Two highly virulent Magnaporthe oryzae fungal isolates collected from Agriculture Research Station (ARS), Nellore and Andhra Pradesh Rice Research Institute (APRRI), Maruteru, Andhra Pradesh, India were used to screen the donor and recurrent parents along with ICF3 lines for blast resistance under in vivo conditions following uniform blast nursery method at ARS and APRRI. The pathogen strains were cultured and stored as described by Prasad et al (2011). The young seedlings at the four-leaf stage were inoculated with the fungal conidial suspension at a concentration of 1 × 105 cfu/mL, and high relative humidity was maintained for disease development. Inoculated seedlings were monitored for the development of blast lesions one week after inoculation. The plants were scored and evaluated on a 0–9 scale as per IRRI-SES scale (IRRI, 1996).
Evaluation of agro-morphological characters
Thirty-day-old seedlings of the selected ICF3 lines were transplanted in the main field at a spacing of 20 cm × 15 cm along with the donor and recurrent parents. Standard agronomic practices were followed to raise a healthy crop and the progenies were evaluated during the Rabi season, 2012–2013. Days to 50% flowering, days to maturity, plant height (cm), number of productive panicles per plant, panicle weight (g), panicle length (cm), grain yield per plant (g), 1000-grain weight (g) and grain type were recorded in three replications and the replicated data was calculated for the mean, coefficient of variation (CV) and critical difference (CD).
RESULTS
Confirmation of marker polymorphism for gene-specific markers and identification of parental polymorphic markers
The DNAs from the recurrent parent MTU1010 and the donor parents B95-1/ISM (for Xa21 and xa13) and NLR145 (for Pi54) were used to determine marker polymorphism. The primer pair pTA248 amplified fragments of 900 bp in the resistant parent (ISM), while that from the susceptible parent MTU1010 was 650 bp. With respect to the primer pair, xa13-prom, ISM amplified a 500 bp fragment, while MTU1010 amplified a 250 bp. Similarly, with respect to the marker Pi54-MAS, a fragment of 210 bp was amplified in NLR145, while a 350 bp fragment was amplified in MTU1010. Thus, all the markers were able to distinguish resistant lines from susceptible ones in a co-dominant fashion.
Parental polymorphism survey was carried out using 617 SSR markers (Supplemental Table 2). Among them, 82 markers showed polymorphism between MTU1010 and ISM, while 83 markers showed polymorphism between MTU1010 and NLR145. Parental polymorphism ranged from 5 markers on chromosome 9 to 12 markers on chromosome 8 (Supplemental Table 2). The average physical distance between each polymorphic marker was 4.1 Mb.
Marker-assisted introgression of BB resistance into MTU1010
F1s generated from the cross C1 were screened for the presence of Xa21 and xa13using pTA248 and xa13-prom to identify the ‘true’ F1s showing heterozygous amplification pattern (Table 1). Of 125 F1s screened, 101 were identified to be true heterozygotes and further used as male parent and backcrossed with MTU1010 to generate BC1F1. Out of a total of 293 BC1F1 plants generated, 55 were identified to be positive for Xa21, 68 were positive for xa13 and 8 were double positive for both Xa21and xa13. These heterozygous plants were then subjected for background selection using 82 SSR markers, which were earlier identified to be polymorphic between MTU1010 and ISM. A solitary ‘positive’ BC1F1 plant (C1-BC1F1-34) possessing the maximum recovery of recurrent parent (MTU1010) genome (72%) was selected and after that backcrossed with MTU1010 to generate BC2F1s. A similar marker-assisted selection procedure was followed for selection of BC2F1 (534) plants and a solitary ‘positive’ BC2F1 plant (C1-BC2F1-23) possessing the maximum recovery of recurrent parent (MTU10101) genome (90%) was selected and utilized for intercrossing with the selected BC2F1 plant from the cross ISM/MTU1010// MTU1010///MTU1010.
Marker-assisted introgression of blast resistance into MTU1010
The F1s generated from the cross C2 were screened for the presence of the target resistance gene, Pi54 using the functional marker Pi54-MAS to identify the ‘true’ F1s showing heterozygous amplification pattern (Table 1). Of 110 F1s, 74 plants were observed to possess target resistance gene in heterozygous (Pi54pi54) condition, which were then used as male parent and backcrossed with MTU1010 to generate BC1F1 plants. Of the 80 BC1F1 plants screened, a total of 35 were identified to be positive (i.e. heterozygous), when screened with Pi54-MAS and they were then subjected for background selection using 83 parental polymorphic SSR markers. A single ‘positive’ BC1F1 plant (C2-BC1F1-17) possessing the maximum recovery of recurrent parent genome (79%) was selected and then backcrossed with MTU1010 to generate BC2F1 plants. A similar marker-assisted selection procedure was followed for selection of BC2F1 (268) plants, wherein a single ‘positive’ BC2F1 plant (C2-BC2F1-4) possessing 92% recurrent parent genome was selected and intercrossed with the best backcross plant generated from the cross NLR/MTU1010//MTU1010///MTU1010.
Marker-assisted introgression of BB and blast resistance genes into MTU1010 through intercrossing
C1-BC2F1-23 possessing Xa21xa21Xa13xa13 was used as a female parent and crossed with C2-BC2F1-4 possessing Pi54 in heterozygous condition, and a set of 360 ICF1 seeds were generated (Table 1). A total of four such ‘triple heterozygous positive’ ICF1 plants (Xa13xa13Xa21xa21Pi54pi54) were identified and then screened with parental polymorphic SSR markers. A single ICF1 plant (ICF1-16), which possessed the maximum-percentage of recurrent parent genome recovery (90%) was identified and 2216 ICF2 seeds were produced. They were then grown under field conditions at Professor Jayashankar Telangana State Agricultural University, Hyderabad, India, during wet season in 2012. A total of 880 ICF2 plants were genotyped and 7 plants possessing all the three target resistance genes in homozygous condition (xa13xa13 Xa21Xa21Pi54Pi54) (Fig. 1) were identified.
Background genome analysis of backcross derived BB and blast resistant lines of MTU1010
Background analysis was carried out among the seven three-gene homozygous ICF2plants using the polymorphic SSR markers by GGT2 or Graphical Genotyper software (van Berloo, 2008). The analysis revealed an average recovery of 87% of MTU1010 genome, with a residual heterozygosity of 5.95%. Four plants had a recovery of more than 85% of MTU1010 genome. Chromosome-wise analysis of the background showed complete recovery of chromosomes 3, 5, 6, 7, 9 and 10 from MTU1010 genome in all the recombinants. A single ICF2-16-59 showed the highest recovery of MTU1010 genome (92%), while two ICF2 plants, ICF2-16-235 and ICF2-16-521 plants scored the lowest recurrent genome recovery (82%). A small genomic region spanning 1.0 Mb around xa13 on chromosome 8 and 3.5 Mb region around Xa21 and Pi54 on chromosome 11, to be introgressed from the donor parent. Interestingly, ICF2-16-59 inherited chromosomes 1, 3, 5, 6, 7, 9 and 10 completely from the recurrent parent, and possessed only short segments from donor parent in the telomeric end of chromosomes 2, 4, 8 and 12, and relatively longer donor segments from the long arm of chromosome 11 (Supplemental Fig. 2).
Fig. 2. Screening of selected improved lines of MTU1010 against bacterial blight (A) and blast (B) diseases under controlled conditions.
A, With respect to screening for bacterial blight resistance, the recurrent parent MTU1010 was highly susceptible, while the donor parent and the selected gene pyramided lines at ICF2generation (1, ICF2-16-59; 2, ICF2-16-231; 3, ICF2-16-235; 4, ICF2-16-282; 5, ICF2-16-521; 6, ICF2-16-786; 7, ICF2-16-837) were highly resistant to the disease. B, When the selected ICF3 plants were screened for blast resistance through uniform blast nursery method, the susceptible check NLR34242 and recurrent parent MTU1010 were highly susceptible to blast disease, while the resistant donor NLR145 along with gene pyramided line ICF3-16-59 showed high level of resistance.
Screening of ICF2 lines for bacterial blight resistance
All the three-gene positive ICF2 plants (those possessing Xa21, xa13 and Pi54 in homozygous condition) were screened for BB resistance. The donor genotype ISM showed an average lesion length of 0.77 cm with disease scoring scale value ‘1’, while the recurrent parent MTU1010 possessed an average lesion length of 12.23 cm (90% diseased leaf area) with disease scoring scale of ‘9’. All the three-gene pyramid ICF2 plants showed highly resistant against the disease with a lesion length of only 0.53–2.28 cm and 1%–5% diseased leaf area with disease scoring values of ‘1’ or ‘3’ (Fig. 2 and Table 2).
Screening of ICF3 families for blast resistance
The seven ICF2 plants possessing the three resistance genes in homozygous condition were selfed to generate ICF3. ICF3 lines were then screened during dry seasons in 2012 and 2013 for blast resistance under uniform blast nursery at APRRI and ARS, which are blast epidemic areas, along with the resistant parent NLR145, the susceptible parent MTU1010 and the susceptible check NLR34242. NLR145 showed high level of resistance against blast, with an average disease score of ‘3’ and MTU1010 showed presence of disease lesions in more than 50% leaf area with an average disease scoring scale ‘7’ and NLR34242 showed presence of disease lesions in more than 75% leaf area with an average disease scoring scale ‘9’. Most of the improved rice breeding lines derived from homozygous ICF2 lines displayed a high level of resistance for rice blast. ICF3-16-59 and ICF3-16-521 showed small brown specks of pinhead size without sporulating center on the leaves and with a disease scoring scale of ‘1’ similar to NLR145 (Fig. 2 and Table 2). The remaining lines showed small roundish to slightly elongated necrotic grey spots, which were about 1–2 mm in diameter with a distinct brown margin, with lesions mostly found on the lower leaves with a disease score of ‘2’ and categorized as resistant.
Agro-morphological evaluation of seven ICF3 lines
Seven ICF3 lines were evaluated for yield and agro-morphological characters during dry seasons in 2012 and 2013 at APRRI (Table 3). ICF3-16-59, ICF3-16-521 and ICF3-16-235 were identified to be promising based on their high level of resistance to both blast and BB, long-slender grain type, high yield, and were further evaluated in wet season, 2014. ICF3-16-59 and ICF3-16-521 exhibited grain yield on par with MTU1010 (0.62 kg/m2) with marginal differences. No significant variation was observed among the plants of the two elite lines with respect to the number of productive panicles per plant, panicle weight, panicle length and grain yield per plant when compared with MTU1010. Interestingly, ICF3-16-235 exhibited higher yield (0.64 kg/m2) as compared to MTU1010.
Table 3. Mean values of agro-morphological characters of three resistant gene pyramided ICF3lines.
No comments:
Post a Comment