Abstract
The BC2F1 population was derived from a cross between rice variety, MR219 (susceptible to blast) and Pongsu Seribu 1 (resistant to blast). The objectives of this research were to know the inheritance pattern of blast resistance and to identify the linked markers associated with blast resistance in BC2F1 population. Sixteen microsatellite markers were found as polymorphic between the parents related to blast resistant genes (Pi-genes). Among the selected blast resistant linked markers, two markers RM6836 and RM8225 showed expected testcross ratio (1:1) for single-gene model in the BC2F1 population with the association between resistant and susceptible progeny. A total of 333-BC2F1 plants were challenged with the most virulent pathotype P7.2 of Magnaporthe oryzae. Chi-square (χ2) analysis for phenotypic segregation in single-gene model showed goodness of fit (P = 0.4463) to the expected segregation ratio (1:1). In marker segregation analysis, two polymorphic markers (RM6836 and RM8225) clearly showed goodness of fit to the expected segregation testcross ratio (1:1) for the single-gene model. The marker RM8225 and RM6836 showed significant R2 values higher than 10 for the trait of the blast lesions degree (BLD). The positions of RM6836 and RM8225 markers on rice chromosome 6 and the distance between these two markers is 0.2 cM. We conclude that single dominant gene control the blast resistance in Pongsu Seribu 1 located on chromosome 6, which is linked to RM8225 and RM6836 microsatellite markers. This information could be useful in marker-assisted selection for blast resistance in rice breeding involving Pongsu Seribu 1.
blast inheritance; microsatellite markers; BC2F1 population; rice variety
1 INTRODUCTION
The evolution of new biotypes of pests and diseases, as well as the pressures of
climate change, pose serious challenges to rice breeders, who would like to increase
rice production by introducing resistance to multiple biotic and abiotic stresses
(Miah et al., 2013Miah, G., Rafii, M. Y., Ismail, M. R., Puteh, A. B., Rahim, H. A.,
Islam, KhN., & Latif, M. A. (2013). A review of microsatellite markers and
their applications in rice breeding programs to improve blast disease
resistance. International Journal of Molecular Sciences, 14, 22499-22528.
http://dx.doi.org/10.3390/ijms141122499. PMid:24240810
http://dx.doi.org/10.3390/ijms141122499...
). Among the biotic
stresses, blast disease is the most harmful threat to high productivity of rice
(Li et al., 2007Li, Y. B., Wu, C. J., Jiang, G. H., Wang, L. Q., & He, Y. Q.
(2007). Dynamic analyses of rice blast resistance for the assessment of genetic
and environmental effects. Plant Breeding, 126, 541-547.
http://dx.doi.org/10.1111/j.1439-0523.2007.01409.x.
http://dx.doi.org/10.1111/j.1439-0523.20...
). Rice blast caused by
Magnaporthe oryzae (M. oryzae) is the most
devastating diseases of rice worldwide (Khush &
Jena, 2009Khush, G. S., & Jena, K. K. (2009). Current status and future
prospects for research on blast resistance in rice (Oryza
sativa L.). In G. L. Wang, & B. Valent (Eds.), Advances in
genetics, genomics and control of rice blast disease (p. 1-10). Dordrecht:
Springer. http://dx.doi.org/10.1007/978-1-4020-9500-9_1.
http://dx.doi.org/10.1007/978-1-4020-950...
; Liu et al., 2010Liu, J., Wang, X., Mitchell, T., Hu, Y., Liu, X., Dai, L., &
Wang, G. L. (2010). Recent progress and understanding of the molecular
mechanisms of the rice-Magnaporthe oryzae interaction. Molecular Plant
Pathology, 11, 419-427. http://dx.doi.org/10.1111/j.1364-3703.2009.00607.x.
PMid:20447289
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).
Rice blast severely reduces production in both irrigated and water stressed upland
ecosystems of tropical and temperate countries (Suh
et al., 2009Suh, J. P., Roh, J. H., Cho, Y. C., Han, S. S., Kim, Y. G., &
Jena, K. K. (2009). The pi40 gene for durable resistance to rice blast and
molecular analysis of pi40-advanced backcross breeding lines. Phytopathology,
99, 243-250. http://dx.doi.org/10.1094/PHYTO-99-3-0243.
PMid:19203276
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). The incorporation of blast resistance genes into cultivars
is the most preferential strategy in rice breeding program to prevent this disease.
Most of the major resistance genes follow gene-for-gene interaction model (Kumbhar et al., 2013Kumbhar, S. D., Kulwal, P. L., Patil, J. V., Gaikwad, A. P., &
Jadhav, A. S. (2013). Inheritance of blast resistance and identification of SSR
marker associated with it in rice cultivar RDN 98-2. Journal of Genetics, 92,
317-321. http://dx.doi.org/10.1007/s12041-013-0255-x.
PMid:23970091
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). Utilization of genetic
resistance is the most effective and environmentally friendly strategy for control
of the disease (Zhu et al., 2004Zhu, M., Wang, L., & Pan, Q. (2004). Identification and
characterization of a new blast resistance gene located on rice chromosome 1
through linkage and differential analyses. Phytopathology, 94, 515-519.
http://dx.doi.org/10.1094/PHYTO.2004.94.5.515. PMid:18943771
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).
Knowledge on the inheritance of disease resistance would facilitate the adoption of
appropriate breeding strategies and improve the efficiency of selection procedures.
Extensive studies have been conducted on inheritance of blast resistance using
Japanese races and identified 13 dominant resistance genes at eight different loci
(Kiyosawa, 1981Kiyosawa, S. (1981). Gene analysis for blast resistance. Oryza, 18,
196-203.). The inheritance of
resistance in cultivars against two races of M. oryzae was studied
and 11 dominant genes were identified (He &
Shen, 1990He, Z. H., & Shen, Z. T. (1990). Studies on blast resistance
genes in indica rice. International Rice Research Newsletter, 15,
4.). Expression of resistance is altered by modifiers or multiple
alleles. There is very little information on the inheritance and nature of
resistance utilizing tropical isolates of M. oryzae (Filippi & Prabhu, 1996Filippi, M. C., & Prabhu, A. S. (1996). Inheritance of blast
resistance in rice to two races, IB-1 and IB-9. Pyricularia
griseaBrazilian Journal of Genetics, 19, 599-604.
http://dx.doi.org/10.1590/S0100-84551996000400012.
http://dx.doi.org/10.1590/S0100-84551996...
). The nature of
resistance and susceptibility is influenced by inoculation techniques, environmental
conditions, human mistakes in scoring and the virulence of M.
oryzae (Srinivasachary et al.,
2002Srinivasachary , Hittalmani, S., Shivayogi, S., Vaishali, M. G.,
Shashidar, H. E., & Kumar, K. G. (2002). Genetic analysis of rice blast
fungus of souther Karnataka using DNA marker and reaction of popular rice
genotypes. Current Science, 82, 732-735.).
Most of the blast R genes are dominant, and some of them are quantitative in nature
(Xu et al., 2008Xu, X., Chen, H., Fujimura, T., & Kawasaki, S. (2008). Fine
mapping of a strong QTL of field resistance against rice blast, Pikahei-1(t),
from upland rice Kahei, utilizing a novel resistance evaluation system in the
greenhouse. TAG. Theoretical and applied genetics. Theoretische und angewandte
Genetik, 117, 997-1008. http://dx.doi.org/10.1007/s00122-008-0839-7.
PMid:18758744
http://dx.doi.org/10.1007/s00122-008-083...
). Furthermore, many R
genes are located as gene clusters with focuses on chromosomes 6, 9, 11, and 12
(Ashkani et al., 2013Ashkani, S., Rafii, M. Y., Rahim, H. A., & Latif, M. A. (2013).
Genetic dissection of rice blast resistance by QTL mapping approach using an F3
population. Molecular Biology Reports, 40, 2503-2515.
http://dx.doi.org/10.1007/s11033-012-2331-3. PMid:23203411
http://dx.doi.org/10.1007/s11033-012-233...
; Ballini et al., 2008Ballini, E., Morel, J. B., Droc, G., Price, A., Courtois, B.,
Notteghem, J. L., & Tharreau, D. (2008). A genome-wide meta-analysis of rice
blast resistance genes and quantitative trait loci provides new insights into
partial and complete resistance. Molecular plant-microbe interactions: MPMI, 21,
859-868. http://dx.doi.org/10.1094/MPMI-21-7-0859.
PMid:18533827
http://dx.doi.org/10.1094/MPMI-21-7-0859...
). Disease resistance is
controlled by one or two (Padmavathi et al.,
2005Padmavathi, G., Ram, T., Satyanarayana, K., & Mishra, B. (2005).
Identification of blast (Magnaporthe grisea) resistance genes
in rice. Current Science, 88, 628-630.; Sharma et al., 2007Sharma, R. C., Shrestha, S. M., & Pandey, M. P. (2007).
Inheritance of blast resistance and associated microsatellite markers in rice
cultivar ‘Laxmi’. Journal of Phytopathology, 155, 749-753.
http://dx.doi.org/10.1111/j.1439-0434.2007.01298.x.
http://dx.doi.org/10.1111/j.1439-0434.20...
), three
(Mohanty & Gangopadhyay, 1982Mohanty, C. R., & Gangopadhyay, S. (1982). Testing of blast
resistance in F rice seedlings in different doses of nitrogen and seasons.
2Annals of the Phytopathological Society of Japan, 48, 648-658.
http://dx.doi.org/10.3186/jjphytopath.48.648.
http://dx.doi.org/10.3186/jjphytopath.48...
) or
more pair of genes (Flores-Gaxiola et al.,
1983Flores-Gaxiola, J. A., Nuque, F. L., Crill, J. P., & Khush, G.
S. (1983). Inheritance of blast Pyricularia oryzae resistance
in rice. International Rice Research Newsletter, 8, 5-6.). The traditional rice cultivars have one or two dominant resistance
genes, which are effective against each fungal isolate (Mackill et al., 1985Mackill, D. J., Bonman, J. M., Suh, H. S., & Srilingam, R.
(1985). Genes for resistance to Philippine isolates of the rice blast pathogen.
Rice Genetics Newsletter, 2, 80-81.).
Normally, DNA markers are used to detect resistance genes (Ballini et al., 2008Ballini, E., Morel, J. B., Droc, G., Price, A., Courtois, B.,
Notteghem, J. L., & Tharreau, D. (2008). A genome-wide meta-analysis of rice
blast resistance genes and quantitative trait loci provides new insights into
partial and complete resistance. Molecular plant-microbe interactions: MPMI, 21,
859-868. http://dx.doi.org/10.1094/MPMI-21-7-0859.
PMid:18533827
http://dx.doi.org/10.1094/MPMI-21-7-0859...
). Identification of resistance genes in
genetically diversified rice material is important for identification of new sources
of blast resistance. Pongsu Seribu 1 is a Malaysian traditional rice variety which
is resistant to blast diseases, can be used as a blast resistant donor in rice
breeding programs. It has mid-late maturity, tall plant stature with short grain
type, developed by Malaysian Agricultural Research and Development Institute
(MARDI). The commercial Malaysian indica rice variety MR219 has
been classified as highly productive (Fasahat et
al., 2012Fasahat, P., Kharidah, M., Aminah, A., & Ratnam, W. (2012).
Proximate nutritional composition and antioxidant properties of Oryza
rufipogon, a wild rice collected from Malaysia compared to
cultivated rice, MR219. Australasian Journal of Crop Science, 6,
1502-1507.) but became susceptible to blast. Grain weight is as high as
28–30 mg, and 200 grains/panicle (Alias,
2002Alias, I. (2002). MR219, a new high-yielding rice variety with
yields of more than 10 mt/ha. Taipei: Food and Fertilizer Technology
Center.). A short maturation period of 105–111 days is the additional good
feature of this variety. The aims of this study were to know the inheritance pattern
of blast resistance in BC2F1 rice population against pathotype
P7.2 isolate and to identify microsatellite markers linked to blast resistance.
Inheritance of this highly effective source of blast resistance Pongsu Seribu 1
should determine for its efficient use in the rice-breeding programs targeted for
improving blast resistance.
2 MATERIAL AND METHOD
MR219 is a high yielding, good eating quality and wide adaptability rice variety. Unfortunately, this cultivar is very susceptible to blast (Figure 1). The MR219-rice cultivar was used as the recurrent parent while the cultivar Pongsu Seribu 1(PS1) was used as donor for the blast resistance (Figure 2). Among the F1 plants, two heterozygous F1 plants were selected and backcrossed with “MR219” to generate the BC1F1s. In the BC1F1 plants, marker-assisted foreground selection was carried out and the markers RM6836 and RM8225 showed heterozygous plants. The heterozygous plants were used to estimate the recurrent genome recovery using background marker. Out of 70-polymorphic microsatellite background markers, at least four SSR background markers per rice chromosome were used for analysis of recurrent genome recovery in each generation. The highest recurrent genome recovery plants were subjected to phenotypic selection. The best four plants (i.e those that have phenotypically resemblance to the recurrent parent with maximum recurrent genome recovery) were backcrossed with MR219 to develop the BC2F1seeds. The BC2F1 plants were inoculated with the most virulent pathotype P7.2 and also subjected to foreground selection followed by phenotypic selection to identify best plants heterozygous for blast resistance with maximum recovery for recurrent parent genome (RPG).
Rice cultivars Pongsu Seribu 1 (PS1) (resistant) and MR219 (susceptible) inoculated with pathotype 7.2 of M. oryzae. Severe blast lesions were observed in MR219 and no lesions in PS1.
One of the most virulent Malaysian rice blast pathotype P7.2 of M. oryzae was collected from the Malaysian Agricultural Research and Development Institute (MARDI). Currently, this pathotype is the most virulent pathogen in Malaysia (Rahim et al., 2013Rahim, H. A., Bhuiyan, M. A. R., Saad, A., Azhar, M., & Wickneswari, R. (2013). Identification of virulent pathotypes causing rice blast disease ( population derived from Pongsu Seribu 2 × Mahshuri. Magnaporthe oryzae) and study on single nuclear gene inheritance of blast resistance in F2Australian Journal of Crop Science, 7, 1597-1605.). Potato dextrose agar (PDA) was used as a media for growing the selected pathotype P7.2 of M. oryzae. PDA was prepared by mixing 39g of PDA in 500 ml of distilled water and boiled for 30 minutes in order to dissolve properly. After that, distilled water up to 1.0 L was added into the solution and was autoclaved at 121 °C for 20 minutes. Before plating PDA media, 10 mg of streptomycin was added for every 250 ml media to avoid bacterial contamination. The solution was then poured in Petri dish under the laminar flow cabinet and sealed with parafilm to avoid contamination. The blast conidial suspensions were filtered through nylon gauze mesh and adjusted to a concentration of 1.5 × 105 conidia’s mL–1 by haemocytometer using deionized water. Before inoculation, 0.05% Tween 20 was added to the suspension to increase the adhesion of the spores to the plant leaves.
The BC2F1 seeds were soaked in water for one day and germinated
on moist Whatman filter paper in Petri dishes for 3 days in a 30 °C dark incubator.
The germinated seeds were transferred in plastic trays (60 cm × 60 cm x 50 cm)
containing 15 kg of soil with NPK (10 g of 15:15:15) as described by Prabhu et al. (2003)Prabhu, A. S., Castro, E. M., Araujo, L. G., & Berni, R. F.
(2003). Resistance spectra of six elite breeding lines of upland rice to
Pyricularia grisea.Pesquisa Agropecuaria Brasileira, 38,
203-210. http://dx.doi.org/10.1590/S0100-204X2003000200006.
http://dx.doi.org/10.1590/S0100-204X2003...
. Plants were grown in a
glasshouse at 25-30 °C for 3 weeks, until they were at the four-leaf stage. A total
of 333 BC2F1 seedlings were inoculated with highly virulent
pathotype P7.2 of M. oryzae to investigate the segregation patterns
of blast resistance phenotypically. Twenty one-day-old plants were inoculated by
spraying with aqueous spore suspension onto the leaves until run-off. The relative
humidity (RH) was maintained at above 90% by covering them with black netting, as
well as watering them two to three times during the daytime. Disease scoring was
carried out nine (9) days after inoculation based on the Standard Evaluation System
(SES) of the International Rice Research Institute (IRRI, 1996International Rice Research Institute – IRRI1996Standard Evaluation
System for Rice4thedManilaIRRI). The blast lesion degrees (BLD) were scored using a scale
0-9 as follows: 0= no evidence of infection; 1= brown specks(<0.5 mm in
diameter); 2= brown lesions of 0.5–1 mm in diameter; 3= 1-3 mm in diameter with gray
centers and brown margins; 4= typical spindle-shaped blast lesion 3 mm or longer,
less than 4% of the leaf area infected; 5= typical blast lesion, 3 mm or longer in
diameter, infected 4-10% leaf area; 6= typical blast lesions, 3 mm or longer in
diameter, infected 11-25% leaf area; 7= typical blast lesions, 3 mm or longer in
diameter, infected 26-50% leaf area; 8= typical blast lesions, 3 mm or longer,
infected 51-75% leaf area; and 9= typical blast lesions, 3 mm or longer, infected
more than 75% leaf area. In the case of single-gene model analysis, the rice plants
showing lesion types 0 to 3 were considered as resistant and the plants showing
lesions type 4 and above were considered to be susceptible to the selected pathotype
P7.2 in BC2F1 population. Plant disease reaction was
categorized according to Singh et al. (2012)Singh, A., Singh, V. K., Singh, S. P., Pandian, R. T. P., Ellur, R.
K., Singh, D., Bhowmick, P. K., Gopala Krishnan, S., Nagarajan, M., Vinod, K.
K., Singh, U. D., Prabhu, K. V., Sharma, T. R., Mohapatra, T., & Singh, A.
K. (2012). Molecular breeding for the development of multiple disease resistance
in Basmati rice. AoB PLANTS, 2012, 1-13.
http://dx.doi.org/10.1093/aobpla/pls029. PMid:23125910
http://dx.doi.org/10.1093/aobpla/pls029...
with some modification.
Total genomic DNA was extracted from fresh leaves of 4-week-old individual plants using the modified CTAB (hexadecyltrimethylammonium bromide) method as described by Doyle & Doyle, (1990)Doyle, J. J., & Doyle, J. L. (1990). Isolation of plant DNA from fresh tissue. Focus (San Francisco, Calif.), 12, 13-15.. DNA was quantified by using nano-drop spectrophotometry (ND1000 Spectrophotometer). The diluted DNA samples diluted with 1xTE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0) to get a concentration of 70 ng/μl and kept in the refrigerator at –20 °C for PCR analysis.
Total sixteen microsatellites with known chromosomal positions distributed on rice
chromosomes were selected from the Gramene database (www.gramene.org) related to blast resistance genes
(McCouch et al., 2002McCouch, S. R., Teytelman, L., Xu, Y., Lobos, K. B., Clare, K.,
Walton, M., Fu, B., Maghirang, R., Li, Z., Xing, Y., Zhang, Q., Kono, I., Yano,
M., Fjellstrom, R., DeClerck, G., Schneider, D., Cartinhour, S., Ware, D., &
Stein, L. (2002). Development of 2240 new SSR markers for rice. DNA Research, 9,
199-220. http://dx.doi.org/10.1093/dnares/9.6.199.
PMid:12597276
http://dx.doi.org/10.1093/dnares/9.6.199...
; Temnykh et al., 2000Temnykh, S., Park, W. D., Ayers, N., Cartinhour, S., Hauck, N.,
Lipovich, L., Cho, Y. G., Ishii, T., & McCouch, S. R. (2000). Mapping and
genome organization of microsatellite sequences in rice ( L.). Oryza
sativaTheoretical and Applied Genetics, 100, 697-712.
http://dx.doi.org/10.1007/s001220051342.
http://dx.doi.org/10.1007/s001220051342...
). Parental lines were
used to identify polymorphic markers related to rice blast resistance gene. PCR
reactions with microsatellite markers were carried out in 15-μl reactions containing
1 μl (70 ng) of genomic DNA, 1.0 μM of each primer, 7.4 μl master mix and 4.6 μl
nuclease-free water. PCR amplification was carried out in a thermocycler
(T100TM, Bio-Rad) using an initial denaturation at 94 °C for 5
minutes followed by 35 cycles of denaturation at 94 °C for 30 seconds, 55 °C for 30
seconds, 72 °C for 30 seconds, the final extension at 72 °C for 5 minutes, followed
by rapid cooling to 4 °C prior to analysis. For electrophoresis, 3.0%
metaphorTM agarose (Lonza) gel was prepared containing 1μl Midori
green in 1X TBE buffer (0.05 M Tris, 0.05 M boric acid, 1 mM EDTA, pH 8.0). The gel
was run at constant voltage of 80V for 80 minutes and visualized using Molecular
Imager® (GelDocTM XR, Bio-Rad).
For marker genotyping, the clearly SSR bands were scored manually. The plants that showed a pattern similar to the susceptible parent alleles were scored as “aa” and those with a banding pattern similar to the resistant parent alleles were scored as “AA”, and the heterozygous plants were scored as “Aa”.
Chi-square (χ2) test was performed to test the goodness of fit of the
BC2F1 population for the phenotypic and marker data by
comparing the observed frequency distribution with an expected one. Chi-square
analysis for goodness of fit to the backcross type of segregation was performed as
mentioned by Tartarini (1996)Tartarini, S. (1996). RAPD markers linked to the Vf gene for scab
resistance in apple. TAG. Theoretical and applied genetics. Theoretische und
angewandte Genetik, 92, 803-810. http://dx.doi.org/10.1007/BF00221891.
PMid:24166544
http://dx.doi.org/10.1007/BF00221891...
. The
chi-square analysis for the genotypic and phenotypic ratio was calculated by using
the formula, χ2 = (O - E)2 / E, where O is the observed value,
and E is the expected value. For the single-gene model, the chi-square value was
considered as significant (p≤0.05) if its value was higher than 3.84. Frequency of
disease reaction trait was analyzed using SPSS ver. 16.0. Single-marker analysis was
carried out according to Divya et al. (2014)Divya, B., Robin, S., Rabindran, R., Senthil, S., Raveendran, M.,
& John Joel, A. (2014). Marker assisted backcross breeding approach to
improve blast resistance in Indian rice (. Oryza sativa)
variety ADT43Euphytica, 200, 61-77.
http://dx.doi.org/10.1007/s10681-014-1146-9.
http://dx.doi.org/10.1007/s10681-014-114...
to know the association between the markers and trait of blast incidence.
3 RESULTS AND DISCUSSION
The frequency distribution of blast disease evaluation for the trait of the blast
lesions degree (BLD) is shown in Figure 3. The
susceptible parent MR219 showed highly susceptible reaction with lesions type 5 to 7
score; whereas the parent Pongsu Seribu 1 was found to be resistant producing
lesions type 0 to 2 under artificial inoculation in the glasshouse (Figures 1-3). Among the 333 BC2F1 plants, 159 plants showed
resistant reaction and 174 plants showed susceptible reaction (Table 1). The observed frequencies, when
tested for goodness of fit with chi-square (χ2) test for single-gene
model, showed goodness of fit (P = 0.4463) to the expected segregation testcross
ratio (1:1) (Table 1). Therefore, resistance
to blast pathotype P7.2 in Pongsu Seribu 1 is most likely controlled by a single
dominant gene. The testcross progeny phenotypically segregated into a ratio of
1R:1S. Present studies are in agreement with findings of Bhatt et al. (1994)Bhatt, J. C., Singh, R. A., Mani, S. C., & Shoran, J. (1994).
Inheritance of blast resistance in rice. Indian Journal of Genetics, 54,
142-148., Mackill
& Bonman (1992)Mackill, D. J., & Bonman, J. M. (1992). Inheritance of blast
resistance in near-isogenic lines of rice. Phytopathology, 82, 746-749.
http://dx.doi.org/10.1094/Phyto-82-746.
http://dx.doi.org/10.1094/Phyto-82-746...
for inheritance of blast resistance in rice and Beyer et al. (2011)Beyer, B. M., Haley, S. D., Lapitan, N. L. V., Peng, J. H., &
Peairs, F. B. (2011). Inheritance of Russian wheat aphid resistance from
tetraploid wheat accessions during transfer to hexaploid wheat. Euphytica, 179,
247-255. http://dx.doi.org/10.1007/s10681-010-0299-4.
http://dx.doi.org/10.1007/s10681-010-029...
for inheritance of Russian
wheat aphid resistance in wheat. Several scientists reported that blast resistance
is governed by dominant genes (Bhatt et al.,
1994Bhatt, J. C., Singh, R. A., Mani, S. C., & Shoran, J. (1994).
Inheritance of blast resistance in rice. Indian Journal of Genetics, 54,
142-148.; Yamasak & Kiyosawa,
1966Yamasak, Y. I., & Kiyosawa, S. (1966). Studies on inheritance of
resistance of rice varieties to blast. I. Inheritance of resistance of Japanese
varieties to several strains of the fungus. Bulletin of the National Institute
of Agricultural Sciences, 14, 39-69.), but in a few cases the resistance was also reported due to
recessive genes (Bhatt et al., 1994Bhatt, J. C., Singh, R. A., Mani, S. C., & Shoran, J. (1994).
Inheritance of blast resistance in rice. Indian Journal of Genetics, 54,
142-148.; Yu et al., 1987Yu, H. Z., Mackill, D. J., & Bonman, J. M. (1987). Inheritance
of resistance to blast in some traditional and improved rice cultivars.
Phytopathology, 77, 323-326.
http://dx.doi.org/10.1094/Phyto-77-323.
http://dx.doi.org/10.1094/Phyto-77-323...
). This situation is in
agreement with the statement that the ability of a plant to express resistance is
also dependent on the genotype of the pathogen. A rice plant cannot be resistant to
an isolate of M. oryzae unless the pathogen has a gene that makes
it virulent to the rice plant. An isolate of M. oryzae cannot be
avirulent on the rice plant unless the rice plant has genes that make it resistant
to that isolate (Ellingboe & Chao,
1994Ellingboe, A. H., & Chao, C. C. T. (1994). Genetic interactions
in Magnaporthe grisea that affect cultivar specific
avirulence/virulence in rice. In R. S. Zeigler, S. A. Leong, & P. S. Teng
(Eds.), Rice blast disease (p. 51-64). Wallingford: CAB
International.). The findings of monogenic inheritance of a dominant nature resistance
are in agreement with the results of several scientists (Orellana et al., 1980Orellana, P., Martinez, J., & Hernandez, A. (1980). Some aspects
of the inheritance of resistance of rice () to Pyricularia oryzae. Oryza
sativaCiencia Y Ticnica en la Agricultura Arroz, 3,
23-33.; Xue
& Chen, 1987Xue, Q. Z., & Chen, H. S. (1987). Genetic study of disease
resistance in Dan 209, a rice cultivar bred by anther culture. Acta Genetics
Sinica, 14, 349-354.). The expression of the gene depends on the virulent
gene(s) present in the fungus. Previous studies showed resistance to blast is
governed either by a single gene or a polygenic system, depending on the genotypes
or cultivars, as well as their specificity to M. oryzae isolates,
where resistance to blast disease is host specific and effective against only
specific strains of M. oryzae (Zhou et al., 2007Zhou, E., Jia, Y., Singh, P., Correll, J. C., & Lee, F. N.
(2007). Instability of the Magnaporthe oryzae avirulence gene AVR-Pita alters
virulence. Fungal genetics and biology: FG & B, 44, 1024-1034.
http://dx.doi.org/10.1016/j.fgb.2007.02.003. PMid:17387027
http://dx.doi.org/10.1016/j.fgb.2007.02....
). However, studies conducted in IRRI revealed that
most of the traditional varieties have one or two dominant genes (Mackill et al., 1985Mackill, D. J., Bonman, J. M., Suh, H. S., & Srilingam, R.
(1985). Genes for resistance to Philippine isolates of the rice blast pathogen.
Rice Genetics Newsletter, 2, 80-81.). Our result is in
agreement with a blast research done at IRRI in the Philippines, which indicated
that one or two dominant genes present in the cultivars confer complete resistance
against each fungal isolate (Yu et al.,
1987Yu, H. Z., Mackill, D. J., & Bonman, J. M. (1987). Inheritance
of resistance to blast in some traditional and improved rice cultivars.
Phytopathology, 77, 323-326.
http://dx.doi.org/10.1094/Phyto-77-323.
http://dx.doi.org/10.1094/Phyto-77-323...
).
Frequency distribution of blast lesions degree (BLD) in BC2F1 population inoculated with rice blast pathotype P7.2. The mean scores of two parents are indicated by arrows.
Phenotypic segregation of blast resistance in BC2F1 population obtained from a cross between rice cultivars MR219 × Pongsu Seribu 1 inoculated with pathotype P7.2 of M. oryzae
A total of 16 polymorphic markers were found as the linked marker for blast
resistance (Table 2). All linked markers
were tested in F1 population. In BC1F1 generation,
two markers (RM6836 and RM8225 markers) showed heterozygous plants. Using other
foreground markers none of plants were found as heterozygous condition which
indicates that some blast resistant gene disappeared due to the backcrossed with
MR219. This statement is more coincide with the findings of Suh et al. (2009)Suh, J. P., Roh, J. H., Cho, Y. C., Han, S. S., Kim, Y. G., &
Jena, K. K. (2009). The pi40 gene for durable resistance to rice blast and
molecular analysis of pi40-advanced backcross breeding lines. Phytopathology,
99, 243-250. http://dx.doi.org/10.1094/PHYTO-99-3-0243.
PMid:19203276
http://dx.doi.org/10.1094/PHYTO-99-3-024...
who found that some genes were lost during
the recombination process in segregating generations of advanced backcross lines.
The two polymorphic (RM6836 -238bp and RM8225 -212bp) linked markers were used to
evaluate BC2F1 progenies. These two markers located on
chromosome 6 of rice showed linkage with resistance and susceptibility in
BC2F1 progeny. The banding patterns of two polymorphic
markers RM6836 and RM8225 linked with Pi genes in F1 and
BC2F1 population for 14 samples along with two parents are
shown in Figure 4 and Figure 5, respectively. The position of RM6836 (54.3 cM) and
RM8225 (54.1 cM) markers are shown in Figure
6. These two markers are 0.2 cM apart from each other on chromosome 6 of
rice. Fjellstrom et al. (2006)Fjellstrom, R., McClung, A. M., & Shank, A. R. (2006). SSR
markers closely linked to the locus are useful for selection of blast resistance
in a broad array of rice germplasm. Pi-zMolecular Breeding, 17,
149-157. http://dx.doi.org/10.1007/s11032-005-4735-4.
http://dx.doi.org/10.1007/s11032-005-473...
and Rathour et al. (2008)Rathour, R., Chopra, M., & Sharma, T. R. (2008). Development and
validation of microsatellite markers linked to the rice blast resistance gene of
Fukunishiki and Zenith. PizEuphytica, 163, 275-282.
http://dx.doi.org/10.1007/s10681-008-9646-0.
http://dx.doi.org/10.1007/s10681-008-964...
mentioned that markers
RM8225 and RM6836 are tightly linked with Piz gene located on
chromosome 6, whereas Rathour et al. (2008)Rathour, R., Chopra, M., & Sharma, T. R. (2008). Development and
validation of microsatellite markers linked to the rice blast resistance gene of
Fukunishiki and Zenith. PizEuphytica, 163, 275-282.
http://dx.doi.org/10.1007/s10681-008-9646-0.
http://dx.doi.org/10.1007/s10681-008-964...
found that these two markers located at distance of 1.2-4.5 cM from the
gene. Results indicate that individuals of the
BC2F1 population (derived from Pongsu Seribu 1) had the
alleles linked with these two microsatellite markers resistant against pathotype
P7.2 of M. oryzae. This finding has potential use in
marker-assisted selection to develop rice cultivars with blast resistance genes in
rice breeding programs. Because these markers had high-selection accuracy for
resistant plant sources, they can be used in MAS for the resistant gene.
Genotyping with markers RM6836 and RM8225 linked to blast resistance genes in F1 population of rice derived from MR219 × Pongsu Seribu 1 (PS1). Running on 3% metaphor agarose gel stained with midori green, only 14 samples plus the two parents for each marker are shown (M=50 bp ladder).
Genotyping with markers RM6836 and RM8225 linked to blast resistance genes in BC2F1 population of rice derived from MR219 × Pongsu Seribu 1 (PS1). Running on 3% metaphor agarose gel stained with midori green, only 14 samples plus the two parents for each marker are shown (M=50 bp ladder).
A total of 333 plants of BC2F1 population were evaluated with the linked markers. The observed segregation ratio for resistance and susceptibility in BC2F1 lines for 16 polymorphic microsatellite markers is shown in table 3. The chi-square (χ2) analysis for RM6836 and RM8225 showed a good fit to the expected testcross ratio (1:1) for a single-gene model (d.f. = 1.0, p>0.05) in BC2F1 population (Table 3). The rest of the markers did not fit the expected segregating Mendelian ratios. Results indicate that RM6836 and RM8225 have an association with blast resistance gene against to pathotype P7.2 of M. oryzae in rice.
Marker segregation analysis in BC2F1 population derived from a cross between rice varieties MR219 × Pongsu Seribu 1
The segregation ratio was not in agreement with the expected Mendelian ratio for other polymorphic markers in BC2F1 population. This is due to the fact that, none of the plants found as heterozygous condition using other foreground markers. In chi-square analyses, two microsatellite markers showed an expected testcross segregation ratio of 1:1, inherited in simple Mendelian fashion. Phenotypic data for disease reaction of resistance and susceptibility to blast pathotype P7.2 also segregated in 1R:1S ratio in the BC2F1 population. So, we can conclude that resistance to blast pathotype P7.2 in Pongsu Seribu 1 is controlled by a single dominant gene. The plants resistant to blast pathotype P7.2 from BC2F1 lines had genotypes with microsatellite markers RM8225 and RM6836, these markers could be used for marker-assisted selection. The existence of Pongsu Seribu 1 with individual blast resistance genes provides a powerful tool for future studies on the rice blast disease. The virulence patterns of blast races (pathotype P7.2) will be easier to study with lines possessing known resistance genes. Moreover, it should be easier to identify additional resistance genes. This blast resistant Pongsu Seribu 1 is currently being used to tag the resistance genes using microsatellite markers. This information can be useful in practical breeding programs as well as in the eventual cloning of the resistance genes.
Marker trait association was performed using SPSS ver. 16.0 software to identify the
association of resistance component trait with linked polymorphic markers of the
blast resistance gene. The genotypic segregation data set of the linked
microsatellite markers generated from the BC2F1 population
combined with phenotypic segregation data for blast resistance trait. This data was
subjected to linear model regression analysis and significance of R2 was
identified using F value comparison. The marker RM8225 and RM6836 showed significant
R2 values higher than 10 for the trait of the blast lesions degree
(BLD) (Table 4). We conclude that RM6836 and
RM8225 are two linked microsatellite markers to blast resistance locus in
BC2F1 generation. The deployment of major gene resistance
will minimize selection pressure and thereby prevent evolution of resistance in the
pathogen population (Bonman et al., 1992Bonman, J. M., Khush, G. S., & Nelson, R. J. (1992). Breeding
rice for resistance to pests. Annual Review of Phytopathology, 30, 507-528.
http://dx.doi.org/10.1146/annurev.py.30.090192.002451.
http://dx.doi.org/10.1146/annurev.py.30....
).
This approach will help breeders to expedite breeding research in crops by enabling
a selection based on the genotype rather than on the phenotype. The markers reported
here provide rice breeders and geneticists a valuable tool for marker-aided
selection of a disease resistance gene.
ACKNOWLEDGEMENTS
The authors would like to acknowledge Long-term Research Grant Scheme (LRGS), Food Security Project, Ministry of Education, Malaysia, for the financial support to conduct research on rice breeding and the Malaysian Rice Research Centre, MARDI, for helpful discussions on this research.
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Publication Dates
-
Publication in this collection
Mar 2015
History
-
Received
11 Sept 2014 -
Accepted
09 Oct 2014