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This article was downloaded by: [North Dakota State University]On: 09 October 2014, At: 17:28Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,UK
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Improvement of BacterialBlight Resistance in RiceCultivars Jyothi and IR50 viaMarker-Assisted BackcrossBreedingS. Bharathkumar a , R. S. David Paulraj a , P. V.Brindha a , S. Kavitha a & S. S. Gnanamanickam aa Centre for Advanced Studies in Botany , Universityof Madras , Guindy Campus, Chennai, 600 025, IndiaPublished online: 03 Oct 2008.
To cite this article: S. Bharathkumar , R. S. David Paulraj , P. V. Brindha , S. Kavitha& S. S. Gnanamanickam (2008) Improvement of Bacterial Blight Resistance in RiceCultivars Jyothi and IR50 via Marker-Assisted Backcross Breeding, Journal of CropImprovement, 21:1, 101-116, DOI: 10.1300/J411v21n01_07
To link to this article: http://dx.doi.org/10.1300/J411v21n01_07
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Improvement of Bacterial Blight Resistancein Rice Cultivars Jyothi and IR50
via Marker-Assisted Backcross Breeding
S. BharathkumarR. S. David Paulraj
P. V. BrindhaS. Kavitha
S. S. Gnanamanickam
ABSTRACT. In pathogen population analysis of 208 Xanthomonasoryzae pv. oryzae (Xoo) strains that were assembled from different partsof India, 21 pathotypes were identified on the basis of disease reactionson near-isogenic lines (NILs) and 13 pathotypes, on rice differentials.Rice cultivars, Jyothi and IR50, which are high yielding but highly proneto bacterial blight (BB) caused by pathogen populations of Xantho-monas oryzae pv. oryzae in India, were chosen. To improve the BBresistance of these two varieties, a pyramid line, NH56, containing fourR-genes, Xa4, xa5, xa13, and Xa21, was selected as the R-donor basedon resistance to existing pathogen population. The four R-genes were
S. Bharathkumar, R. S. David Paulraj, P. V. Brindha, S. Kavitha, and S. S.Gnanamanickam are affiliated with the Centre for Advanced Studies in Botany, Uni-versity of Madras, Guindy Campus, Chennai-600 025, India.
Present address for correspondence to: S. Bharathkumar, Plant Pathology Division,National Institute of Agricultural Science and Technology, 249 Seodun-dong, Suwon444707, Korea (E-mail: [email protected]).
The authors thank the Rockefeller Foundation and Bayer CropScience for financialsupport. The authors also thank the Director, CAS in Botany, University of Madras forthe encouragement and the Associate Director, RARS, Pattambi, Kerala, for providingnet-house and field facilities.
Journal of Crop Improvement, Vol. 21(1) (#41) 2008Available online at http://jcrip.haworthpress.com
© 2008 by The Haworth Press. All rights reserved.doi:10.1300/J411v21n01_07 101
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successfully transferred to cultivars through a traditional backcrossmethod and their presence was documented with marker-aided selec-tion (MAS). Thirty BC4F2 plants derived from JxNH56 (cv. Jyothi)and 45 BC4F2 plants derived from IR50xNH56 (cv. IR50) had all fourresistance genes (Xa4, xa5, xa13, and Xa21), which should be usefulresistance donors for breeding other BB-resistant elite indica varieties.doi:10.1300/J411v21n01_07 [Article copies available for a fee from TheHaworth Document Delivery Service: 1-800-HAWORTH. E-mail address:<[email protected]> Website: <http://www.HaworthPress.com>© 2008 by The Haworth Press. All rights reserved.]
KEYWORDS. Disease resistance, bacterial blight, RFLP, STS markers,traditional backcrossing, near-isogenic lines, Jyothi, IR50
INTRODUCTION
Rice (Oryza sativa L.) is the principal food crop of half of the world’spopulation and the present world population of 6.5 billion is likely toreach 8 billion by 2020. Thus, rice production must be increased by 50percent to satisfy the growing demand of ever-increasing population.To meet this goal, rice varieties with higher yield potential, durable re-sistance to diseases and insects and tolerance to abiotic stresses areneeded. Bacterial blight (BB), caused by Xanthomonas oryzae pv.oryzae (Xoo), has been one of the most serious diseases of rice, affect-ing production in irrigated and rain-fed lowland ecosystems throughoutAsia, northern Australia, mainland Africa, the southern part of theUnited States, and Latin America (Mew, 1987). Ou (1985) reported thatthe disease caused yield losses of 20-30%, but the intensive cultivationof high-yielding susceptible cultivars led to frequent appearance of newraces of the pathogen and reduced the effectiveness of resistant cultivarsin the field. During 1975, the disease had reached epidemic proportionsin Bihar and other neighboring states of India (Rangaswami, 1975). Thedisease is prevalent in almost all the states of the country (Sattar, 1996)and it is known to result in yield losses ranging from 74 to 81% in sus-ceptible cultivars (Veena et al., 1996).
To manage BB disease, more than 23 resistance genes have beenidentified so far (Zhang et al., 1998), two of which, Xa1 and Xa21,have been cloned from rice (Song et al., 1995; Yoshimura et al., 1998).Some of these resistance genes have been used in rice breeding for BB
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resistance, and many useful cultivars have been released in Asiancountries (Khush et al., 1989). In rice, single-gene resistance has beenthe primary means of control of BB, but unfortunately, due to continu-ous and large-scale use of single-gene resistance, there has been a shiftin the virulence pattern of the strains, leading to breakdown of resis-tance (Mew et al., 1992). For example, the highly resistant BB locus,Xa21, was found to confer resistance to all known Xoo races in Indiaand the Philippines (Ikeda et al., 1990; Khush et al., 1990). However,recent studies have shown that Nepalese strains were virulent on Rgene Xa21 present in rice line IRBB21 (Adhikari et al., 1999). In Indiaalso, a sub-population of Xoo virulent to rice line IRBB21was isolatedduring a BB epidemic that occurred in 1998 in Kerala (Venkatesanand Gnanamanickam, 1999). Hence, pyramiding of R-genes isthought to delay the virulence shifts. According to Kinoshita (1995),the pyramiding of multiple resistance genes into rice varieties is oneway to develop durable resistance to BB. Several workers have startedto pyramid lines with different R-gene combinations and these lineshave also been included in screening effective gene/gene combina-tions. Sridhar et al. (1999) screened 20 near-isogenic lines consistingof 11 lines with a single gene for resistance and the remaining ninepossessing various combinations of R-genes. Among those lines car-rying R-genes Xa1, Xa3, Xa4, Xa7, Xa10, Xa11, and Xa14 individu-ally were distinctly susceptible to BB. On the other hand, they observedthat xa8 and Xa21 were singly effective against bacterial blight, exhib-iting a score of 1. All the gene combinations were resistant, presum-ably because of interaction or quantitative complementation betweenresistance genes (Yoshimura et al., 1995; Huang et al., 1997). Similarstudies to select effective R-gene combinations were carried out else-where (Adhikari et al., 1999; Veracruz et al., 1999; Shanti et al., 2001;Singh et al., 2001). Different resistance genes often confer resistanceto different isolates, races or biotypes. Combining these resistancesbroadens the number of races or biotypes that a variety can resist(Mackill and Ni, 2001). Furthermore, combining major and minorgene resistances may lead to increased durability of resistance (Wanget al., 1994).
Marker-assisted selection allows the identification of plants withmultiple resistance genes in a population. Markers have been used topyramid several BB resistance genes. Yoshimura et al. (1995) usedmarkers to pyramid R-genes Xa10 + Xa4 and Xa4 + xa5. Huang et al.(1997) pyramided four resistance genes, Xa4, xa5, xa13, and Xa21, us-
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ing PCR-based markers. This four-gene pyramid was found to be effec-tive against much of the pathogen population. To facilitate selection ofprogenies containing more than one R-gene and with the advantage ofavailability of near-isogenic lines, several genes for BB resistancehave been tagged with RFLP and PCR-based markers (McCouch etal., 1991; Ronald and Tanskley, 1991; Ronald et al., 1992; Yoshimuraet al., 1992, 1995; Blair and McCouch, 1997). In rice breeding pro-grams at IRRI and national rice improvement programs in the Philip-pines, Indonesia and India, resistance genes Xa4, xa5, Xa7, and Xa21were targeted for transfer to commercially important rice varieties(Nelson et al., 1996). The genes xa5 and Xa7 were pyramided intoIR64 via MAS (Suwarno et al., 2000). Sanchez et al. (2000) trans-ferred three BB resistance genes into three susceptible rice lines pos-sessing desirable agronomic characters. Genes from rice cv. PR106,widely grown in Punjab, India, were introgressed with pyramided re-sistance genes xa5, xa13, and Xa21 from pyramid line IRBB62 usingMAS (Singh et al., 2001). Recently, major genes conferring diseaseresistance in several crop species have been mapped with linkedDNA markers, facilitating MAS for disease resistance in these crops(Melchinger, 1990; Penner et al., 1995; Miklas et al., 1996). Marker-assisted selection has been successfully used in selecting for resis-tance in the absence of pathogens (Melchinger, 1990), pyramidingmultiple genes for durable resistance against rice bacterial blight(Huang et al., 1997) and for development of multiple disease-resistantgermplasm (Kelly, 1995; Narayanan et al., 2002; 2004).
In this study, our objective is to show the successful improvement oftwo local elite rice cultivars (cv. Jyothi and IR50) for BB resistancethrough a traditional backcrossing method. We confirm the presence oftransferred R-genes with MAS.
MATERIALS AND METHODS
BB Pathogen Strains and Their Virulence Characteristics
A collection of BB-infected rice leaf samples from farmers’ fields aswell as research stations of major rice-growing areas of Haryana,Punjab, Uttaranchal, Uttar Pradesh, Bihar, West Bengal, Andhra Pradesh,Tamil Nadu, and Kerala states of India, was made. Five to six samplings
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were made from the four corners and areas infected in-between the cor-ners of the field. Bacterial blight pathogen strains of X. oryzae pv.oryzae were isolated from infected samples collected from differentstates of India. These Xoo strains were subjected to pathotype analysisin field plots at Regional Agricultural Research Station (RARS), Kerala,to evaluate the virulence spectrum of X.oryzae pv. oryzae on near-isogenic lines (NILs) (IRBB4, IRBB5, IRBB7, IRBB10, IRBB13,IRBB21, IRBB52, IRBB54, IRBB55, IRBB57, IRBB58, IRBB59,IRBB60), differential cultivars (DV85, Cas 209, IR20, IR8, IR24) andlocal varieties, cv. Jyothi and IR50. Plots of 3-by-1-m size were con-structed with levees. Ten plants from each rice line were planted 10 cmapart from each other in a row and row spacing was 15 cm. On the 45thday, pathogen inoculation was carried out with a bacterial suspension ofXoo adjusted to 0.1 OD (optical density) at 600 nm, which is equivalentto 106 cfu/mL (Kauffmann et al., 1973). Evaluation for resistance wasmonitored 15 d after inoculation by measuring BB lesion length (LL).Plants with LL of < 6 cm were scored as resistant and those with LL of> 6 cm were scored as susceptible.
Selection of Resistant Donor
To select a resistant donor for the breeding program, dominantR-genes in near-isogenic lines IRBB4 (Xa4), IRBB21 (Xa21) and a pyr-amid line NH56 (Xa4, xa5, xa13, and Xa21) were evaluated with the ex-isting pathogen strains prevalent in India. Cultivar IR24 was used as aBB-susceptible check. Disease reactions were documented 15 days af-ter inoculation and a BB-resistant, near-isogenic line was selected as theR-donor parent for backcrossing in a net-house experiment.
Backcross Breeding
To improve BB resistance in cv. Jyothi and IR50, introgression offour resistance genes (Xa4, xa5, xa13, and Xa21) in the pyramid lineNH56 was carried out in the net-house at RARS, Pattambi, Kerala. Inthe backcrossing, cv. Jyothi and IR50 were female parents and NH56was male parent, as shown in the protocols below:
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Marker-Assisted Selection (MAS)
Molecular markers closely linked to the R-genes were used to selectresistant progenies with all four R-genes. Both RFLP markers (for Xa4)and PCR-based STS markers (for R-genes xa5, xa13, and Xa21) wereused. Primers for PCR amplification were synthesized (Integrated DNATechnologies, Inc., IA, USA) following the primer sequences to mark-ers RG556, RG136 (Huang et al., 1997) and pTA248 (Chunwongse etal., 1993). The RFLP clone G181 was obtained as a gift from Dr. T.Sasaki, Rice Genome Project, Japan. DNA from leaf tissues of parentsand progenies was extracted following the method of Tai and Tanskley(1990).
The primer pair for Xa21 (pTA248 F-5�AGACGCGGAAGGGTGG-TTCCCGGA3�) and (R-5� AGACCGGTAATCGAAAGATGAAA3�)
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was previously designed from the sequence of a genomic clone RAPD248. The STS primers for xa5 (RG556 F-5�TAG CTC CTG CCG TGCTGT GC 3�) and (R-5� AAT ATT TCA GTG TGC ATC TC 3�) weredesigned based on end sequences of RFLP clones RG556 and RG207.The PCR products digested with DraI detected polymorphism betweenparents. The STS primers for xa13 (RG136 F-5’TCC CAG AAA GCTACT ACA GC 3�) and (R-5� GCA GAC TCC AGT TTG ACT TC 3�)were designed based on the most closely linked markers (Zhang et al.,1996). The amplified PCR products digested with HinfI detected poly-morphism between parents. The RFLP marker G181 present at a dis-tance of 1.7 cM from Xa4 on rice chromosome 11 was used to detect thepresence of the R-gene. The plasmid clone of G181 was transferred andmultiplied in Escherichia coli DH5a cells. Insert from the transformedplasmid was released using restriction enzyme PstI to check transfor-mation of clone G181. PCR amplification was done to multiply theclone using primers (952-1-5�AGC GGA TAA CAA TTT CAC ACAGG3�) and (953-1-GCA AAA TGT TGC ACT GAC CC3�). In restric-tion digestion of plant DNA to 3 μg of plant DNA, 1X BSA, 0.3U of en-zyme and 1X buffer were added and allowed to digest overnight at37ºC. Restriction enzymes BamHI, Bgl II, Dra I, EcoR I, EcoR V, HindIII, Nae I, Apa I, Xba I, Xho I, and kpn I were used to detect polymor-phism between the parental lines. The digested DNA was blotted.
RESULTS
The BB Pathogen Population
The resistance of NILs and rice differentials harboring single and twoR-genes ranged from resistance to susceptibility to pathogen strains(Xoo) in the field. The lines containing more than two genes were highlyresistant to all pathogen strains. Based on the disease reactions of NILs,21 virulence groups (pathotypes) were observed among the 208 patho-gen strains pathotyped. On rice differentials, 13 pathotypes were identi-fied.
Reactions of Rice Donors to Xanthomonas oryzae pv. oryzaeRaces and Local Strains
The donor, NH56, showed a high level of resistance to two races andall 140 Indian BB pathogen (Xoo) strains. The recurrent parents (Jyothi
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and IR50), inoculated with pathogen Races 1 and 6 and local strains ofXoo, were susceptible to Race 1, highly susceptible to Race 6 and alsosusceptible to local pathogen strains. The resistance of NILs, IRBB4(Xa4) and IRBB21 (Xa21), ranged from susceptible to highly suscepti-ble to Races 1 and 6 and to all local strains of Xoo. Some BB pathogenstrains of Xoo were virulent also to R-gene Xa21 (Table 1).
Marker-Assisted Selection for Presence of R-Gene in the Progenies
In each and every generation of the backcross, resistant progenieswere selected by phenotypic (Figure 1) and genotypic selection (Figure2a). After four BC generations, the selected BC4F1 plants for each of therecurrent parents were selfed once to produce BC4F2 progeny.
In MAS, 60 BC4F2 plants (cv. Jyothi) and 80 BC4F2 plants (cv. IR50)showing the parental phenotype and presumably possessing one to fourtarget resistance genes in various combinations were obtained through
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TABLE 1. Reaction of Parental Rice Cultivars to Two Races and Local Strainsof Bacterial Blight Pathogen Xanthomonas oryzae pv. oryzae
*-local strains that breakdown resistance of Xa21-gene; S = Susceptible; R = Re-sistant; HS = Highly susceptible
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their STS marker genotypes (Table 2). Of these, 30 BC4F2 plants fromJxNH56 (cv. Jyothi) and 45 BC4F2 plants from IR50xNH56 (cv. IR50)were derived via molecular analysis and the selected BC4F2 plants hadall four resistance genes (Xa4, xa5, xa13, and Xa21). The selectedBC4F2 plants, together with the parents, were genotyped for the STSmarkers linked to the four resistance genes (Table 2). Among the re-striction enzymes used for R-gene Xa4, EcoRI, DraI, and NaeI showedpolymporphism between parents for the presence of the R-gene (Figure2b). The parental characters (cv. Jyothi and IR50) of BC4 progenieswere more or less similar to the donor, NH56. Plant characters, such asnumber of productive tillers, plant height, culm length and panicle exer-tion, were similar to those of their parents.
DISCUSSION
This comprehensive study took into account the pathotypic and ge-netic diversity among populations of Xanthomonas oryzae pv.oryzaethat was prevalent in the country and linked the information on patho-gen diversity to BB-resistance breeding in India. In this approach, majorgenes (R-genes) of rice were implicated and pyramiding of 4 BB resis-tance genes via backcross breeding was shown to improve the BB resis-
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FIGURE 1. Elite rice cultivars IR50 and Jyothi (susceptible to BB) with an im-proved progeny of IR50 showing BB resistance (middle)
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tance in cultivar Jyothi and IR50 (Davidpaulraj 2003; Bharathkumar,2004).
Brindha (2002) analyzed populations of Xoo from Kerala. The pres-ent study has applied the same tools for assessment of pathotype and ge-netic diversity to a larger collection of X. oryzae pv. oryzae from outsideof Kerala. In 1997, DNA fingerprinting with different kinds of markersand microsatellites was used to analyze about 100 Indian strains of X.oryzae pv. oryzae. This work was carried out in collaboration with re-searchers at the National Chemical Laboratory, Pune, India (Rajebhosaleet al., 1997). Those efforts indicated the prevalence of a number ofpathotypes or races. From our previous studies and from the contribu-
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FIGURE 2b. Restriction polymorphisms between rice elite cultivars and pyra-mid line NH56 (Xa4 + xa5 + xa13 + Xa21) for RFLP probe G181.
FIGURE 2a. PCR analysis for parental polymorphism with primers for theR-genes Xa4, xa5, xa13, and Xa21
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Bharathkumar et al. 111
TABLE 2. Reaction of Selected BC4F2 Rice Plants Containing 1 to 4 BacterialBlight [BB; Xanthomonas oryzae pv. oryzae (Xoo)] Resistance Genes.
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tions that emerged from the Central Rice Research Institute (CRRI),Cuttack and the Centre for Cellular and Molecular Biology (CCMB) atHyderabad, we discovered that the Indian population of BB pathogenwas diverse. This study corroborates some of the previous information onmajor genetic groups of X. oryzae pv. oryzae prevalent in the country.
Results presented in Table 1 have been useful to identify 21 virulencegroups in the 208 pathogen strains that were pathotyped on the rice NILs.Data clearly revealed also that almost all the strains were able to over-come only those NILs that harbored a single R-gene for BB resistance,but not those NILs (IRBB52 to IRBB60) that harbored either 2 gene,3-gene or 4-gene combinations. The utility of the international rice differ-entials in identifying pathotypes/races of X. oryzae pv. oryzae was less ascompared with the rice NILs. Only 13 pathotypes could be identifiedfrom among the 208 pathogen strains. The valuable information thatemerged from the pathotyping was that resistance in rice cultivars thatcarried single R-genes was likely to breakdown in the field and that a pyr-amid of R-genes for BB resistance was likely to be more durable.
A gene pyramiding approach was followed to incorporate four Rgenes for BB in rice plants of two popular, high-yielding rice cultivars,cv. Jyothi and IR50. Although BB is known to be a severe rice produc-tion constraint in parts of Kerala, its management has mainly beenthrough breeding disease-tolerant cultivars, such as Jyothi and a host ofothers with high yield potential. In recent years, despite the fact thatJyothi and IR50 had gotten progressively more prone to the disease,farmers continued to plant Jyothi and IR50 as preferred cultivars.
To check if the cloned BB-resistance gene Xa21 (the first rice genethat was cloned by Song et al., 1995) alone was able to resist the patho-
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TABLE 2 (continued)
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gen strains prevalent in epidemic areas of Kerala and other regions ofIndia, inoculations were made. When the results indicated very clearlythat about 14% of the Kerala strains could breakdown Xa21 (Table 1), agene pyramid, rather than a single R-gene, was introgressed throughbackcross breeding. Resistant plants in the progeny were identified bypathogen inoculation (phenotypic evaluation) (Figure 1; Table 2).Therefore, a resistant donor, NH56, was identified after the inoculationassays. This rice line was not attacked by any of the 140 strains ofXanthomonas oryzae pv. oryzae. Most of these strains were fromKerala and their avirulence to NH56 indicated durability of resistancefor this rice-growing region of the country. Therefore, the 4-gene com-bination (xa4, xa5, xa13 + Xa21) pyramid carried by NH56 should theo-retically impart durable resistance to BB in Kerala and other regions inIndia. Backcross breeding strategies were devised to introgress thesegenes from NH56 into cv. Jyothi. Brindha (2002) and Davidpaulraj(2003) used MAS to locate BB-resistant plants in BCprogenies. In thepresent study, however, all BC (BC1to BC4) progenies were subjectedto a careful phenotypic evaluation with appropriate strains of the patho-gen (Xoo) and with markers. Improved IR50 and Jyothi seeds are beingdistributed by Kerala Agricultural University to rice farmers in Kerala.
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