Pod borer, caused by Helicoverpa armigera, is an important agronomic pest of pigeon pea. Genetic analysis for resistance to H. armigera in an interspecific F2 population and a set of F3 families both derived from a cross between C. cajan cv. ICP-26 (susceptible) and C. scarabaeoides acc. ICPW-94 (Resistant) revealed that the resistance is controlled by a dominant allele at a single locus. Pod borer resistance is associated with non glandular short trichomes, which is also governed by a dominant allele at a single locus. The resistance locus (PPB1) was mapped by linkage analysis with 32 random amplified polymorphic DNA (RAPD) loci, and six inter simple sequence repeat (ISSR) loci segregating among the F2 population. The PPB1 locus was mapped to the linkage group 2 (LG-2) and was flanked by ISSR marker loci UBC8731270 (15.9 cM), and the non glandular short trichome (NGST) locus (12.0 cM) and OPA18565 (26.3 cM). The presence of only few double recombinants between UBC8731270 and NGST (4.31%) and PPB1 and OPA18565 (10.34%) in the F2 population indicated that the simultaneous use of both the markers along with morphological trait NGST would be useful in introgression of pod borer resistance gene into C. cajan background and pigeonpea breeding programme.
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Pigeonpea is the second important grain legume crop next to chickpea growing predominantly in the subtropical countries of the world including India. Among the countries growing this crop, India has the largest area (3.53 million hectares) under pigeonpea cultivation, and also contributes a major share about 75% of world production with an average yield of 0.78 tons/hectare (Varshney et al., 2010). Since this is a crop of warm humid tropical environments conducive to the growth and multiplication of various insects and pests, the yield ceiling was constrained with several biotic stresses. Among the biotic stresses, insect pests feeding on flowers, pods, and seeds are the most important biotic constraint affecting pigeonpea yields (Gnanesh et al., 2011; Grover and Pental, 2003).
The pod borer complex comprises of Helicoverpa armigera (Pod borer), Maruca testulalis (Spotted pod borer), Lampides boeticus (Blue butter-fly), Exelastis atomosa (Plume moth) and Melanogromyza obtuse (Pod fly) causes 30-80% yield losses (Sharma et al. 2010). These pests reduce yield of 2.5 million tons per annum worth more than 3750 million rupees per year (Banu et al., 2005). Among these pod destructive pests the loss (70-80%) caused by lepidopteran pest H. armigera is the main constraint for yield in pigeonpea (Banu et al., 2005; Saxena, 2008). Again, most or often this loss was well supplemented by M. obtuse (70-80%), L. boeticus (4-10%) and E. atomosa (5-10%) for yield loss in pigeonpea (Sharma et al., 2010). The involvement of these polyphagus pests in pod feeding led to the pod borer became a complex problem in pigeonpea. Chemicals and pesticides are partly effective to control pod borer problem, ecologically harmful and financially not viable. Again these uses of pesticides have negative impact on environmental pollutions. The best approach to overcome this problem is to develop a resistance cultivar, and the host plant resistance provides an efficient, economical and safe means of crop protection against these pod borers.
The source of host resistance to H. armigera is not observed in the primary gene pool of pigeonpea. Hence, the genetic control of resistance to this pest has been studied in pigeonpea using interspecific mapping population (Verulkar et al., 1997; Aruna et al., 2005) raised by involving C. cajan and C. scarabaeoides. All the studies reported that, resistance to H. armigera (Pod borer) is controlled by a dominant allele of a single gene. Again, it was also reported that low density of glandular long and high density of non glandular short trichomes is associated with the pod borer resistance (Aruna et al., 2005).
The advent of molecular markers and its use in genetic linkage analysis is highly useful for mapping, monitoring and cloning of agro-economic genes (Varshney et al., 2005 and Kole and Gupta, 2004), and finally the availability of a tightly linked marker for the pave the way for introgressive marker assisted breeding. Identification of a molecular marker associated with pod borer resistance would allow rapid screening of cultivars, advanced breeding lines and segregating populations at seedling stage. There is no report available for the identification of molecular marker associated with pod borer and its allied traits or localization of any corresponding gene loci on linkage map. Only few reports pertaining to identification of simple inherited trait (plant type, fusarium wilt and sterility mosaic diseases) specific markers including RAPD, AFLP and SSR were available in pigeonpea (Kotresh et al., 2006; Ganapathy et al., 2009; Dhansekar et al., 2010). More recently Gnanesh et al. (2011) established an intraspecific linkage map in pigeonpea and locate the QTLs controlling SMD resistance for different isolates.
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The present study was undertaken because of the importance of tightly linked flanking markers in designing marker-assisted selection schemes for the efficient selection of pod borer resistant lines. In this communication, the identification of flanking markers to pod borer resistance locus employing RAPD and ISSR markers and interspecific F2 mapping population is reported.
2. Material and method:
Mapping population development:
Single plant of C. cajan cultivar ICP-26, exhibiting pod borer susceptibility and low density non glandular short trichome, and C. scarabaeoides accession ICPW-94, exhibiting pod borer resistance and high density non glandular short trichome, were self pollinated and cross hybridized. A single F1 hybrid plant was self pollinated to generate the F2 population (116 lines). F2 plants were individually self pollinated to develop 78 F3 families.
Evaluation of F2 and F3 lines for field resistance to pod borer:
In the first experiment, 116 F2 plants and 10 plants from each parent were evaluated for field resistance to pod borer. Twelve plants of the susceptible cultivar C. cajan cv. ICPL-87 has been planted as control. In the second experiment, the parental lines and 78 F3 families were assessed for field resistance to pod borer. All the pods of individual plants were examined for the presence of exit holes at an interval of every seven days. The evaluation entries were then scored on IP scale of 1-9, where 1 = completely resistance (No damage) and 9 = highly susceptible (≥72% pod damage). A rating scale of 1 to 3 was considered as resistant and from 4 to 9 as susceptible.
The presence of non glandular short trichome on the pod surface was observed for both the parents, F1 plant, F2 lines and plants of F3 families. The number of NGS trichome per mm2 were observed using scanning electron microscope (Zeiss 1450EP SEM, Germany), the count exceeding 50 were classified as dense while limiting within 30 as sparse.
Molecular Marker Analysis:
DNA isolated from young leaves of parents and F2 lines using the modified Cetyl-trimethyl-ammonium bromide (CTAB) protocol as described by Shivramakrishnan et al. (1997), and were diluted to a final concentration of 10 ng/μl using T10E1 (10mM Tris-HCl and 1mM EDTA) buffer. DNA concentrations and purity were measured using a UV-Vis spectrophotometer (UV -1; Thermo Fisher Scientific, USA) with T10E1 buffer as the blank. For confirmation, quantification of the DNA was accomplished by analysing the purified DNA on 0.8% (w/v) agarose gels along with diluted, uncut phage lambda DNA as standard.
Equal amounts of DNA from 10 highly resistant (IP score 1) and 10 highly susceptible (IP score of 7 to 9) F2 lines were pooled to constitute the resistant bulk (RB) and susceptible bulk (SB) respectively for carrying out the bulked segregant analysis (Michelmore et al., 1991).
For RAPD and ISSR analysis, PCR amplification of 25 ng of genomic DNA was carried out separately using each of 150 decamer oligonucleotide primers (Operon Technologies, Alameda, CA, USA) and nine ISSR primers from the set 100/9 (University of British Columbia, Vancouver, Canada) respectively. Each amplification reaction (25 µl) contained the 25 ng template DNA, 2.5 µl of 10X assay buffer [100 mM Tris-Cl, pH 8.3; 0.5 M KCl; 0.1% (w/v) gelatin], 1.5 mM MgCl2, 200 µM of each dNTP, 0.25 μM primer, 1.0 units Taq DNA polymerase (Bangalore Genei Pvt. Ltd., Bangalore, India). Amplification conditions for RAPD analysis include an initial denaturation step of 94°C for 5 min, followed by 45 cycles of denaturation at 94°C for 1 min, a primer annealing step at 37°C for 1 min, and an extension at 72°C for 2 min; then a final extension was carried out at 72-C for 5 min (Williams et al. 1990). For ISSR amplification following conditions were used - an initial denaturation step of 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 s, a primer annealing step at 40-60°C for 45 s, and an extension at 72°C for 2 min; then a final extension was carried out at 72°C for 5 min. Both RAPD and ISSR amplification was carried out in a thermal cycler (Veriti-96; Applied Biosystems, Foster City, USA), and PCR products were separated in 1.4% (w/v) agarose gel containing 0.5 μg ml-1 ethidium bromide in TAE buffer (40 mM Tris acetate, pH 8.0; 2 mM EDTA) at a constant 50 V. A gel loading buffer [20% (w/v) sucrose; 0.1 M EDTA, 1.0% (w/v) SDS; 0.25% (w/v) bromo-phenol blue; 0.25% (w/v) xylene cyanol] was used as a tracking dye. Amplified DNA fragments were visualised on the gel documentation system (Geldoc XR system, Biorad, USA) and photographed. The sizes of the amplified products were determined using 250 bp step up ladder (Bangalore Genei Pvt. Ltd.) as standard, and TL-120 software (Non-linear Dynamics, Total Lab Ltd., Newcastle Upon Tyne, UK).
Segregation and genetic linkage using RAPD and ISSR markers:
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RAPD and ISSR primers, which distinguished the parents (ICP-26 and ICPW-94), as well as the bulk samples (RB and SB), were used to screen individual F2 lines. The F2 lines having the `ICP-26' allele were scored as 'A' and those possessing ICPW-94 alleles were identified as 'B'. Linkage relationships among the segregating loci were established using the computer package MAPMAKER/EXP.3.0 (Lander et al., 1987). The linkage group was generated using all possible two point recombination estimates with a minimum LOD score of 4.0, and a recombination frequency < 0.30. The most probable marker order was determined using three point tests followed by the `order' command. The `ripple' command was used to verify the marker order. Map distance were expressed in centimorgans (cM) using the Kosambi map function (Kosambi 1944).
Segregation of the pod borer resistance and non glandular short trichome:
The parent C. cajan cv. ICP-26 was found to be highly susceptible to pod borer with an IP score of 8 and 9, whereas C. scarabaeoides acc. ICPW-94 was highly resistant with IP score of 1 and 2. All the F1 plants from the cross ICP-26 - ICPW-94 were pod borer resistant with IP score 1, suggesting that a dominant gene(s) governance. The frequency distribution of pod borer IP scores in the F2 population (116) gave a good fit for 3 (resistant-90):1 (susceptible-26) ratio (χ2 = 0.413, P-value = 0.520), indicating the oligogenic nature i.e. a single dominant gene might be responsible for resistance to pod borer (Fig.1a; Table-1). Phenotypic trait data for density of non glandular short (NGS) trichome on pod surface were recorded for the F2 population (116), and the frequency distribution showed 3 (89 dense) :1 (27 sparse) ratio (χ2 = 0.183, P-value = 0.668), indicating NGS trichome might also be under simple genetic control (Fig.1b; Table-1). In F3 generation, the segregation pattern of 78 F3 families for pod borer reaction was good fit for 1 (22 non segregating resistant): 2 (42 segregating): 1 (14 Non segregating susceptible) ratio (χ2 = 2.101, P-value = 0.349), indicating monogenic segregation pattern.
Identification of RAPD and ISSR markers linked to Pod borer Resistance:
In the present study, out of 150 random decamer primers (for RAPD) and 12 ISSR primers tested, 143 RAPD and nine ISSR primers distinguished the parental lines and therefore, were used as informative primers. However, only 18 RAPD and five ISSR primers differentiated the resistant bulk from the susceptible bulk (Table-2, Fig. 2). All these markers were found to be reproducible in three repeated RAPD and ISSR assays prior to single plant analysis. These 23 primers (RAPD and ISSR) generated 38 markers which include 32 RAPD and six ISSR markers (Table-2). 14 markers including 11 RAPD and three ISSR markers were linked to pod borer resistance in coupling phase, whereas 24 (21 RAPD and three ISSR) markers were in repulsion phase. Among the markers 18 (14 RAPD and four ISSR) were distorted for their segregation (Table-2). The segregation pattern of the marker UBC8731270 and OPA18565 in a set of F2 lines, together with the reaction to pod borer is given in Fig. 3.
Linkage analysis at a LOD of 4.0 and RF <0.3 grouped six of the 38 markers identified in the present study in one linkage group along with the locus conferring resistance to pod borer (PPB1) and NGS trichome (NGST). This linkage group distributed over 97.96 cM. Two markers, including one RAPD (OPB07860) and one ISSR (UBC8722000) showing segregation distortion remained in this linkage group. The PPB1 and NGST loci were loosely linked (12.0 cM), and flanked by UBC8731270 and OPA18565 at 15.9 cM and 14.3 cM, respectively (Fig.4). The NGST locus was occupied a position between PPB1 locus (12.0 cM) and OPB18565 (14.3 cM). The markers UBC8731270 and OPA18565 were linked to pod borer resistance in coupling and repulsion phase with recombination frequency of 0.154 and 0.169. But, presence of the NGST locus between PPB1 and OPA18565 led to increment of its map distance to 26.3cM away from PPB1 locus.
Considerable efforts have made to identify sources of resistance to pod borer and its allied traits in pigeonpea because of its importance throughout the world. The source of resistance to pod borer are only the allied species present in the secondary gene pool of the domesticated C. cajan (Verulkar et al., 1997; Sharma et al., 2003 and Aruna et al., 2005). The present pod borer resistant source is C. scarabaeoides acc. ICPW-94, the wild species. The present study showed that the pod borer resistance was under the control of single dominant gene. This result was also corroborated with the result of previous studies (Verulkar et al., 1997; Aruna et al., 2005) involving interspecific crosses.
Molecular mapping of the locus conferring resistance to pod borer present in accession ICPW-94 of C. scarabaeoides was accomplished by employing RAPD and ISSR markers in conjunction with bulked segregant analysis involving the interspecific F2 population. This locus, named PPB1, was flanked by one RAPD (OPA18565) and one ISSR marker (UBC8731270). Of the two markers, OPA18565 is linked in repulsion phase whereas UBC8731270 is linked in the coupling phase. Again the trait locus controlling non glandular short trichomes on pod surface was also loosely linked with PPB1. Based on the available literature this is the first report on the identification of DNA markers (RAPD and ISSR) linked to pod borer resistance in pigeonpea based on linkage analysis involving the interspecific F2 mapping population. Only few reports pertaining to identification of simple inherited trait specific DNA markers were available in pigeonpea. Kotresh et al. (2006) tagged a couple of RAPD markers to wilt susceptibility in pigeonpea. Recently, Ganapathy et al. (2009) tagged four AFLP markers to sterility mosaic disease and these markers were linked in coupling phase to susceptible dominant allele. Dhansekar et al. (2010) also identified RAPD markers linked to plant type genes in pigeonpea.
Absence of the 1270 bp DNA fragment amplified with UBC873, which is 15.9 cM away from PPB1 in segregants will show homozygosity for susceptibility in majority of F2 lines. Use of the repulsion phase marker OPA18565 will aid identification of the resistant homozygotes in which the amplified fragment will be absent. Considering that the double crossover frequency is lower than that of single crossovers between the PPB1 and either of the flanking markers, use of both markers simultaneously is expected to provide higher efficiency in selection. Therefore, the resistant and susceptible homozygotes can be distinguished from each other as well as from the resistant heterozygotes in early-segregating generations by employing both the linked markers and phenotyping of NGST. Further analysis of these markers at recombinant inbred level will substantiate its efficacy. However, C. scarabaeoides acc. ICPW-94 can be utilized effectively as a donor of pod borer resistance allele in the introgressive hybridization programme of pigeonpea through marker-aided selection.