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a National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan
b Dep. of Genetics, Univ. of Melbourne Parkvill,Victoria 3052, Australia
* Corresponding author (y_zafar{at}yahoo.com)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: CLCD, cotton leaf curl virus disease • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphism • PCR, polymerase chain reaction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CLCD is transmitted by white fly (Bemisia tabaci Gennadius) (Mansoor et al., 1993; Hameed et al., 1994) and is associated with variable geminiviruses (Zhou et al., 1998; Mansoor et al., 1999b; Sanz et al., 2000; Briddon et al., 2001). The disease is characterized by upward or downward curling, small and large vein thickening, and finally leaf enation (an outgrowth on the lower side of the leaves).
After the appearance of the disease in Pakistani cotton crop and more recently in Indian cotton, many projects were designed to improve the resistance of cotton to CLCD (Ali, 1999). Among those, a diverse genetic base of the cultivars grown in Pakistan was suggested as an important element in controlling the disease (Zhu et al., 2000); a narrow genetic base may predispose the crop to an epidemic (Holley and Goodman, 1989).
Restriction fragment length polymorphisms (RFLPs) have been applied to cotton species to study genetic diversity, population genetics, evolutionary history, and genome mapping (Shappley et al., 1996; Wang et al., 1995; Yu et al., 1997). For our purposes, this may not be a viable approach because the level of polymorphism in cotton is quite low compared with other plant taxa (Brubaker and Wendel, 2000). Moreover, the analysis is time consuming, requiring large quantities of DNA (2–10 µg) and radioactive material.
RAPD and amplified fragment length polymorphic (AFLP) markers have been used successfully to estimate genetic similarity and for cultivar analysis of various Australian cotton cultivars (Multani and Lyon, 1995), Pakistani cotton cultivars (Iqbal et al., 1997), and wild cotton species (Khan et al., 2000; Abdallah et al., 2001). The genetic distance obtained from RAPD markers of 16 U.S. cotton cultivars was compared with the taxonomic distances measured from morphological features. The classification of cultivars on the basis of the two methods produced similar results (Tatineni et al., 1996).
Most of the newly released disease resistant Pakistani cotton cultivars were developed by crossing exotic resistant parents (primary sources of resistance to CLCD) followed by selection of superior genotypes; a relatively small gene pool for all the Pakistani cotton cultivars was thus created (Iqbal et al., 2001). The tendency to use similar parents extensively in a breeding program has led to concern about the lack of genetic diversity (Fouilloux and Bannerot, 1988). This study was conducted to screen those newly released cotton cultivars for CLCD by conventional methods (field evaluation, whitefly-transmission, and grafting of the material) and by molecular diagnostic tools (multiplex PCR and dot blot hybridization). RAPD analysis was then conducted to assess genetic diversity and genetic relationship among the extremely resistant and resistant cotton lines. The information gathered should be useful in marker-assisted breeding as well as in genome mapping.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evaluation through Grafting
Two-month-old plants (40 of each genotype) were grafted with a highly infected bud by the T-shaped grafting method to ensure the provision of virus inoculum. After insertion, the bud was wrapped with a polyethylene strip to inhibit evaporation. The process was conducted preferentially in the evening. At maturity, disease severity was rated.
Examination by Whitefly-Transmission Studies
Infected cotton plants were kept in cages and inoculated by a large population of whiteflies. The exposure time was 5 d (Hashmi et al., 1993). At the second true leaf stage, the genotypes were confirmed for the absence of virus by polymerase chain reaction (PCR) using specific degenerate primers. The plants were sprayed with a pesticide to kill any adult whiteflies. Then the virus-free cotton plants were exposed to the viruliferous whiteflies. These plants were shifted to elevated temperature (40°C). After 7 wk of exposure, symptoms (vein thickening and curling) were observed.
Evaluation by DNA Diagnostic Test (Multiplex PCR)
Total genomic DNA of the whitefly-infected plants was extracted by CTAB (hexadecyltrimethylammonium bromide) method (Doyle and Doyle, 1987). The multiplex PCR analysis was carried out as described by Mansoor et al. (1999a). The multiplex PCR was repeated twice.
Dot-Blot Hybridization
For dot-blot hybridization, 10 µL of total genomic DNAs (approximately 5 µg) of all the genotypes were denatured by adding 10 µL of 1 M NaOH followed by incubation at 25°C for 15 to 20 min. The DNA samples were then neutralized by mixing 10 µL of 3 M sodium-acetate (pH 4.8). A vacuum manifold was used to spot the samples in duplicate on nylon membrane (Hybond N, Amersham Biosciences Corp., Buckinghamshire, UK). A PCR product (510 bp) was amplified from a cotton plant infected with CLCuV (a geminivirus) (Mansoor et al., 1999b) and was radioactively labeled by the oligo-labeling method with the Ready Prime DNA labeling kit (Amersham, UK). The membranes were placed in hybridization tubes and prehybridized for 2 h before addition of the denatured probe. The probe was hybridized overnight at 65°C in a hybridization oven. The blots were first washed with 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS) and secondly with 0.5x SSC, 0.1% SDS at 65°C for 30 min duration, respectively. The blots were also exposed to X-ray film at -70°C and developed after overnight exposure.
Disease Scoring
To assess the severity of the disease infection, the percentage of the total infected plants in each genotype was scored by a scale of 0 to 10 (10 = 100%, 9 = 90%, ... , 1 = 10% and 0 = no infection) for field evaluation, grafting, and whitefly-transmission studies, respectively. This method was less tedious and more accurate than the conventional method of scoring (0–6). However, positive (present) and negative (absent) was used to count for dot-blot hybridization and multiplex PCR analysis (Table 2)
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PCR with Random 10-mer Primers
For RAPD analysis, a total of 50 primers were used in a PCR reaction. These primers belonged to Operon kits OPA (20 primers), 10 primers of OPB series (OPB-7, OPB-8, OPB-9, OPB-10, OPB-12, OPB-13, OPB-14, OPB-15, OPB-19, and OPB-20), and OPJ (20 primers) (Operon Technologies, Inc., Alameda, CA). PCR was performed in volumes of 50 µL containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 100 µM each of dATP, dCTP, dGTP, dTTP, 0.2 µM primer, 0.001% (w/v) gelatin, 30 ng of genomic DNA, and 1 unit of Taq polymerase. Taq polymerase, together with buffer, MgCl2, dNTPs, and gelatin were from Perkin Elmer, Norwalk, CT. Amplification was performed with Perkin Elmer DNA thermal cycler 480 programmed for a first denaturation step of 5 min at 94°C followed by 40 cycles of 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C. The reaction was kept at 72°C for 10 min and then held at 4°C until the tubes were removed.
Scoring and Analysis of RAPD Data
Amplification products were analyzed by electrophoresis in 1.2% (w/v) agarose gels and were detected by staining the gel with ethidium bromide (10 ng/100 mL of agarose solution in TBE). All visible and unambiguously scorable fragments amplified by primers were scored under the heading of total scorable fragments. Amplification profiles of all the 20 cotton genotypes were compared with each other and bands of DNA fragments were scored as present (1) or absent (0). The data of the primers were used to estimate the similarity on the basis of the number of shared amplification products (Nei and Li, 1979). Similarity coefficients were utilized to generate a dendrogram by means of unweighted pair group method of arithmetic means (UPGMA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In multiplex PCR analysis, two products of 360 and 510 bp were detected in many cotton genotypes. The positive control in the multiplex analysis was the infected DNA of radish plant, Raphanus sativus L. (the most susceptible to the disease). Two DNA segments were amplified from the cotton varieties CIM-240, NF-801, CIM-435, BH-36, NIAB-Karishma, MNH-93, and S-12 and radish (positive control), while a DNA segment of 510 bp was amplified from the genomic DNA of FH-900, MNH-552, NIAB-98, BH-118, RH-500, VH-137, and CIM-482. The intensity of the amplified bands varied among the different cotton genotypes. No viral DNA was detected in the remaining cotton genotypes (Fig. 1a and b) . By dot-blot hybridization, the intensity of signal varied with different cotton genotypes. No signal was detected in the total genomic DNA of genotypes CIM-482, CIM-473, LRA-5166, Cedix, CP-15/2, CIM-443, FH-945, FH-930, Ravi, MNH-554, FH-900, FH-901, VH-137, CIM-448, MNH-552, CIM-1100, BH-118, NIAB-98, CIM-443, and RH-500. The dot-blot hybridization signals were detected in the genomic DNA of S-12, CIM-240, NF-801, CIM-435, BH-36, NIAB-Karishma, MNH-93, and radish (Fig. 2) .
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The distinction between the nonsusceptible (ER/resistant) and susceptible genotypes was very clear, but there were variations in the degree of susceptibility. The presence of such types of grades is very difficult to quantitate. According to Tarr (1951), the environment may affect plant resistance or the virus or both. Furthermore, it has been reported that environmental factors such as temperature and rainfall may change the incidence of the disease (Massey, 1934; El-Nur and Abu-Salih, 1966). Also, involvement of minor genes may cause variations in disease expression (Huchinson and Knight, 1950), and susceptible varieties with higher growth rates more easily escape CLCD than can slow growing genotypes (Ali, 1997). Finally, varieties resistant or tolerant to whitefly (vector of the virus) escape because whiteflies feed on them less frequently. Further work is needed to elucidate the causes of variation in disease expression among susceptible genotypes.
Molecular tests have been used to assess traces of geminiviruses. Genotypes such as FH-900, NIAB-98, NF-801, VH-137, RH-500, CIM-482, CIM-448, and MNH-552 were found resistant by conventional methods; however, viral DNA was detected by multiplex PCR. This technique was more sensitive than the other techniques (Mansoor et al., 1999b). Dot-blot hybridization was used as an additional method and as a check of multiplex PCR, the results of which can be compromised by compounds such as host plant polysaccharides and phenolics, which precipitate with DNA. In dot-blot hybridization, the intensity of the signal is directly proportional to the quantity of viral DNA; however, the technique can detect the presence of the virus only when the titre is high enough (Maule et al., 1983).
In this study, we wanted to use the molecular and conventional diagnostic methods to evaluate the cotton genotypes under highly stringent procedures. It is quite hard to define different levels of the resistance that is displayed by the plants to virus infection in discrete classes. The term "immunity" for host-plant resistance (HPR) was coined to address the failure of virus replication in the infected cell (Waterhouse et al., 2001). In resistant plants, it has been reported that replication occurs but virus movement out of infected cells is suppressed. However, in susceptible plants, the virus replicates and is translocated to other parts of the plant by phloem loading, which results in disease expression (Pappu et al., 1995). In the present study, the PCR-negative genotypes were called extremely resistant (ER) genotypes instead of immune because the multiplex analysis was conducted against two viruses (CLCuV Pak-1 and CLCuV Pak-2). The genotypes that were symptomless but harboring viral particles were recognized as resistant genotypes. By pooling the data, 20 ER and resistant genotypes were selected to study the genetic kinship and divergence.
In early 1970s, high-yielding tetraploid cotton varieties of American origin were introduced into Pakistan, and of these, the varieties that were better adapted were released directly for general cultivation. Those that were less adapted were crossed with local breeding lines. The same gene pool was used repeatedly and resulted in a narrow genetic base (Iqbal et al., 1997). In RAPD analysis, there was no single primer that could differentiate all genotypes. Genetic relatedness ranging from 81.51 to 93.41% was found among the elite Pakistani cotton cultivars that mostly had been released before the CLCD epidemic (Iqbal et al., 1997); the resistant cultivar CIM-1100 was the most genetically divergent (57.02% similar) among the tetraploids assessed in an earlier study (Iqbal et al., 1997). However, in the present study, CIM-1100 was quite genetically similar to the newly released CLCD resistant cultivars (86.23–92.85%). Moreover, CIM-448 is the sister line of CIM-1100. The newly released CLCD resistant cultivars were developed by a cross of either LRA-5166/CP-15/2 (primary source of resistance) or the locally adapted resistant cultivars (developed also from the same parental lines) with local genotypes and then selection that was comparable to that used to develop the adapted parents. Thus, the need to breed for CLCD resistance resulted in the loss of genetic diversity.
In the present study, 81.41 to 94.90% (with an average of 89.55%) genetic similarity was observed. Multani and Lyon (1995) studied a number of Australian cotton cultivars and found 92.1 to 98.9% genetic relatedness. Tatineni et al. (1996) assessed genetic diversity among 19 cotton genotypes with eight primers and compared the RAPD data with the taxonomic data. In their studies, 33.8% of the primers did not produce any polymorphisms, while Iqbal et al. (1997) found that 98% of the primers amplified the polymorphic pattern; but, the level of genetic divergence was quite low. Brubakar and Wendel (1994) also reported that the level of RFLP diversity was low in G. hirsutum cultivars as compared with the other reported taxa. The cotton genotypes used in our study are elite cultivars in addition to few standard genotypes (LRA-5166, CP-15/2, and Cedix). Hence, the results are indicative of their genetic relationships and are in accordance with earlier studies.
The close genetic kinships are quite alarming and may impede further plant improvement. It has been very well documented that plant improvement is based on the information about the genetic relationships among accessions within and between species (Thormann et al., 1994). Moreover, plant breeders select breeding material to breed for elite lines on the basis of genetic relationship among the breeding material (Hallauer and Miranda, 1988). The tendency to use similar parents extensively in a breeding program has led to a concern of lack of genetic diversity (Fouilloux and Bannerot, 1988). It has been shown that genetic uniformity of U.S. cotton cultivars is greater today than it was 25 yr ago (Esbroeck et al., 1998). Moreover, with the advent of "Green Revolution," the productivity of crop plants has risen tremendously over the last four decades (Mann, 1997). However, this transition in agriculture has resulted in loss of genetic variability (Tilman, 1998) and the gain in productivity has reached a plateau. The narrow genetic base has also created the potential for outbreaks of disease in epidemic form (Holley and Goodman, 1989). The value of genetic diversity for disease control has been well established in grain cereals (Garrett and Mundt, 1999; Zhu et al., 2000). This information underscores the need to avoid genetic uniformity in elite germplasm with its consequent negative effect on gains in long-term productivity (Messmer et al., 1992). A proactive policy demands consolidation of virus resistant varieties with divergence sources for a broader base.
The dendrogram (Fig. 5) assigned the genotypes into groups which correspond well with their centers or subcenters of release and/or pedigree relationships. In Cluster A, three cultivars or genotypes (FH-930, FH-945 and FH-901) were developed at one cotton research institute (Cotton Research Institute, Faisalabad). Similarly, in Cluster B, the cultivars (CIM-473, CIM-1100, and CIM-448) were developed by one breeding center (Central Cotton Research Institute, Multan). Earlier, subclustering had been reported in some elite Pakistani cotton genotypes, developed at one breeding station (Iqbal et al., 1997). The clustering of the varieties might be due to selection of the elite lines from a single population, which is the case with Basmati rice cultivars (Bligh et al., 1999). Moreover, breeders mostly share the elite lines of other breeding stations in cotton improvement programs, thus making the breeding material identical, which ultimately results in close kinship of the varieties or genotypes. Two cultivars (FH-900 and RH-500) are grouped together in a single cluster (D), and the group is relatively more genetically dissimilar than the other groups. The parentage of FH-900 consists of four genotypes (Table 1). Similarly, the parentage of VH-137 (which is not included in any cluster) includes parents that are quite distinct from the other cultivars (Table 1). It has been suggested that conical crosses should be made in breeding programs to increase the genetic diversity in the population (Fouilloux and Bannerot, 1988). Conical crosses would broaden the genetic window and should aid breeding for disease resistance by creating better segregating populations.
In addition, allele transfer by interspecific hybridization between G. barbadense and G. hirsutum has been reported; this is an important source of selectable variation for developing modern cotton cultivars (Percy and Wendel, 1990). However, interspecific hybridization currently has little use in a conventional breeding programs (Reinisch et al., 1994; Yu et al., 1998).
In the present study, the multiplex PCR and dot-blot hybridization were found to be the most sensitive and refined diagnostic tools for screening the breeding material against the CLCD. The RAPD analysis performed to evaluate genetic diversity and relatedness not only correlated well with the breeding data, but also quantified the narrow genetic base. The assessment of genetic diversity will be useful in selecting divergent parents for genome mapping purposes.
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ACKNOWLEDGMENTS |
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Received for publication July 17, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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released after CLCD infestation era. Numbers both in first column and row represent the cotton genotypes of the table.