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Pak. J. Bot., 41(4): 1627-1635, 2009.








1 Department of Botany, GC University, Faisalabad, Pakistan 2 Department of Botany, University of Agriculture, Faisalabad, Pakistan 3 National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan.

* E-mail: iftikhar _pbd @yahoo.com Abstract Cotton (Gossypium hirsutum L.) genotype Rajhans (pilose) was crossed with FH-634 (glabrous) and developed F2 and F3 segregating populations. PCR based techniques RAPD and SSR were used to determine DNA markers linked with the trait of hairiness using 400 RAPD and 54 SSR primers. Prior to analysis, PCR conditions were optimized. Eight RAPD primer markers OPD- 19640, OPF-11630, OPG-06980, OPG-17500, OPH-131100, OPN-14890, OPO-11920, OPO-141200 were consistently detected in duplicate reactions. One SSR primer pair, JESPR-154 amplified a 150 bp DNA fragment in hairiness population. Polymorphic DNA markers were used to develop linkage map. Construction of genetic linkage map using RAPD and SSR markers revealed that the inheritance for leaf hairiness exists, which can be used to improve insect resistance in cotton.

Introduction Damage due to insect pests is among many production constraints leading to decreased yields and profits (Diwakar, 2004). It is affected by increasing population of sucking and chewing insect pests. Among these pink bollworm, spotted bollworm, American bollworm, army worm, white fly, jassid, mealy bug and aphid are very harmful pests. There are several characteristics both in wild and cultivated species of cotton, often collectively called “defence umbrella” that repel insects. These traits include hairiness of leaves, absence of nectars, reddish colour of the stem and leaf, okra leaves and gossypol (Mursal, 1994). Different studies have depicted that hairs on the plant surface are not liked by insects (Alexander et al., 2004).

Research on host plant resistance to insect pests has accelerated in recent years. The inheritance of host plant resistance (HPR) mechanism is known for different traits conferring resistance against the insect pest complex and diseases. Plant breeders have exploited germplasm resources for the evaluation of cotton cultivars resistant to the insect pests and diseases. Many resistant accessions of cotton (Gossypium hirsutum) have been identified: 59 to boll weevil, 98 to pink bollworm, 56 to Heliothis spp., 6 to mites, 11 to tarnish plant bugs, and 33 are high in gossypol (Jenkins, 1995). Resistance against jassids in G. hirsutum genotypes was due to the presence of long and dense hair (Knight & Sadd, 1954; Mursal, 1994; Rahman, 2002). Moreover, the pubescent genotypes provide resistance to boll weevil, pink bollworm and plant bugs, and are neutral for mites (Ahmad et al., 1987). Glabrous genes provide resistance to Heliothis spp. (Lukefahr et al., 1971) and pink bollworm, and are neutral for boll weevil. Increased plant pubescence above the IFTIKHAR ALI ET AL., 1628 degree normally found in the pubescence cultivated varieties are governed by two major genes and a complex of modifiers, H1 gene derived from A genome (Knight, 1955) is found in G. hirsutum, G. barbadense, G. herbaceum and G. arboreum cotton. A stronger gene, H2 gene, identified and derived from Hawaiian wild tetraploid cotton, Gossypium tomentosum (Knight, 1955; Knight & Sadd, 1954) confer finely dense (pilose) pubescence. The presence of H2 gene in the B-genome of wild diploid G. anomalum was reported by Saunders (1964) but Gerstel & Phillips (1958) located it in A-genome which was confirmed by Endrizzi (1963) in a study on cotton monosomics.

Lack of genetic diversity among the elite cotton cultivars is the major concern using the conventional breeding approaches (Rahman et al., 2002). High-yielding cultivated upland cotton types have long been regarded as uniform genetically and thus relatively vulnerable to potential pathogen or insect epidemics (Bowman, 1999). In cotton, the development of the genomic resources and tools has allowed identification and mapping of genes and loci controlling traits of interest. The genomic resources and tools could be used to promote or facilitate cotton genetic improvement in numerous ways (Zhang et al., 2008). Advanced high-density genetic linkage maps (Alonso-Blanco & Koornneef, 2000;

Diwan et al., 2000; Zhang et al., 2002; Mei et al., 2004) established using molecular markers, for a series of economically important crops provide a basis for marker-assisted selection (MAS) of agronomically useful traits, for pyramiding of resistant genes, and the isolation of these and other important genes by map-based cloning strategies (Tanksley et al., 1995). In cotton, genetic linkage maps have been useful in characterizing the conservation of gene order and synteny in the tetraploid Gossypium species relative to their diploid progenitors. The leaf hairiness is an important trait of insect resistance in cotton. The objective of this study was to construct the genetic linkage map for inheritance of leaf hairiness trait in cotton using DNA markers.

Materials and Methods

Ten cultivars of Gossypium hirsutum L. viz., FH-634, FH-901, SL 7-9, HR-103, HRRajhans, FH-900, MNH-552, CIM-443 and NIAB-78, kindly provided by K.N.

Shah, Ayub Agricultural Research Institute, Faisalabad, Pakistan, were sown at Research Farm, National Institute for Genetic Engineering and Biotechnology, Faisalabad, Pakistan. Cultivars were grouped into three categories i.e., glabrous, semi hairy and pilose on the basis of mean trichomes density. Rajhans (pilose), introduced from India, was crossed as female with FH-634 (glabrous), a local genotype. F1 population was raised during off-season in green house to get F2 seed. F2 and F3 segregating populations for hairiness was developed for genomic analysis. The genomic DNA was extracted (Iqbal et al., 1997) from 100 hairiness and 100 hairiless plants. Concentration of the DNA was measured spectrophotometrically. DNA pools contrasting for hairy trait were constructed by mixing the same amount of DNA from each individual to generate bulks.

RAPD analysis and PCR amplifications: The bulks of Hairiness and Hairiless was used for RAPD analysis. A total of 400 primers (Operon kits) were used in PCR reaction. PCR was performed in volumes of 25 µl containing 2.5 µl 10X [(750 mM Tris-HCl (pH 8.8)], 200 mM (NH4)2SO4, 3 µl MgCl2, (25 mM), 2.5 µl 0.001% gelatin, 1µl each of dATP, dCTP, dGTP, dTTP (2.5 mM), 2 µl primer (15 ng/μl), 3 µl of genomic DNA (15 ng/µl),

0.2µl (1 unit) Taq polymerase and 7.8 µl ddH2O. Amplification was performed in Perkin Elmer DNA thermal cycler 480 programmed for a first denaturation step of 5 minutes at


94°C followed by 40 cycles of 1 minute at 94oC, 1 minute at 36°C and 2 minutes at 72oC.

The reactions were kept at 72oC for 10 minutes and then held at 4oC until the tubes were removed.

Agarose gel electrophoresis: Amplification products were analyzed by electrophoresis in 1.2% agarose gel run in 0.5% TBE buffer and detected by staining with ethidium bromide (10 ng/100 ml of agarose solution in TBE). Before loading PCR product in the gel, one drop of 5X RAPD dye (bromophenol blue mixed with 10% glycerol, 0.1 M EDTA, and 2% SDS) was added to the reaction mixture. Only 12 µl of the reaction mixture was loaded on the gel submerged in TBE buffer. Samples were electrophoresed for approximately two hours at 50 volts. After electrophoresis, the amplified products were viewed under ultraviolet transilluminator and photographed using the Stratagene Eagle Eye Still Video System.

Scoring of the RAPD data: Good quality photographs were used to read the amplification profiles. All visible and unambiguously scorable fragments amplified by primers were scored under the heading of total scorable fragments. Bands of less than 250 bp were in some cases difficult to score and were not considered. The primers that amplified polymorphic DNA were used to amplify genomic DNA of individual plants from which the bulks were constructed.

Microsatellite markers/ Simple sequence repeat (SSR) analysis: A total of 54 primer pairs custom-synthesized from GIBCOBRL (Life Technologies, USA) were used in SSR analysis. The PCR condition for amplification was same for all the 54 SSR primer pairs.

The following concentrations of PCR reagents for 20 µl final volumes were used (supplied by Fermentas). Template DNA (15 ng) 2.0 µl, dNTPs (2.5 mM) 6.4 µl, buffer (10 X) 2.0 µl, MgCl2 (25 mM) 1.6 µl, Primer-F(30 ng/µl)1.0 µl,Primer-R(30 ng/µl)1.0 µl,Taq polymerase(5 Uµl)0.2 µl,Double distilled H2O 5.8 µl.

Linkage analysis: Linkage analysis was conducted by Mapmaker 3.0 software (Lander et al., 1987). Map units were computed by applying the Kosambi (1944) function.

Linkage groups were identified at a minimum LOD 3.0 and a maximum distance of 50 cM. Map orders were determined by the “order” command for coupling phase makers and the “compare” command for repulsion phase makers following three point analyses.

Final map orders were tested by the” ripple” command.

Results DNA marker studies for hairiness: Two bulks contrasting for hairiness were constructed by pooling an equal amount of DNA from the 20 hairiness homozygous and 20 hairiless plants to find DNA markers for hairiness.

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Fig. 1. Polymorphic RAPD markers (OPD-19640, OPF-11630, OPG-06980, OPG-17500, OPH-131100, OPN-14890, OPO-11920, OPO-141200) for Hairiness trait. M= Size marker, H= Hairiness, h= Hairiless.

Bulked segregant analysis (BSA) was applied to identify RAPD markers linked with the hairiness trait. The size of DNA fragments amplified was in the range of 300 to 2000 bp. The number of DNA fragments amplified was in the range of 6 to 12 with an average of 8.2. The polymorphism reproduced by the 8 primers were confirmed on the individual samples of each bulk and was also applied on two parents. The polymorphic bands linked to hairiness trait amplified by the three primers (OPF-11630, OPH-131100, and OPN-14890) were present in all the hairiness individuals tested while absent in hairiless individuals.

However, polymorphic markers (OPD-19640, OPG-06980, OPG-17500, and OPO-11920) showed 1.8 % recombination, while the recombination frequency of the OPN-14 was 6.2 cM. The polymorphic DNA fragment amplified by the primer OPO-14 was present in trans-phase with the H2 gene. Thus individual plant analysis with all the polymorphic markers indicated a tight linkage with the H2 gene.

SSR Markers for hairiness: The primer pairs JESPR-153 amplified polymorphic DNA fragments for the hairiness trait, which amplified two loci. The estimated size of the allele at one locus was 150 bp and at the second locus was 130 bp (Fig. 2).

Genetic linkage map for hairiness: Polymorphic markers were used to construct the genetic linkage maps for hairiness trait. One SSR marker and nine polymorphic RAPD markers showed linkage with the hairiness trait (Fig. 3). The preliminary linkage map for the hairiness trait showed that the four RAPD markers viz., OPC-08700, OPF-11630, OPHand OPO-141200 congregate within the locus and exhibited the close linkage to the locus. Three RAPD markers OPG-17500, OPO-11920 OPG-17500 and OPD-19640 were present at 1.8 cM on the either side of the locus. One RAPD marker OPN-14890 was present at 6.2 cM from the locus.The SSR marker JESPR-153 showed the map distance of 15.7 cM from the locus


M P1 P2 B1 B2 H h H h H hH hH h H h H hM 150 bp 130 bp

Fig. 2. SSR marker amplified by the primer pair JESPR-153 linked with the velvet hairiness locus:

P1=Hairiness parents, P2=Hairless parents, B1=Hairiness bulk, B2=Hairiless bulk, H=Hairiness, h=Hairiless, M=1 kb ladder.

–  –  –

Discussion In the present studies, DNA markers were identified using intraspecific population which is more practical to commence MAS. Two cotton genotypes viz., Rajhans and FHwere used to make crosses. Thus, the pedigree of the parents was dissimilar. Bulked segregant analysis (BSA) with RAPD has been exploited to discover DNA markers linked with different traits (Schuster et al., 2001) and allele-specific markers (Bentolila et al., 1998). Several approaches have been suggested to saturate genomic regions of interest with molecular markers. These include near isogenic lines (NILs) and recombinant inbred lines (RILs). The combination of RAPD and BSA has been proved to be faster and less expensive (Mackay & Caligari, 2000). In the present investigation, a total of 4.98 and 6.47 RAPD bands per primer were observed for velvet hairiness. An average of 5.48 RAPD bands per primer in wheat was reported by (Rahman, 1997), while in sugarcane there were 8.7 RAPD bands (Harvey et al., 1995).

IFTIKHAR ALI ET AL., 1632 In this study, RAPD and SSR analysis was conducted to find DNA markers linked with the trait of hairiness. RAPD markers are PCR-based, can be used to identify a single copy gene from a complex genome (Saiki, 1990) and different bands can be visualized on agarose gel under UV light. Other technology like restriction fragment length polymorphism (RFLP) produces low variation in cotton (Yu et al., 1997) and needs high quality DNA. Moreover, it is a difficult task to isolate high quality DNA in Gossypium due to the presence of polysaccharides and polyphenolic compounds.

The stability of RAPD markers and the repeatability of the PCR reaction have been a source of concern in many studies (Jones et al., 1997). Thus the RAPD reaction was repeated for polymorphism to ensure the reproducibility of the marker. Further, only those major bands whose presence or absence could be easily distinguished were used to follow the segregation patterns in the progenies.

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