DNA marker studies in cotton

Globally, cotton production is seriously hampered by the infestation of diseases and insect pests. Among diseases, recent epidemic of cotton leaf curl virus disease (CLCD) is the major threat to the cotton crop, in Pakistan. Epidemics of the CLCD have compelled to the development of new strategies in cotton breeding programs of both long and short-term nature. On long term basis, genetic control is inexpensive and durable strategy to overcome the problem. The cotton breeders are engaged to evolve resistant varieties, however, it is quite difficult to select for resistance/ susceptibility in field and especially in greenhouse conditions due to uneven distribution of the disease. Thus, marker-assisted selection (MAS), a relatively new tool for plant breeders, can be applied to replace evaluation of a trait (e.g. virus resistant) which is difficult and expensive to evaluate. When a linked marker is found that co-segregates with a gene or QTLs, it may be easier to screen a given population. For genome mapping purposes or for DNA marker studies, knowledge about the nature of resistance against the disease is necessary. At the commencement of the inquiry and continuing until now, reports on the genetic analysis of host plant resistance (HPR) mechanism against the disease are scanty. Hence, firstly, in this research the numbers of gene(s) conferring resistance were determined. A total of 22 genotypes were screened against the disease with conventional and DNA based tests. Direct and reciprocal crosses were made between the extremely resistant (ER) genotypes (LRA-5166, CP 15/2 and CIM-443) and the most susceptible genotype, S-12. The segregating population of all the crosses exhibited a ratio of 10 (resistant): 3 [extremely resistant (ER)]: 3 (highly susceptible). Conducting multiplex PCR analysis differentiated the resistant and ER plants. On the basis of F3 progeny test, three genes R1CLCuVhir, R2CLCuVhir and SCLCuVhir were proposed to control resistance against the disease and were dominant in nature. Moreover, the role of extrachromosomal inheritance was ruled out by observing similar kind of results in reciprocal crosses. DNA marker studies were conducted on the population raised to study the inheritance of the disease. Random amplified polymorphic DNA (RAPD) technique was applied to find DNA markers for CLCD resistance. A total of 300 available random decamer primers were surveyed using bulked segregant analysis (BSA). With the BSA, none of the detected polymorphic DNA fragments were linked to the CLCD resistance in an F2 population segregating for resistance derived from LRA-5166. Upon reducing the bulk size, again none of the fragments were found to be linked with the disease resistance. The analysis was continued on the parents of the population. Thirteen percent of the total amplified DNA fragments were found to be polymorphic among the parents (LRA-5166 and S-12). However, none of the polymorphic DNA fragments was linked with the disease resistance trait. Subsequently, the parents of the second cross (CP-15/2 x S-12) were screened with the available random primers. A polymorphic DNA fragment of about 1180 by was linked in trans phase with a recombination frequency of around 14%. The polymorphic DNA fragment was designated as OPN12(1180). With this marker, within cultivar variation was observed in individual plants of LRA-5166. Total genomic DNA was extracted from 10 individual plants of LRA-5166. The marker was present in 40 % of the plants. The analysis was also continued on an F2 population segregating for resistance derived from LRA-5166. A recombination frequency of 40% was estimated. The cotton parents LRA-5166, CP-15/2 and S-12 were also screened with five SSRs. The primer pairs CM-43 and CM-162 amplified polymorphic DNA fragments among the cotton genotypes; LRA-5166, CP-15/2 and S-12. The primer CM-43 amplified two loci, which were polymorphic a mong the parents. The estimated size of alleles at one locus (a) was 101 (a1) and 109 by (a2) resolved on 6% denaturing polyacrylamide gel. Two alleles b2 and b2 of around 125 by and 150 bp long, respectively, were detected at the second locus (b). The recombination frequency -of around 12.5% at `a' and 14% at `b' locus with the CLCD resistance were estimated on an F2 population segregating for resistance derived from CP-15/2. Similarly, the recombination frequency of 33% at both the loci with the CLCD resistance were calculated on the population with resistance from LRA-5166. The amplified products with CM-162 primer pair was easily resolved on 2.5% agarose gel. The alleles detected were a1 (227bp) and a2 (200 bp). The S-12 genotype amplified allele at (227) and the ER genotypes CP-15/2 and LRA-5166 amplified a2. Two alleles of 200-227 bp were amplified from the F2 population derived resistance from CP-15/2. A total of 28 each ER and susceptible homozygous F2 plants were su r veyed. The recombination frequency at this locus was 16%. A new allele `a3' of approximately 180 bp long was amplified from 2 homozygous non-susceptible and 1 susceptible homozygous F2 plants, which derived resistance from LRA-5166. No definite linkage was found with this SSR locus with the disuse resistance trait in this population. The linked DNA markers were surveyed on the available germplasm to detect the efficiency of these markers for marker-assisted selection (MAS). A total of 28 cotton genotypes were screened with the OPN12(1180) DNA marker. Most of the genotypes released during post-CLCuV infestation era were included in the analysis. The DNA marker detected non-susceptible cotton genotypes CIM-1100, V3, CIM-482, CIM-473, FH-930, FH-901, FH-945 and CIM-435, which derived resistance from the CP-15/2. However, the DNA marker could not detect cotton genotype CIM-448. Thus out of 9, the marker was absent in 8 cotton genotypes (efficiency is around 89%) with resistance f rom CP-15/2. The DNA marker detected 2/5 genotypes with resistance from LIRA-5166. Similarly, the DNA marker was not amplified in ER/resistant FVH-53, NIAB-98, VH-137 and RH-500, which derived their resistance from other sources. However, the DNA marker could not differentiate the susceptible genotypes: MNH-93, NIAB- Karishma and CIM-240 except BH-36. Thus the marker has limited use because it can detect only the genotypes with resistance derived from CP-15/2. The SSR primer pair CM-162 detected all non-susceptible cotton genotypes that derived resistance from CP-15/2 or its derivatives. Similarly, the primer detected most non-susceptible genotypes with resistance from other sources but could not differentiate the susceptible genotypes. The results suggested that the SSR marker is specific to the source of resistance. The cotton genotypes were also screened with the SSR primer pair CM-43. It was observed that the marker could detect susceptible and non-susceptible genotypes w ith 79% confidence level irrespective of resistance source. Thus the marker is not limited to the source of resistance. The second major threat to cotton production is the infestation of insect pests. Among these, cotton bollworms and sucking pests are very serious. Many control measures have been reported to check or at least keep the damage below threshold level, including varietal non-preference for pests (presence or absence of nectars, hairiness, leaf size, shape etc.), cultivation practices, biological control, management of beneficial insects etc. collectively referred as Integrated Pest Management (IPM). The recombinant DNA (rDNA) technology can overcome the barrier of linkage drags in conventional breeding. For this purpose, gene isolation is required before commencement of rDNA technology. Gene(s) could be isolated through map-based cloning which needs DNA markers in the flanking regions of the gene. A primer OPC-08 produced a DNA marker of around 700 bp for velvet hairiness, identified using BSA. Only one recombinant was found in individual plant analysis with the primer OPC-08 (5'-TGGACCGGTG-3') indicating a tight linkage with the H2 gene. The genetic distance between the locus for velvet hairy and the DNA marker was 1.7 cM. The DNA marker was designated as OPC08(700), which can be used in molecular breeding or to clone hairiness genes for genetic engineering purpose. Similarly, a DNA marker for the extrafloral nectarines trait (one of the components of defense umbrella) was detected by a random primer OPB-12. The RAM reaction performed with the individual plant DNA samples revealed that the only polymorphic DNA fragment of 1400 by amplified by the primer OPB-12 was associated with nectarines. This DNA molecule was designated as OPB-12 (1400). For neap-based cloning, additional DNA markers are needed to saturate the region around the genes for that purpose, new robust techniques such as AFLP and SSR can be exploited to find additiona l DNA markers. The other major control measure (on short-term basis) was to broader genetic base of cotton cultivars/varieties. It was based on the rationale that a divergent genetic base is a potential buffer against the spread of diseases or to combat any natural disaster in advance. The RAPD analysis was applied first on 20 different tetraploid extremely resistant (ER/resistant/tolerant cotton genotypes selected by employing different diagnostic methods. The genetic similarity of the exotic virus resistant germplasm with the elite cultivars was in the range of 81.45% to 90.59%. Similarly, the genetic relatedness among the elite cultivars was in the range of 81.58% to 94.90%. It has been clearly shown that none of the cultivars except variety VH-137 possesses a diverse genetic background. This study will work as an indicator for plant breeders to breed for high genetic diversity by conducting conical crosses or by inter-specific hybridization.