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Up to recent improvements in molecular genetics, breeders have been developing qualitative and quantitative inherited traits through conventional breeding methods based on phenotypic screening and selection. Since 1980s, QTL mapping has been applied regularly; however association mapping is a promising alternative method for discovering and dissecting complex traits (Chan., et al., 2011). Improved mapping resolution, reduced study time and more allele numbers detected as main advantage above traditional QTL mapping (Zhu, C. et al,. 2008). Molecular markers technology developed through the last era has opened opportunity of directly statistical associations study (linkage disequilibrium, LD) among genetic markers and phenotypic traits in populations, hence called as association genetics (Nordborg and Weigel, 2008). The obtainability of suitable genetic markers has become easier, so it is possible to associate genome regions covering these markers to difference in complex traits. Association mapping methods was first successfully used for the dissection of alleles at loci participating to susceptibility to human diseases (Goldstein et al., 2003). Currently, this methodology has been used in diverse plant species such as in barley (Kraakman et al. 2006) and potato (Ivandic et al. 2003), wheat (Breseghello and Sorrells 2006), and rice (Agrama et al., 2007), to recognize markers and inheritable genes related with a diversity of phenotypes. association studies In cotton (Gossypium sp.), are limited ,therefore, such studies on phenotypic traits under drought stress condition were essential for cotton breeding, and important to determine the potential of the association mapping method in this crop (Abdurakhmonov et al., 2007). New computational approaches give more accurate discovery of markerââ‚¬"trait associations in complex-pedigree populations by accounting for population structure and kinship, additionally also the extent of linkage disequilibrium (LD) can determines marker resolution in a specified population. By these methods cloning of genes of interest will be facilitated (Pritchard et al. 2000, Yu et al. 2006).
The Use of SSR markers to know population structure and diversity results in good resolution than use of other kinds of markers because of the great level of polymorphism at SSR loci (Cho et al.2000). Currently, a very SSR markers are powerfully used in MAS programs in cotton because most of marker material has been obtained from populations of bi-parental crosses with limited genetic background, covering a few meiotic events since experimental hybridization.
In cotton (Gossypium sp.), this is the first ever report of association mapping for drought tolerance. In this study, a considerable number of morpho-physiological, plant architecture and yield traits were studied and some significant marker-trait associations were found.
Phenotypic variations of cotton Germplasm for Morpho-physiological traits
The screening for identification or development of drought tolerant genotypes is of high importance for improving cotton production. The first objective of the present study was to evaluate 90 Pakistani cotton germplasm collected from different research centers for Morpho-physiological traits at seedling stage at control and drought stress conditions. Similar procedure was use previously in cotton crop by Azhar and Raza (2000), Saqibet al. (2002). The obtained phenotypic data of seedling traits under water stressed were used for our association mapping study.
Different scientists studied the plant growth at early stages of plant development (Noor et al. 2001, Bhatti et al. 2006, Azhar et al. 2007, Sharifi 2008). Shoot and root growth of cotton cultivars after a short drought and subsequent recovery period was studied by Pace et al. (1999) and found that all trait values declined in water stressed plants than that of control.
the resulted means square from analysis of variance for all seedling traits revealed significant variation (P<0.05) with respect to water levels and genotypes However, the interaction between water levels and genotypes was significant for shoot length, fresh shoot weight and dry shoot weight and non-significant for root length, fresh root weight and dry root weight. Mean value of all genotypes indicated reduction in all morpho-physiological traits. Correlation coefficients between the relative values of most of the seedling traits also revealed positive and highly significant (P< 0.01). Similar adverse effect of water stress on cotton seedling traits have been noted in previous studies (Pettigrew, 2004 and Basel et al., 2005.
For the assortment of water stress tolerant genotypes, selection index was performed by operating the well watered, water stressed and relative means of all seedling traits. Single trait and multi-trait selection using GGE biplot was performed. Data indicated that CIM-482occupied the top rank at water limited while MNH-636 showed overall best performances for relative value of drought stress condition. Similarly, the top 16 varieties that promoted as best performance for water limited are as fellow CIM-482, FH-900, MS-40, FH- 901, S-11, SLS.1, 1021 (Kivi), NIBGE-4, 2616, MNH-636, 841/52, L.S.S, 299 F, CIM-109, 65090, CIM-1100; and the top 16 varieties according to their relative means of all seedling traits are MNH-636, FH-900, MS-40, CIM-482, CIM-496, 3996, 299 F, 2616, CRS-2007m 841/52, XiaoVemian, SLS-1, 6040, NIBGE-4, CIM-1100, GR-156 which can be selected as drought-tolerant for further investigation. However, the lowest five genotypes (CIM-446, SLH284, CRSM.83, UA-7-25/46, TH-35/99,), which showed low performance for water limited and relative means were selected as drought susceptible.
The differing measurement of Morpho-physiological traits provided strong indication of varietal differences among G.hirsutum genotypes for the adverse effect of water and salt stress as shown in our results as well as previous studies in Pakistan (Abbas et al. 2011, Iqbal et al. 2011,Mahmood et al. 2006). Thus, seedling traits may be useful for association mapping study with the objective of improving drought tolerance in cotton under water-limited environments.
Phenotypic variations of plant architecture and yield traits
The second objective of this study was to evaluate Pakistani cotton Gossypium hirsutum germplasm collected from different breeding research centers for Plant architecture and yield traits, especially (plant height, number of nodes, Cotton Yield per Plant (g), Individual boll weight (g) and total number of bolls. In order to achieve such evidence in G. hirsutum, 90 genotypes were evaluated. This method distinguished tolerant and non-tolerant genotypes and we can use the phenotypic data for molecular breeding programs like genetic diversity and association mapping in our study. Different scientists studied the plant growth at different stages of plant and development. Seed cotton yield per plant is determined by two basic traits boll number and boll weight. Water stress circumstances reduced the boll weight; Bolls per plant and eventually seed cotton yield were seriously affected in water stress as compared to normal irrigation conditions (Anonymous. 2009). Maurer (1991) also observed reduced bolls per plant in stress conditions. In present study, the varieties revealed considerable diversity for growth and productivity traits in relation to water regimes. The performance of cotton genotypes under water stress condition revealed that MNH670 and MNH552 had maximum numbers of bolls, seed cotton yield and plant heights. MNH807 had the highest boll number. Plant height also regulates the yield in the sense that as plant height rises, both the number of fruiting branches and number of bolls also increases, accordingly yield also increases. However in stress and non-stress conditions, the cultivar MNH670 showed high water stress tolerance as compared to other cultivars. At the same time the cultivar Australia-407721 revealed low water stresses susceptibility in both conditions. The cultivar MNH670 and MNH807 has exhibited high stress tolerance because of their small yield differences under both stress and non-stressed conditions. (Ullah et al. 2008) assessed genotypic variability for drought tolerance in cotton using physiological attributes with productivity traits under well watered and water stress regimes in field experiment and reported that seed cotton yield was markedly affected under water stress conditions in all cultivars studied. In another study, based upon the observations on the physiological parameters, cotton genotypes were tested for variability under water stress at flowering and boll development stage and high level of variability was found in almost all the characters studied (Singh et al., 1996). Morphological traits of cotton are severely affected by drought stress and they are easy to be quantified. The morphological traits are easy to be measured and there are less chances of error in their phenotyping. This fact makes them a better selection criterion for evaluating drought tolerance of cotton genotypes.
Genetic diversity assessments
Knowledge of genetic diversity is important for successful genetic improvement programs. Thus, the genetic diversity in cotton cultivars was studied using 95 simple sequence repeat (SSR) primer pairs The pairwise genetic distances between individuals were calculated by the percentage disagreement method, and the resulting matrix showed a mean genetic similarity of 0.315 among the 50 genotypes, revealing high level of genetic relatedness. Regarding the pairwise combinations, the genetic similarities between genotypes varied from 0.1 between CIM-446 and CIM-448 to 0.53 between CIM-482 and MNH-6070 and STAPM-82 and AYT-85094. A UPGMA cluster analysis revealed inconsistencies in the clustering patterns, but did not precisely reflect the origins of the populations, as each cluster consists of cultivars released from different breeding origins. The inconsistencies between the cultivar clustering and known parentage of these cotton cultivars in this study revealed a narrow genetic base, in agreement with the results of Iqbal et al. (2001). However, different recent studies have reported a relatively higher genetic diversity, with an average genetic distance of up to ~37-77% in G. hirsutum, as based on the analysis of some germplasm resources from Pakistan (Khan et al., 2009), Brazil (Bertini et al., 2006), China (Liu et al., 2011; Zhang et al., 2011) and India (Chaudhary et al., 2010), results obtained from SSRs or a combination of SSR and RAPD markers. Sapkal et al. (2011) reported genetic diversity (up to 57%) for 91 Upland cotton accessions with genetic male sterility maintainer and restorer properties that was similar to the finding in our study (57.5%). These results suggested the presence of useful genetic diversity both in exotic and breeding line resources. The current study showed that microsatellite markers are efficient for measuring the genetic diversity and relatedness and for identifying varieties of cotton. The results revealed relatively low genetic diversity of Pakistani cotton cultivars and also generate information about levels of genetic relatedness. We suggest the further evaluation of the molecular genetic diversity in efforts to conserve the maximum diversity level by applying additional markers to improve the resolution of these tools for use in breeding programs. This is because the narrowness of the genetic diversity in the germplasm was associated with the recent and potentially future declines in G. hirsutum L. production and its quality, serving as a timely warning to increase the speed of the efforts to widen the genetic base of the germplasm resource by mobilizing new genetic variations from the gene pool. The wide ranges of the genetic diversity of the genus, particularly the diversity of exotic wild cotton germplasm, are key resources to improve cotton cultivars and address the various essential problems associated with the abiotic stresses and fiber quality.
Association mapping of Morpho-physiological, plant architecture and yield
For molecular studies, there should be a reasonable degree of variability present among the organism of interest, only then the molecular approaches can identify the genetic case underlying this variability. As there was a significant variability shown in our experimental material in the green house as well as in the field as mentioned above, so the results of our molecular outcomes are of future impacts?. The tests for associations between the 95 markers using general linear methodology (GLM) revealed significant associations between cotton morphological and productivity trait and SSRs in G. hirsotum germplasm. In this manuscript, a total of twenty three marker loci main effect were significantly associated (P < 0.05) with the morpho-physiological traits phenotyped under the drought treatment (Table 4.14), with Phenotypic variance explained (R2) value ranged from 4.7% to 19%. The highest phenotypic variance explained (R2) was ascribed to NAU3011 D13 (Ch.18) associated with root length. Marker NAU3011 found to be associated (P= 0.0012) with root length in drought treatment appeared to be a major locus as it ascribed with highest phenotypic variance explained (R2) value of 19%. Majority of the markers were found to be associated with more than one trait. NAU3414 located on A9 (Ch.9) was associated with maximum (7) traits (shoot length, shoot fresh weight, fresh root weight, fresh plant weight, dry shoot weight, and dry plant weight). Out of total 23 markers associated with drought treatment, 9 markers were found to be common in control and drought treatment (Table 4.16). These markers will be helpful in future endeavors to explore the complex nature of drought tolerance in cotton and It is advised that these markers can successfully employed for MAS for drought stress in future molecular breeding programs. However From field experiments twenty marker loci were significantly associated (P < 0.05) with the plant architecture and yield traits phenotyped under the drought treatment (Table 4.31). Phenotypic variance explained (R2) value ranged from 4.26% to10.14%. BNL946D10 (ch.20) reported highest phenotypic variance explained (R2) value of 10.14% associated with seedcotton yield. There were 12 markers which found to be common in control and drought treatment (Table 4.33). BNL946D10 (ch.20) was found to be associated with maximum number of traits (plant height, number of nodes, numbers of bolls, boll weight) this marker locus is of importance. JESPR274 (Ch.9) is also another important locus which imparts high phenotypic variance explained (R2) value of 17.8% under control treatment. The promising results achieved from this study would encourage the additional experiment of these techniques in different genetic organizations, as we used these ways for cotton. The idea to study the markers-traits associations in two ways, significance of the association between marker and trait (P-values) by means of TASSEL software and markerââ‚¬"trait associations presents in different QTLs studies. Implementation of association mapping to plant breeding and genetic improvements is appears to be encouraging means of resolving the limitations of conventional linkage mapping (Stich et al,. 2005). SSR markers linked with the main morpho-physiological, growth and productivity traits in our study were identified across the genetic background of diverse cotton accessions form Pakistan, allowing us to utilize several meiotic events happened since history of germplasm development. Therefore, these markers should have more possibility to be effective markers for MAS programs and offer valuable information beneficial for genetic enhancement of Upland cotton. The reasonably large extent of LD in the cotton genome shows the significant possible for LD based association mapping of complex traits in cotton with a comparatively small number of markers.