Introduction
Cotton (Gossypium L.) is a leading fiber crop in the world. Although the genus Gossypium L. has approximately 50 species, only four of them are cultivated, which include two diploids (2n = 2x = 26): G. arboretum L. (A2A2) and G. herbaceum L. (A1A1), as well as two allotetraploids (2n = 4x = 52): G. Hirsutum L. (AADD) and G. barbadense L. (AADD). It was believed that the tetraploid cotton was originated from an interspecific hybridization of an old world diploid species that was closely related with G. arboreum or G. herbaceum (A genome donor) and a new world diploid species relative to G. raimondii Ulbrich or G. gossipioides Standley (D genome donor), which occurred about 1~2 million years ago (Beasley, 1940).

Use of heterosis in cotton production might be one of the key approaches to increase seed cotton yield. Heterosis for yield in F₁ hybrids cotton has been extensively analyzed in the past decades. Useful heterosis for yield in F₁ hybrids during 1947 and 1972 ranged from 7% to 50% in interspecific hybrids and from 10% to 138% in intraspecific hybrids (Davis, 1978). In any hybrid programme, a large number of crosses need to be made, while only few of the hybrids will show good performance over the standard check. This process is extremely labour intensive, time-consuming and tedious. Molecular markers increasingly detect locus differences among genotypes and represent a powerful tool for the assessment of genetic diversity in plant species (Tanksley, 1983). Selection of desirable parents is an important task to initiate a hybrid-breeding programme. Because heterosis is associated with the interaction of different alleles at a locus (Jones, 1945), it has been suggested that molecular marker diversity may be used to select parents for hybridization.

In cotton, a number of efforts have been made to investigate the relationship between DNA marker- based genotype variation of the parents to be used in a hybrid- breeding programme and heterosis with varying results. For example, Diers et al. (1996) reported that marker- based genetic distance was not consistently correlated with heterosis for inbred diallels and for cultivar diallels in rape seed. Sheng et al. (2002) reported significant correlation between genetic distance and seed yield but the determinative coefficient was very low (0.1024). However, Riaz et al. (2001) found that the genetic distance of sequence-related amplified polymorphism (SRAP) in American. B. napus inbred lines was significantly correlated with hybrid yield performance and heterosis.

Meredith and Brown (1998) studied the relationship between genetic distance estimated by restriction fragment length polymorphic (RFLP) markers among 15 cultivars and one strain from the USA and yield heterosis of 120 F₂ hybrids produced by a half _ diallel genetic design and found that the correlation were very low (r=0.08). Wu et al. (2002) studied the correlation between genetic distance measured by random amplified polymorphic DNAs (RAPD), inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers among six domestic and two exotic cultivars and interspecific F₁ and F₂ hybrids, and found the correlation between these was low.

Gutierrez et al. (2002) used five US, four Australian cultivars and two day- neutral converted lines of G.hirsutum to analysis the association between genetic distance based on SSR markers and performance of agronomic and fiber traits of F₂-bulk populations and deduced that significant correlations ranged from negative to positive depending on the traits, genetic background and environment.

Zhang et al. (2007) studied the relationship between parental molecular marker diversity and hybrid performance in both intra and inter specific hybrids of cotton to evaluate the feasibility of predicting hybrid performance using molecular markers. Three cytoplasmic male sterile (CMS) lines were crossed with 10 restorer lines to produce 22 F, hybrids during 2003. Of 22 F(1) s, 14 hybrids were intraspecific (G. hirsutum × G. hirsutum) and eight interspecific (G. hirsutum × G. barbadense). These 22 F, hybrids and their parents were evaluated for yield and fiber quality traits at Zhejiang University, Hangzhou, China during 2004 and 2005. Genetic distances (GD) among the parents were calculated from 56 random-amplified polymorphic DNAs (RAPD) and 66 simple sequence repeat (SSR) marker data, and their correlation with hybrid performance and heterosis were analysed.

Mohammadi et al. (2008) investigated the correlation between the potential of molecular markers and hybrids performance in Maize. Significant correlation was found between GD value of parental lines and hybrid performance for the testcross and diallel data. In diallel analysis significant correlation was observed between total grain yield per ear (TGW) and genetic distance based on SM coefficient, whereas the correlation of GD and specific combining ability of hybrids for this trait was not. Through the stepwise multiple regression analysis a total of 19 informative SSR markers distributed over all chromosomes, except chromosomes 7 and 8, were detected. GD values based on informative markers in general were grater compared to that of based on all markers and significant improvement was observed in the correlations between GD estimates based on informative markers and TGW as well as SCA.

The objectives of the present study were 1- To investigate the relationship of genetic distance, based on SSR markers, with hybrid performance and heterosis and to determine whether these markers would be useful for predicting hybrid performance and heterosis in cotton. 2- To improve the yield and fiber quality using interspecific hybridization (G.hirsutum × G. barbadense) in cotton.

1 Results and Discussion
1.1 Marker polymorphism
Analysis of microsatellites (SSR’s) in 32 parents (28 barbadense lines and 4 hirsutum testers) using 40 primers. Of these, 23 primers revealed a high DNA polymorphism among parents, these 23 primers produced a total of 134 amplified profiles (Table 1). Among these, 93 were polymorphic with an average of 68.65 per cent polymorphism. Primers viz., BNL 3871, BNL 3867 and BNL 1611 gave highest (100%) polymorphism. The number of bands ranged from two (BNL 3871, BNL 1034, BNL 1227 and BNL 3867) to ten (BNL 2655, BNL 3145, BNL 1440, BNL 3171 and BNL 3994) with an average of 3.35 bands per primer. The primers viz., BNL 1034, BNL 1227, BNL 1059 and CIR 246 showed the least polymorphism (50%). DNA amplification pattern of 32 parents is shown in Figure 1.

Table 1 Analysis of SSR patterns generated using 40 primers in cotton genotypes

Figure 1 DNA amplification pattern of 32 parents genotypes

1.2 Molecular marker diversity among the parents
The similarity coefficients (Table 2) involved in the line x tester study ranged from 57% to 96 %, with an average of 81%. Among the parental lines, the lines DB 533 × DB 534 F₄ IPS 8 and DB 533 × DB 534 F₄ IPS 1 showed highest similarity coefficient value (96%). While, the lines DB 533 × DB 534 F₄ IPS 48 and DB 533 × DB 534 F₄ IPS 16 exhibited lowest similarity coefficient value (57%). All the 32 genotypes showed diversity among themselves indicating that there is a considerable amount of variation, which can be exploited through appropriate breeding programme.
 

Table 2 Similarity coefficients for the 32 parents computed from SSR molecular marker data

 
The dendrogram constructed from the pooled data is presented in Figure 2, revealed three distinct clusters. One cluster involved testers and in other clusters all barbadense lines were placed which are already having proven record in giving good hybrids. 

Figure 2 Dendrograms derived from an unweighted pair group method analysis (UPGMA) cluster analysis by using Nei’s similarity coefficient based on SSR markers

 
The similarity coefficient values between the line DB 533 × DB 534 F₄ IPS 49 and the tester DH 98-27 showed 67%. It revealed that DB 533 × DB 534 F₄ IPS 49 was closely related to DH 98-27 with 67% similarity between parents. The hybrid between DB 533 × DB 534 F₄ IPS 49 and DH 98-27 exhibited the highest yield of 2884.26 kg/ha. Similarity coefficient (88%) value between lines and testers showed between the line DB 533 × DB 534 F₄ IPS 52 and the tester ZCH8, the hybrid between these recorded an yield of 2040.757 kg/ha. Lowest similarity coefficient value was noticed between the line DB 533 × DB 534 F₄ IPS 16 and tester DH 98-27 which revealed that they are far distinct from each other. This combination exhibited 2384.62 kg/ha yield.

1.3 Correlation between genetic distance and hybrid performance and heterosis
Genetic distance (GD) based on SSR markers were computed in Table 3. Genetic distance (GD) ranged from 0.041 to 0.429, with an average of 0.183. The result implied that each cluster dendrogram substantially reflected its own genetic relationship among parents. Overall, a low significant correlation of GD with hybrid performance and heterosis was detected in Table 4 and Figure 3. Highly significant positive correlation were found between genetic distance (GD) and ginning outturn for F₁ performance (0.277) and heterosis over MRC 6918 (0.279) and DCH 32 (0.279), while significant positive correlation were found between genetic distance (GD) and ginning outturn for mid parent heterosis (0.237). Highly significant positive correlation were found between genetic distance (GD) and seed cotton yield for F₁ performance (0.359) and heterosis over Bt check MRC 6918 (0.336) and over non Bt check DCH 32 (0.362), while significant positive correlation were found between genetic distance (GD) and seed cotton yield for mid parent heterosis (0.226). Significant positive correlation were found between genetic distance (GD) and lint index for mid parent heterosis (0.227), F1 performance (0.251) and heterosis over MRC 6918 (0.250) and DCH 32 (0.250), while significant positive correlation were found only between genetic distance (GD) and fiber micronaire value for F₁ performance (0.241).

Table 3 Genetic distance for the 32 parents computed from SSR molecular marker data

Table 4 Correlation coefficients of genetic distance (GD) with F1 performance and heterosis

Figure 3 Relationship between genetic distance (GD) and interspecific F1 performance, mid parent heterosis and heterosis over MRC 6918 and DCH 32 for seed cotton yield (kg/ha)

G. hirsutum and G. barbadense are allotetraploid (2n=4x=52) cottons, which together represent the most extensively cultivated species worldwide. While G. hirsutumis the most widely-cultivated species well-known for its higher yield ad wider environmental adaptation. It was recognized that the two species cross easily and produce vigorous F₁ hybrids (Loden and Richmind, 1915). Useful heterosis in interspecific F₁ hybrids which combined productivity and quality has been reported by many researcher (Davis and Palomo, 1980, Roupakias et al. 1998, Galanopoulou-Sendouca and Roupakias, 1999; Zhang and Wang, 2005). 

DNA based molecular markers acted as a versatile tool to study variability and diversity in different plant species. The development of DNA based markers represent an alternative procedure of the identification of promising parental lines for superior performances of hybrids. The microsatellite (SSR’s) markers have been widely used for the estimation of variation among closely related individuals due to its multiallelic nature and high polymorphism. Molecular markers based on polymorphism of DNA are especially useful for this purpose because they are not affected by environment (Tatineni et al., 1996; Saghai-Maroof et al., 1984). Several examples of the application of molecular markers to estimate genetic distances have been reported in maize (Smith et al., 1990) and rice (Zhang et al., 1995). Thus, molecular markers like SSR’s (microsatellite) could be used for germplasm classification and clustering to derive valuable information for heterosis prediction.

Therefore, they were useful for heterosis prediction in seed cotton yield, lint index, ginning outturn and fiber micronaire. According to Bernardo (1992) inadequate genome coverage, random dispersion of molecular markers (unlinked to QTLs) and different levels of dominance could be the reason for low correlation between molecular distance and heterosis and/or F₁ performance. The existence of multiple allelism and epistasis could also cause the low correlation of GD and F₁ performance/heterosis.

An assessment of the usefulness of molecular markers in breeding cotton for yield and fiber quality improvement may therefore need further consideration. More molecular markers covering all 26 chromosomes and at higher densities and molecular markers that are linked to QTL for agronomic traits and fiber properties are needed for further studies.

2 Materials and Methods
2.1 Plant materials and field evaluation
During 2010 the twenty eight F₄ lines of (Gossypium barbadense L.) (Table 5) cross (DB 533 × DB 534) depending on the highest of fiber strength, are proposed to be crossed with the four common diverse (Gossypium hirsutum L.) viz., DH 98-27 (T₁), ZCH8 (T₂), 178-24 (T₃) and DH 18-31 (T₄) selected based on the earlier study. The crossing programme was taken up during 2010. The F₄ lines and four common testers were sown on staggered dates. To obtain derived F1s seed, the flower buds of the proper size from testers (used as female) were hand emasculated in the evening between 3.00 to 6.00 pm. The emasculated flowers were covered by butter paper packets for avoiding out crossing as well as ensuring their easy identification at the time of crossing. The emasculated flowers were pollinated during the next day morning between 9.30 am to 11.30 am by brushing the pollen from one of the F₄ lines (used as male) on the stigmatic surface. The pedicel of each pollinated flower was tied with price label bearing date and lines number for identification of crossed bolls. In this manner derived F1s seeds were obtained. Simultaneously, two populations of F₄ lines were selfed and material was advanced to F₅ generation during the same season.

Table 5 List of F4 barbadense line parents involved in the study

 
The experimental material was planted on a medium black soil at College of Agriculture, Dharwad under irrigated condition. The F₅ lines, derived F1s of two populations along with the straight crosses and ruling commercial check (MRC6918 and DCH 32) were sown during kharif 2011 in all a randomized block design with two replications and a spacing of 90 cm between rows and 60 cm between the plants within a row. Recommended fertilizer doses were applied and other cultural practices were carried out at regular interval. Plant protection measures were taken at appropriate time to control pests and diseases. Each set of 28 F₄ lines thus was involved in 112 crosses (refer to as derived F1s), which were subjected to L×T analysis. The observations were recorded for number of bolls (no/plant), mean boll weight (g), seed index (g), ginning outturn (%), lint index (g), seed cotton yield (kg/ha), fiber length (mm), fiber strength (g/t), fiber micronaire value (µg/inch), fiber uniformity ratio (%), fiber maturity ratio and fiber elongation (%). Fiber quality traits were measured with the High- Volume Instrument.

2.2 SSR molecular marker analysis
Leaf tissue of each parents was harvested and total genomic DNA was extracted from young leaves using the hexadecyl-trimethyl ammonium bromide (CTAB) method described by Saghai-Maroof et al., (1984). SSR assays were performed using 40 oligonucleotide primers from Sigma Aldrich Chemicals Pvt. Ltd., Co. Amplification reactions were carried out in 20 mL volumes containing 2.0 mL 10× assay buffer, 2.0 mL dNTP mix (2.5 mM each), 0.5 mL forward primer (5 pM/mL), 0.5 ml Reverse (5 pM/mL), 0.5 mL Taq DNA polymerase (3U/mL), 2.0 mL Template DNA (15 ng/mL) and 7.5 mL Sterile double distill water. The amplification programmed for 5 min at 94℃ Denaturation (initial) of genomic DNA by one cycle followed by 25 cycles of 1 min at 94℃, 1 min at 48 ± 5℃ and 1 min at 72℃. This was followed by a final extension at 72℃ for 5 min. Amplification products were analysed by Non-Denaturing gel electrophoresis (PAGE) and viewed by silver staining.

2.3 Scoring the amplified fragments
The amplification of DNA profiles for all the primers were compared with each other and the bands of DNA at each amplification level of every primer were scored as present (1) or absent (0) thus generating the 0, 1 matrix.

Per cent polymorphism (%) = (Total No. of polymorphic bands)/(Total No. of bands generated by 40 primers) ×100%

2.4 Analysis of SSR profiles
Pair similarity coefficients were calculated for all pairwise combinations of the parental lines according to the method developed by Nei and Li (1979): Sij= 2Nij / (Ni+ Nj), where Sij is the similarity between parents i and j; Nij is the number of bands present in both parents; Ni is the number of bands present only in parent I; Nj is the number of bands present only in parent j. GD (genetic distance) was calculated as GD= 1- Sij. The similarity matrix from SSR markers, which were computed using NTSYS-PC version 2.1 (Rohlf, 2001) were used to construct dendrograms based on UPGMA (the unweighted pair- group method with arithmetic means). Using the same NTSYS software, a cophenetic value matrix was calculated to test the goodness of fit for the cluster analysis to the original distance matrix.

For studying the relationship between SSR molecular maker and hybrids performance and heterosis, the mid parent heterosis (MPH) was computed using the formula 100 × (F₁-MP)/MP, where F₁ is the hybrid performance and MP is the mid-parent mean. Per cent heterosis in F₁ over commercial check (CC) was computed using the formula 100 × (F₁-CC)/CC, where CC commercial check mean.

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