Phenotypic, Cytological and Molecular (AFLP) Analyses of the Cotton Synthetic Allohexaploid Hybrid ( G. hirsutum × G. longicalyx )²

The wild cotton diploid species (2n = 2x = 26) are important sources of useful traits such as high fiber quality, resistance to biotic and abiotic stresses etc., which can be introgressed into the cultivated tetraploid cotton Gossypium hirsutum L (2n = 4x = 52), for its genetic improvement. The African wild diploid species G. longicalyx Hutchinson and Lee could be used as donor of the desirable traits of fiber fineness and resistance to reniform nematode. However, hybridization of wild diploid species and cultivated tetraploid cotton encounters a sterility problem of the triploid (2n = 3x = 59), mainly due to ploidy. The restoration of the fertility can be done by creating an allohexaploid (2n = 6x = 78) through the doubling with colchicine of the sterile triploid chromosomes. With this method, a synthetic allohexaploid hybrid (G. hirsutum × G. longicalyx) 2 has been obtained. This genotype was studied using phenotypic, cytological and molecular (AFLP) analyses in order to confirm nomic traits from wild species to varieties of G. hirsutum.


Introduction
Cotton, from the genus Gossypium, is the most important natural fiber source for the textile industry in the world [1]. The Gossypium genus is composed of 53 species among which 7 are tetraploid (2n = 4× = 52) and 46 are diploid (2n = 2x = 26) [2]. Diploid cottons are classified into 8 genome types, denoted A-G and K, based on chromosome pairing relationships [3] [4]. The tetraploid cottons have a genome designated by AD, which resulted from the ancestral allopolyploidization of progenitor A-genome and D-genome diploids about 1-2 million years ago [2] [5].
Two cotton diploids species (G. arboreumn L and G. herbaceum L) and two tetraploids species (G. hirsutum L and G. barbadense L) are cultivated for their spinnable fiber [6] [7] [8]. G. hirsutum, known as Upland cotton, provides more than 90% of the world's cotton production due to its high yield [8]. The remaining cotton fiber supply is produced from the other three cultivated cottons [9].
Apart from these four species, the other 46 species of Gossypium are wild.
Genetic improvement of the main cultivated cotton G. hirsutum can be done using wild species as donor of traits of interest [10]. Indeed, wild diploid species are important sources of several desirable genes to improve fiber quality, resistance to diseases and insect pests, or tolerance to abiotic stress of Upland cotton [4] [11]. The African wild species G. longicalyx Hutchinson and Lee (F1 genome) represents an interesting source of genes that can potentially be transferred to the main cultivated cotton species. This wild diploid species could be used as donor of the desirable trait of fiber fineness, which is very important to textile industry [4] [12] [13], and also as donor of the resistance to reniform nematode [14] [15].
Technically, the use of wild diploid species to improve cultivated tetraploid cotton faces the problem of the sterility of the triploid (2n = 3x = 59) obtained, mainly due to ploidy. To overcome this problem, a strategy used involves the creation of an allohexaploid hybrid as a bridge genotype. This method begins with the hybridization of G. hirsutum with a diploid wild species to obtain a sterile triploid (2n = 3x = 59). The next step consists in doubling the chromosome number of the sterile triploid with colchicine to give a fertile allohexaploid (2n = 6x = 78). Such a synthetic allohexaploid hybrid could subsequently be used to introgress the alien genes through trispecific hybrid or monosomic addition lines [16] [17] [18]. In cotton breeding, the successful use of this method has

Cytological Analysis
To check the chromosome numbers of the hexaploid and its parental species, mitotic chromosome preparations were carried out using root tips. Fast-growing root tips were collected in 0.

Morphological observation and plant fertility evaluation
The appearance, shape and size of the hexaploid seeds were observed and compared to those of its parental species and their germination rates were assessed. The test of germination was conducted on 44 seeds of the hexaploid and 30 seeds of each of its parental species. The seeds were placed to germinate in Petri dishes with moist filter paper at 28˚C. The criterion for germination was a radicle length of > 1 mm. The germination rates were calculated as the percentage of seeds that germinated from the total number of seeds placed in the Petri dish.
The morphological observations carried out on the plants concerned: the as- Only fully stained and large pollen grains were scored as viable and non-aborted.
The quantity of viable pollen was estimated as the percentage of stained pollen.
The self-fertility was assessed by determining the average number of seeds obtained per self-pollinated flower. The cross-fertility was assessed by counting the average number of seeds obtained per cross-pollinated flowers.

Fiber fineness analysis
For fiber fineness analysis, cotton fibers were harvested at full maturity and used for the analysis. The fibers were combed and a tuft of parallel fibers was cut from the seed. Their free points were also cut and the median region was placed on a slide and covered with a cover glass. We let one or two drops of 18% NaOH

Molecular Analysis
DNA isolation Total genomic DNA of G. hirsutum, G. longicalyx and two synthetic allohexaploid (G. hirsutum x G. longicalyx) 2 plants were isolated from young fresh leaf tissues following the CTAB method as described by [22]. to EcoR I and Mse I adaptors with T4 DNA ligase to generate template DNA for amplification by PCR. Two consecutive PCR were performed: a pre-selective and selective PCR. In the pre-selective reaction, DNA was amplified using an AFLP pre-amp primer pair complementary to the adaptors and each having one selective nucleotide. Pre-selective PCR amplification was used as template for the selective amplification using AFLP primers, each containing three selective nucleotides. The PCR amplification products were run on 6% denaturing polyacrylamide gel using the ALF-Express (Pharmacia Biotech, Freiburg, Germany), which is an Automated Laser FLuorescence DNA sequencer. The obtained digital image of the profiles was analyzed. The scoring of bands was done as present (1) or absent (0) for AFLP marker loci and data were entered in a binary data matrix as discrete variables.

Mitotic Chromosome Analysis
Analysis of the mitotic metaphase plates showed 52 chromosomes for G. hirsutum, 26 chromosomes for G. longicalyx and 78 chromosomes for the putative (G. hirsutum × G. longicalyx) 2 hexaploid hybrid (Figure 1). This number of 78 chromosomes proves the hexaploid status of the material studied because it is in Open Journal of Genetics

Seed Aspect, Germination Rate and Seedling Abnormalities Analysis
The seeds of the (G. hirsutum × G. longicalyx) 2 hexaploid hybrid had normal appearance and shape but they were all larger than the seeds of the parental species ( Figure 2). This result is in accordance with [24] and [26] who reported that polyploids have usually larger seeds than parental species. It is probably a direct consequence of large cell size in polyploids [27], since genome duplication increases cell volume by increasing genome size [28]. This is in line with the expectation that the sizes of seed are a function of cell size, which is larger in polyploids [29].
In the germination test, the seeds of the hexaploid hybrid showed the relative lowest germination rate with 38 germinated seeds on 44 (86.36%) compared to its parental species G. hirsutum and G. longicalyx, which respectively presented 100% and 96.67% (29 germinated seeds on 30) germination rates. The germination rate gives an estimate of the viability of the seeds. The result obtained suggests a problem of viability of about 13% of the seeds produced by the allohexaploid hybrid. [24] working on synthetic polyploidy in Hylocereus monacanthus also reported problems of seed viability. The genome of newly formed polyploid plants usually undergoes extensive genetic and epigenetic changes which can alter gene expression and generate physiological changes that can affect seed viability [28] [30] [31].

Analysis of Morphological Observations of the Plants
The height of the hexaploid plants varied from 152 to 236 cm with an average of Open Journal of Genetics

Fertility Analysis
The mean proportion of stainable pollen grains (pollen fertility) of the hexaploid plants was 83% while the pollen fertility of G. hirsutum and G. longicalyx ap- is relatively lower than that of the parental species. By selfing, the hexaploid gave a mean of 4.39 seeds per pollinated flowers while G. hirsutum and G. longicalyx produced respectively 34 and 6 seeds per capsule on average (Table 1). These results show the restoration of fertility at the hexaploid level by the doubling of the chromosomes of the sterile triploid hybrid, even if the self-fertility of the parental species was higher. The results of cross-pollinations between the hexaploid and G. hirsutum (Table 1) gave practically no seed per capsule, i.e 0.018 and 0.3 seed per capsule when the hexaploid was used as female and male respectively. This very low success rate of cross-pollination, despite the good level of pollen fertilities (for both hexaploid hybrid and G. hirsutum) is probably due to the presence of incompatibility barriers between the hexaploid hybrid and G. hirsutum [36] [37].

Expression of Fiber Fineness and Resistance to Reniform Nematode Traits
The results of the fiber fineness analysis are presented in  [13], and its remarkable potential to improve the fiber fineness of G. hirsutum, with regard to the expression of this interesting trait in the hexaploid (G. hirsutum × G. longiclayx) 2 .
The data concerning the evaluation of the resistance to the reniform nematode are presented in Table 3. The parental species G. hirsutum presented the greatest number of eggs per gram root (205.8 eggs/g root) while the number of eggs per gram root of G. longicalyx and the hexaploid were very low, 0 and 3.8 eggs per gram root respectively. Compared to the susceptible G. hirsutum species, G. longicalyx (0% eggs/g root) and the hexaploid (1.96% eggs/g root) were very resistant. This finding confirms the high resistance to the reniform nematode of the wild African cotton species G. longicalyx [14] [15] and shows the inheritance and expression of this interesting trait in the hexaploid.

Molecular Analysis with AFLP Markers
The results of the AFLP analysis are presented in Table 4.    efficiently discerned differences between the two parental species and distinguished them distinctly from each other. This is consistent with [38] who also highlighted such differences between G. hirsutum and G. longicalyx, using SNP markers.
All 44 specific loci of G. hirsutum and 38 of 42 specific loci of G. longicalyx were found in the hexaploid. In total, all the loci revealed in the hexaploid come from G. hirsutum and G. longicalyx, which confirms the hybrid status of the hexaploid as indicated by cytological analysis. However, 4 of the 42 specific loci Open Journal of Genetics of G. longicalyx were missing in the hexaploid (Figure 4). [20] studying the synthetic allohexaploid (G. hirsutum × G. anomalum) 2 also found missing SSR alleles of the wild species G. anomalum in the hexaploid. Generally, the main reasons used to explain parental band missings in hybrids are loss of chromosomes or rearrangements of chromosomes. In the present study, the loss of chromosomes cannot explain the missing AFLP loci because the synthetic allohexaploid had the expected number of chromosomes (2n = 78). The explanation of chromosomal rearrangements is also unlikely since the two different hexaploid plants used for the molecular analysis had exactly the same missing loci. It is unlikely that there will be exactly the same recombination in two different plants. A slight genetic differentiation between the G. longicalyx plant used to develop the hexaploid hybrid and the G. longicalyx plant used in this molecular analysis could likely be the reason for the missing loci in the hexaploid hybrid.
Indeed, intraspecific differentiation exists in wild cotton species. For example, [39] using AFLP data, reported intraspecific variation (in terms of percentage of polymorphic fragments) in four species of cotton (G. aridum, G. laxum, G. lobatum, and G. schwendimanii). Anyway, phenotypic analysis showed that the current synthetic allohexaploid hybrid had the characteristics of interest (resistance to reniform nematode and fineness of the fibers) sought in G. longicalyx.
Therefore, this hexaploid material is perfectly suited for use in a cotton improvement program.

Conclusion
The present study demonstrated the hybridity and hexaploid status of the genotype studied using cytological and molecular marker methods. Moreover, this synthetic allohexaploid hybrid exhibited the useful traits of the African wild diploid species G. longicalyx with regard to the fineness of the fibers and the resistance to reniform nematode. This synthetic allohexaploid hybrid represents very interesting genetic stocks that can be used as a bridge for the transfer of useful agronomic traits from wild species to upland cotton varieties.

Conflicts of Interest
The authors declare that there is no conflict of interest.