Production of New Cotton Interspecific Hybrids with Enhanced Fiber Fineness

To improve cotton fiber fineness, the (Gossypium hirsutum L. × Gossypium longicalyx Hutch. & Lee)2 allohexaploid and the [(Gossypium hirsutum L. × Gossypium thurberi Tod.)2 × G. longicalyx] allotetraploid were backcrossed to G. hirsutum to produce introgressed genetic stocks. The ribbon width (RW) of 600 swelled fibers produced by the hybrids, their parents, and their backcross progeny were analyzed for each compared genotype using an optical microscope. The RWs varied between 6.41±2.15 μm for G. longicalyx to 17.45±2.98 μm for the G. hirsutum parent cultivar C2. Fibers produced by the trispecific hybrids and their progeny were finer than the bispecific hybrid material. For the introgressed stocks, the lowest RWs were observed for the trispecific hybrid (10.79±2.14 μm) and certain backcross progenies (between 11.98±1.27 μm to 12.71±1.61 μm). The allohexaploid RW was 13.58±1.41 μm. One of its tetraploid progeny produced approximately the same value (13.94±2.48 μm). These results show that G. longicalyx is a potential genetic stock for cotton fiber fineness improvement. The genetic stocks produced are valuable materials for improve the fineness of cotton fiber. Keyswords: Gossypium, interspecific hybridization, fiber fineness, RW


Introduction
Cotton fiber is an important raw material for the textile industry.The world production of cotton fiber was estimated at 119.7 million bales in 2012/2013 (USDA, 2013).The cotton fiber price is determined based on the fiber quality.The quality is defined by parameters, such as length, strength, fineness, maturity, and color.These parameters are established during fiber development.Among the fiber physical properties, fineness is considered as a major trait that determines the cotton fiber quality.This character influences the spinning limit, yarn strength, yarn uniformity and productivity.The fiber fineness determines the number of fibers required to spin a yarn at a particular thickness.The finer a fiber is, the more significant the quantity of fibers in the yarn cross-section.Spinning finer fibers produces stronger and uniform yarns (May, 1999).Long et al. (2010) found that the strongest yarns were produced from genotypes with the longest and finest fibers.
Cotton fiber fineness is expressed as the perimeter, diameter or RW, cross sectional area, and standard fiber weight (Gordon & Hsieh, 2006).Most cotton breeders and geneticists estimate cotton fiber fineness in terms of linear density (tex) values, which are conventionally determined using a gravimetric fineness method or AFIS, and micronaire values are measured using HVI.More recent methods have been verified for determining fiber fineness.The methods are based on an image analysis system (Huang et al., 2002), electromagnetic scattering using a laser system (Thomasson et al., 2009), light-scattering experiments (Aslan et al., 2003;Adedoyin et al., 2011) and vibroscopic techniques (Delhom et al., 2010).An instrument was recently developed using polarized light microscopy and image analysis, wherein a cottonscope measures the fiber RW, maturity and linear density (Rodgers et al., 2010(Rodgers et al., , 2012)).
The cotton genus Gossypium (Malvaceae) contains 45 diploid species (2n = 2x = 26) and five tetraploid species (2n = 4x = 52) (Fryxell et al., 1992).The diploid species are differentiated into eight genome groups designated by the capital letters A, B, C, D, E, F, G and K (Stewart, 1995).The sole F genome species, G. longicalyx, is separated from the remaining genome groups based on its distinctive geography, morphology and ecology (Fryxell et al., 1992).All diploid Gossypium genomes contain an exclusive wild species, except for the A genome.The tetraploid species contain two distinct subgenomes related to the A and D diploid genomes (Wendel & Cronn, 2003).The tetraploid species G. hirsutum L., which is cytogenetically known as (A h D h ) or (AD) 1 , is the main cotton cultivated throughout the world; it accounts for 95% of the world lint production.The level of genetic diversity among agriculturally elite types of G. hirsutum is low (Gingle et al., 2006a(Gingle et al., , 2006b)).Diploid species are an important reservoir for alleles of interest that can be exploited to improve cotton fiber qualities (Mergeai, 2006).The results generated thus far in numerous interspecifc breeding projects in different parts of the world show cryptic, beneficial alleles in wild germplasms and that multigenic traits can be introgressed through interspecific breeding using the main cultivated cotton species, irrespective of the donor species performance.According to Demol et al. (1978), cotton fiber fineness can be improved by using the African wild diploid species G. longicalyx Hutch & Lee.To verify this hypothesis, the (Gossypium hirsutum L. x G. longicalyx)² allohexaploid and [(G.hirsutum × G. thurberi Tod.)² × G. longicalyx] allotetraploid were created at GemblouxAgro Bio-Tech (University of Liège).The objective of this study was to evaluate the possibility of enhanced fineness through introgression in the backcross progenies of these two interspecific hybrids.
ii).Selfed progenies of MAALS (Monosomic Alien Addition Lines) obtained by Koto (1983) from the bi-specific hexaploid (G.hirsutumcv.C2 × G. longicalyx)² (G 368) were created in Gembloux using the scheme illustrated in Figure 1. 10 out of 13 possible MAALs of G. longicalyx on G.hirsutum numbered from I to XIII provided by the CIRAD (France) gene bank were used in this study.
All plant materials were grown at the same time in Gembloux Agro Bio-tech greenhouses.The seed-cotton was randomly harvested without regard to the boll position.

Mitotic Observation
The karyotype of MAALS progenies likely to carry a supernumerary chromosome was determined based on their phenotype using mitotic cells and the method described by D' Hont et al. (1995).Roots were collected from growing seeds germinated in Petri dishes on filter paper at 30 °C for 48 hours.First, the roots were pre-treated in 0.04% Hydroxyquinoline at room temperature for 4 hours in the dark and were fixed at metaphase in Carnoy's I solution (glacial acetic acid 1: ethanol 3) for 48 hours.Thereafter, the roots were stored in 70% ethanol at 4 °C, the roots tips were digested in an enzyme solution (5% cellulose Onozuka R-10 and 1% pectolyase Y-23 in citrate buffer) at 37 °C for 1 hour, and they were compressed onto slides in a fresh fixative (3:1 ethanol: acetic acid).Chromosome preparations were visualized and counted under a fluorescent light microscope after staining with 4',6-diamidino-2-phenylindole (DAPI)/Vecta-shield.

Ribbon Width Measurement
RW measurements were performed on fibers produced by three seeds randomly harvested without regard to the boll position and opening date.Certain HTL progenies were quasi sterile and did not produced sufficient seeds; thus, the measurements were performed on 2 seeds.For each seed, the diameters of 200 swollen fibers were measured using the software NIS-Elements BR 2.30 and a Nikon Eclipse E800 microscope equipped with a digital Nikon camera.To perform the measurements, the fibers were straightened and paralleled through combing.The median region of the fibers was used for the observations.Each parallel fiber sample was drawn along a glass slide.The fibers were moistened and swollen in an 18% NaOH solution.The measurements were performed on one side of the slide, and the slide was moved across the field of vision to avoid repeating the measurements on any fiber based on Roehrich's (1947) recommendations.The fiber RWs were determined by dividing the mean of the measured diameters by the 1.3 Summers coefficient (Roehrich, 1947).

Statistical Analysis
The mean and standard deviation were calculated.The kurtosis and skewness of the fiber fineness frequency distributions (Pearson, 1895(Pearson, , 1905) ) were calculated using Minitab 17 Statistical Software 2010.ANOVA was performed to compare the RW means between and within genotypes.The multiple comparisons were examined for significance at P < 0.05 using Tukey's test, and the output was condensed into letter grouping.

Results and Discussion
Monosomic alien addition lines (MAALs) with only one chromosome from G. longicalyx in the genetic background of G. hirsutum and HTL trispecific hybrids containing the two wild diploid species genomes G. longicalyx and G. thurberi in a G. hirsutum genetic background were used in this study.
Seed setting and seedlings were obtained through backcrossing and selfing the MAALs progenies as well as the HTL trispecific hybrid to G. hirsutum (Table 1).The material morphological traits are also reported in Table 1.
The HTL plants presented no phenotypic segregation, and the BC1 plants exhibited segregation based on morphological characters.The leaf shape varied, and the number of lobes per leaf ranged from 1 to 5. The BC1 plant leaf size varied, but all were larger than the G. thurberi and G. longicalyx leaves and similar to G. hirsutum.We examined the normalities of the fiber RW distributions for all genotypes, and skewness and kurtosis were used to characterize the symmetry and peakedness of the fiber RW distributions.The skewness coefficient measures the degree of symmetry in the variable distribution.A positive or negative skewness, respectively, indicates a long asymmetric right tail and a long asymmetric left tail (Doane & Seward, 2011;Hall, 2014).A skewness value of zero indicates normal distribution, whereas a skewness value greater than 1 indicates that the distribution differs significantly from a normal distribution.

Most
Genotypes that present a negative skewness exhibit a distribution with more coarse fibers, while genotypes with a positive skewness exhibit more fine fibers.
Kurtosis measures the degree to which a distribution is more or less peaked compared with a normal distribution.
A high kurtosis value indicates a relatively peaked distribution with a long tail, and a low kurtosis signifies a relatively flat distribution with thinner tails (Zenga, 2014).A high kurtosis indicates a narrow fineness range for the fibers.
The parental species G. hirsutum var.C2 and G. longicalyx exhibited positive skewness and opposite kurtosis.
(Table 2).The G. hirsutum var C2 presented a negative skewness with larger fibers than the wild parental species G. longicalyx.
The RW distributions for these cotton samples are shown in Figure 3 wherein the x-axis represents the fiber RW, and the y-axis represents the fiber frequencies.Despite exhibiting the same mean, the three genotypes presented different distributions.We presume that MAAL III # 2 contains more fine fibers than the remaining genotypes, which indicates that the mean cannot be used to differentiate the cotton fineness levels.

Figure 3. RW distribution of some MAALs
Statistical parameters such as skewness and kurtosis, are necessary to further characterize the RW of different cottons presenting the same mean.A fiber RW distribution that is symmetrical (skewness = 0) around the mean and as peaked (kurtosis > 0) as possible is necessary.
The HTL and the genotypes BC2-5 and BC1-6 exhibit a negative skewness and positive kurtosis value, which indicates a narrow range of fiber RWs with more coarse fibers than the mean.The mean RWs of the fibers were similar for the three seeds of G. hirsutum var C2 and HTL F1 BC2-16 plants (Tables 2 and 3).Bradow et al. (1997) observed significant variations in Pee Dee cotton (G.hirsutum L.) fiber diameters, cross-sections, and circularities at the boll and locule levels measured on 2500 fibers with the AFIS lengths and diameter modules (Zellweger-Uster, Knoxville, TN).In this study, the overall average Pee Dee 3 fiber diameter was 12.7±0.9µm.Studying the influence of seed location on fiber quality, Davidonis and Hinojosa (1994) found that fiber perimeters of apical seeds were thinner in the micropylar region than the chalazal regions, and, conversely, the fiber perimeter of basal seeds were larger in the micropylar region than the chalazal regions.These authors hypothesized that each fiber cell receives a certain allotment of assimilates for development.
According to Tukey's test, MAAL-III # 2 (2n = 53) and three introgressed stocks (2n = 52) produced through the bi-specific introgression method did not exhibit significantly different fiber RWs compared to the hexaploid (G.hirsutum L. × G. longicalyx)².Note.* Significant at the 0.05 probability level; † Means followed by the same letter are not significantly different from each other; Chr Nb: Chromosome number; SD: Standard deviation.
The HTL and HTL F1 BC2-5 RWs were, respectively, 10.79±2.14 and 12.71±1.61µm.These values were lower than the average hexaploid RW; these genotypes carry several G. longicalyx and G. thurberi chromosomes in a G. hirsutum background.Tukey's test showed a significant difference between the progenies of [(G.hirsutum × G. thurberi.)²× G. longicalyx] hybrids and their parental species.The coefficients of variation ranged from 9.26 to 21.44%; this value ranged between 21.04 and 21.44% for the wild species G. longicalyx and was lower at 12% for the hexaploid MAAL-F l -I-6, MAAL-III # 2, and HTL F1-BC1.The high variation observed in this study is first due to the large variation within the cotton seed.A source of error with a lower effect may the microscope measurement, but the reliability of the fiber RW measurements greatly increased through mercerization of the fibers before the measurements.Huang et al. (2002) performed longitudinal measurements of 25 cotton fiber samples from one bale; they analyzed 1.000 to 4.000 fibers for each sample using an imaging analysis method, and the coefficient of variation for the mean fiber width from one cotton bale was 2.91%.Adedoyin et al. (2011) reported that the coefficient of variation from laser diffraction system measurements on 20 fibers from one bale of cotton was 9.35%.
Few studies have focused on cotton fiber quality measurements at the seed level because modern instruments require a large sample size, and non-instrumental measurement methods are tedious and time consuming.As a result, the full differences in fiber quality within a single plant and seed have not been estimated.The cotton scope was recently developed and only requires 50 mg of lint per replication, which would be useful for fiber fineness characterization at the seed level.
Baezinger et al. ( 2007) reported that to enhance the probability of genetic improvement for a specific trait of interest, the trait must be satisfactorily measured and heritable, and sufficient genetic variability must exist to ultimately realize long-term genetic improvement for the trait through plant breeding.Cotton fineness measured as perimeter, linear density or micronaire is moderately to highly heritable (May, 1999).Enhanced fineness is particularly important because selecting a smaller perimeter could facilitate selection of a low-micronaire, longer, and stronger fiber (Kloth, 1998).
According to Long and Bange (2012), RW provides the best average indication of fiber fineness or coarseness regardless of how the external fiber architecture is influenced by other components, such as convolutions, level of fullness affected by fiber maturity, and fiber perimeter.RW is an attribute that relates to how fibers will best pack together in yarn.RW is more related to yarn strength than either micronaire or gravimetrically determined linear density; that is, fibers with smaller RWs spin stronger yarns (Bange et al., 2010).These authors found that less mature cotton from a cooler growing season produced stronger yarns and fabric partly due to the fiber's smaller RW, which had a greater effect on fiber packing density and inter-fiber friction.RW measurements through microscope analysis remains a reliable and accurate means for highlighting the broad differences observed in the analyzed material.
Although a genetic basis for the fiber fineness is unknown, many studies have localized the QTL associated with this trait to 1 to 8 different chromosomes (Nacoulima & Mergeai, 2014), which indicates that the cotton fiber fineness is a trait controlled by several genes.The tri-specific introgression pathway is more suitable for introgression of genes located on several chromosomes (Mergeai et al., 1997).Under this approach, all of the chromosomes for the wild diploid species encountered the G. hirsutum chromosomes.The recombination frequency for homoeologous chromosomes between the donor species and the sub-genomes A h and D h is typically higher for trispecific hybrids than bispecific hybrids (Mergeai, 2006).
However, exploiting trispecific hybrids is difficult because this genetic material is highly unbalanced with low fertility.Linkages among desirable and undesirable traits by the donor and bridge species also complicate successful use of this genetic material.Several backcrossing and selection schemes are necessary to produce fertile offspring and eliminate uninteresting traits as well as fix the selected agronomic traits.
Within the (G.hirsutum × G. longicalyx)² allohexaploid progeny, three introgressed stocks (2n = 52) present enhanced fineness, which suggests that multiple introgressions likely occurred in these plants; thus, they are valuable material for fineness introgression into Upland cotton.
The introgressed stocks with enhanced fiber fineness are from three different MAALs.Presumably; one or several QTLs from the G. longicalyx chromosomes that influence fineness were integrated into the G. hirsutum background.Another hypothesis is that the different MAALs carry the same alien chromosome, which was preferentially transmitted to the pentaploid progenies.This phenomenon was observed in various studies (Ahoton et al., 2004;Benbouza et al., 2008;Sarr et al., 2012).
Recombination may also have occurred at the hexaploid level.G. longicalyx loci from different linkage groups were detected on different MAALs (data not shown), which highlights the importance of using molecular markers to monitor chromosome fragment introgression. Fig

Table 1 .
of the MAALS progeny plants presented G. hirsutum morphological traits.The remaining plants (38.5%) differed morphologically from the G. hirsutum plants.The MAALS progeny karyotypes were determined.RW measurements were performed on plants with 2n = 53 and plants with 2n = 52, presenting a sign of introgression.Seed germination and number of seedlings of plants

Table 2 .
Skewness and Kurtosis values of MAALs and their parental species

Table 3 .
Skewness and Kurosis value of HTL plants and their parental species

Table 4 .
Average RW (µm) of the fibers of the MAALs and their parental species

Table 5 .
Average RW (µm) of the fibers of the HTL progenies and parental species Note.*Significant at the 0.05 probability level; † Means with the same letter are not significantly different from each other; Chr Nb: Chromosome number; SD: Standard deviation.