Genetic divergence of colored cotton based on inter-simple sequence repeat ( ISSR ) markers

The management of colored cotton is an agricultural activity widely adopted by farmers located at Brazilian semiarid region. The fiber colors currently available are still limited to green and shades of brown, however, there is possibility to broaden the variability for this trait by using accessions from Gossypium Brazilian bank in breeding programs. Therefore, it is necessary to know the genetic diversity of available accessions in the collection. Here, the genetic divergence in colored fiber accessions was estimated in order to identify promising candidates for further use in hybridization procedures of cotton improvement. DNA of twelve accessions were extracted from leaves and used in inter simple sequence repeat-polymerase chain reaction (ISSR-PCR) assays, using commercial oligonucleotides. The genetic divergence was estimated by clustering-unweighted pair group method with arithmetic mean (UPGMA) method. Five groups were clustered among them, three were contributive results for further use in hybridization procedures, including Brazilian cultivars and Peruvian accessions. Based on level of divergence, we suggest that lines generated from these materials could generate news shades of fiber colors in further use for selection procedures in cotton breeding.


INTRODUCTION
Plant genetic resources represent a valorous portion of the biological diversity and contribute towards achieving security and sustainable development from preservation of cultivars, landraces, and wild relatives of important *Corresponding author.E-mail: liziane.lima@embrapa.br.Tel: +55 83 3182-4300.Fax: +55 83 3182-4367.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License plant species.Germplasm banks are reservoirs with an important hole to preserve these resources for further use in both applied and basic researches.The primary importance of these banks is that they carry undefined variation that proves to be a valuable resource for breeders to develop new crop cultivars (Sachs, 2009).Maintenance of germplasm banks have generally occurred in regions and by nations associated with crop production and commerce.
Cotton (Gossypium hirsutum L.) represents the most important natural fiber in the world.The genetic resources of Gossypium are extensive, dispersed globally across five continents, and consist of approximately 45 diploid (2n=2x=26) and five allotetraploid (2n=4x=52) species (Harlan and Wet, 1971;Fryxell, 1992;Stewart, 1994;Brubaker et al., 1999).Two allotetraploid species, G. hirsutum L. (upland cotton) and Gossypium barbadense L. (Pima, Sea Island or Egyptian cotton), account for the majority of cotton world production, although the former is widely grown worldwide (>90% of the total area) due to fiber yield and broad adaptation to several environments (Campbell et al., 2010;Percy et al., 2014).The quality of fibers from G. barbadense L. is better than G. hirsutum L., however the transference of fiber traits into upland genotypes provides limited success due to hybrid breakdown and segregation toward either parents (Gore et al., 2012;Gore et al., 2014).
The major cotton collections are located in United States, Russia, Uzbekistan, China, India, Brazil, Australia, and France (Campbell et al., 2010;Percy et al., 2014).The National Cotton Germplasm Collection (NCGC) has nearly 10,000 accessions of Gossypium accessible at the website www.ars-grin.gov.Each one has a single plant introduction number (PI) at the time the accession enters the collection.The first major breeding effort to incorporate the development and maintenance of a cotton germplasm collection was implemented in Trinidad in 1926 by the Empire Cotton Growing Corporation (Frelichowski and Percy, 2015).
The Brazilian collection is maintained by the Brazilian Agricultural Research Corporation (Embrapa) at the National Center for Genetic Resources and Biotechnology and currently has more than 3,000 accessions, several of them used for cotton breeding program to Savanna (Cerrado) and semiarid regions.
White fibers are desirable for most Brazilian textile industries, because they provide a uniform substrate for dyeing and finishing.Onto new market trends, other niches have emerged, such as the naturally colored fibers, that required no or less dying in the textile processing, reducing the pollution to the environment due to minor residual chemical toxicant (Xiao et al., 2007;Yuan et al., 2012;Feng et al., 2013).This technology is a differentiated product and therefore with higher valueadded, representing an alternative model of innovation, to promote social and sustainable transformations (Cavalcanti, 2012).
Colored fibers appear as brown or green during the fiber development process.Generally, the resistance, length and fiber percent are lower in colored than in white accessions due to the pleiotropic effects of fiber color genes (Carvalho et al., 2011;Lacape et al., 2005), but Brazilian breeders have made efforts in order to improve this trait via genetic improvement.According to Kohel (1985), several mutants conditioning fiber color and quality traits were described and mapped.Among the many fiber color variants described, almost all have had a dominant expression over the white fiber color of commercial cottons.
The Brazilian Company of Agricultural Research (Embrapa Cotton) coordinates a robust program to colored cotton, involving improvement to yield, fiber quality and environmental adaptation.Currently, six cultivars are commercially available and others top lines are in progress (Carvalho et al., 2011).Periodically, new different accessions are introduced and evaluated in selection procedures, aiming to identify promising materials to assist the colored fiber breeding.In this work the genetic divergence of new lines of cultivated and wild Gossypium accessions were estimated based on polymerase chain reaction-inter simple sequence repeat (PCR-ISSR) molecular markers.

Genetic resources and ISSR-PCR assays
Seeds of twelve cotton accessions, including wild and commercial cultivars, were used in this work.The genealogy and origin of materials are found in Table 1.DNA from seeds were extracted (Dellaporta et al., 1983) and further used in PCR assays.Twelve ISSR oligonucleotides, from University of British Columbia, were used in reactions (Table 2).

Genetic analysis of cotton accessions
Amplification products were scored as presence (1) or absence (0) of the band, for each accession.A binary data matrix was generated, from which it was calculated genetic similarity index between all individuals compared two by two, using the index agreement Jaccard (Sneath and Sokal, 1973).
The similarities (Sji) were calculated, according to the expression: where a means the presence of bands on both accessions; b, presence of band in first accession and absence in second and c is the presence in second and absence in the former.Clustering was done using symmetric matrix of similarity coefficient.A dendrogram based on Sij values was constructed using clustering technique of unweighted pair group method with arithmetic mean (UPGMA).In order to eliminate the nonhierarchical effects, the cophenetic correlation coefficient was estimated (Sneath and Sokal, 1973).Analysis was performed using the software GENES, version 2013.5.1 (Cruz, 2013).

RESULTS AND DISCUSSION
The ISSR oligonucleotides used for genetic analysis were contributive to identify divergent groups in cotton accessions.An average of 9 bands/oligo was obtained, with polymorphism rate varying from 75 to 12% (Table 2).UBC 866 and UBC 853, both rich in CT repetitions, were highly polymorphics, with rate of 73 and 71%, respectively.The pattern of bands obtained with these oligos is found in Figure 1.
Amplicons generated by ISSR-PCR assays were used to estimate the genetic divergence of cotton accessions by UPGMA method.A detail of fiber colors is found in Figure 2. Five groups were clustered (Figure 3), showing the following composition: Group A-compounded by five G. hirsutum L. accessions: BRS Topázio, BRS Verde and BRS Rubi, all colored fibers, and BRS 286 and V3, both white fiber.The peculiarity of this group is that all accessions are mid-cycle (140 to 160 days) and widely adapted to Brazilian Northeast region.V3 is a land race in pre-breeding proceeding and BRS 286 is full-sib of BRS Rubi.Both have the same parent, the drought tolerant CNPA 7H, developed by Embrapa to semiarid environments (Pedrosa et al., 2009;Carvalho et al., 2011).
About the other groups, the most relevant results were seen in B and D, both clustered wild G. barbadense accessions, from Peru, with fiber shades varying from cream to brown (Table 1 and Figure 3).Group C contained cvs.BRS 336 (white) and BRS 200 (brown), both G. hirsutum, with excellent fiber length.The last group contained only one G. barbadense accession, which remained distant from other Peruvians genotypes, because it is a land race with wild phenotype.
In overall, the use of accessions from groups A, B and D could be contributive to broaden the genetic basis of new lines of colored fibers.The white fiber accessions could contribute to improve the fiber qualities, providing genetic gains in selection procedures, while BRS Topázio and BRS Rubi, two Brazilian colored fibers of high yield and satisfactory fiber traits, could contribute to minimizing the deleterious effects often resulting from interspecific Gossypium crossings (Carvalho et al., 2011).

Conclusion
Groups formed with cotton accessions offer opportunity to generate new colored lines, by using crossing works, with high yield and fiber quality for further use in selection procedures in breeding program.

Table 1 .
Genealogy and origin of cotton accessions used in ISSR-PCR assays.

Table 2 .
Sequence of ISSR oligonucleotides used in genetic analysis of colored cotton fiber.
TNB: Total number of bands; NBP: number of polymorphic bands.