Cotton genetic diversity study by AFLP markers
Keywords: AFLP, cluster analysis, cotton, genetic diversity, PCR.
National Food Research Institute, Kannondai, Tsukuba,
polymorphism (AFLP) markers have been used to ascertain the intensity
of inherent diversity and relatedness in cotton (Gossypium
spp.) plants. The effectiveness of this method to distinguish inter
and intra specific difference in cotton could be handy in cultivar
recognition and in marker assisted parental selection tool for plant
breeders. Twenty cotton cultivars belonging to Gossypium hirsutum
L., and G. arborium L. from the
and cotton products occupy a pivotal position in the world economy.
Polymerase chain reaction (PCR) technology has promoted the development of a range of molecular assay systems which detect polymorphism at the DNA level and offer an alternative to the hybridization-based method of RFLP. In this study we used the most widely adopted PCR-based marker technologies, AFLPs for characterizing the natural variations of cotton cultivars. The AFLP technique is based on the amplification of short restriction endonuclease digested genomic DNA fragments onto which adaptors have been ligated at both ends. Primers complementary to the adaptors and possessing 30 selective nucleotides of one to four bases are used in a selective amplification reaction. The presence or absence of these selective nucleotides in the genomic fragments being amplified provides the basis for revealing polymorphism. AFLPs tend to generate dominant markers due to the differences in the DNA sequence in the selective 30 nucleotides immediately adjacent to the restriction enzyme site.
AFLPs have been used to estimate genetic relationships in many studies including cotton (Pillay and Myers, 1999) lentil (Sharma et al. 1996), soybean (Maughan et al. 1996), and barley (Becker et al. 1995). The objective of this study was to evaluate the genetic diversity in cotton cultivars and hybrids by the use of AFLP marker technology.
used in this study were consisted of 19 accessions of G. hirsutum
and one accession from G. arborium (Table
1). Seed material was obtained from the cotton-breeding program
Rohi old world cotton belonging to G. arborium. AC134 (obsolete local cultivar), B557 (obsolete local cultivar), FH87 (high oil content), S14 (high ginning outturn), SA100 (red leaves), these are belongs to the new world G. hirsutum L.
Laokra 5.5 x SA100, SA100 x Laokra 5.5, S14 x SA100, SA100 x S14, SA100 x Stoneville 857, Stoneville 857 x SA100, SA100 x DPL-7340-424, DPL-7340-424 x SA100, Laokra 5.5 x Stoneville 857, Stoneville 857 x Laokra 5.5, S14 x Stoneville 857, Stoneville x S14, B557 x AC134, AC134 x B557.
were germinated in
AFLP procedure (Vos et al. 1995) and as modified
by Pillay and Myers (1999) was used to access the
genetic diversity. DNA of 250 ng was extracted from all the 20 cultivars.
The DNA was digested with EcoRIand MseRI at
Bands that showed clear polymorphisms were scored visually as present (1) or absent (0). Cluster analysis was performed to provide a statistical basis to establish the number of cluster represented by the 20 genotypes. A clustering procedure (hierarchical cluster analysis) was performed using the unweighted pair group mean with arithmetical averages (UPGMA) method (Sneath and Sokal, 1973) by computer programme of SPSS. The output of this analysis was used to derive a dendrogram, which showed the phylogenetic relationships between all the cultivars.
Four primer-pair combinations were used to assay 20 cotton plants from each of the 20 accessions for AFLP analysis. An average of 40 to 80 scorable bands was detected after selective PCR amplification with each primer combination. The bands ranged in size from 50 to 500 bp. The results of agglomeration schedule (Table 2) and the dendrogram of genetic relationshipsfrom UPGMA cluster analysis (Figure 1) resulted in five major groups. The G. hirsutum genotypes were clustered into four major groups depending upon their origins, while the G. arboreum formed one separate group. The AFLP dendrogram assigned the genotypes into groups corresponding with their origin and pedigree relationships.
This study showed that AFLP is a very sensitive technique for detecting markers for genetic studies in cotton. Banding patterns obtained with AFLP were highly reproducible when the same sample DNA is used in independent experiments (Pillay and Myers, 1999). We used silver staining, the silver stained AFLP gels are considered to produce a larger number of better-defined bands than 32P-labeled gels (Cho et al. 1996).
Figure 1 shows that the cross Stoneville 857 x S14 (18) and cross S14 x Stoneville 857 (17) are reciprocal crosses of the parents Laokra 5.5 and SA100 and combined at the first level. Cultivar SA100 (6) joined this group at level three to form one group. The second group consisted of three sisters groups i.e. cross Stoneville 857 x Laokra 5.5 (16) and cross Laokra 5.5 x Stoneville 857 (15) were reciprocal crosses, while cross B557 x AC134 (19) and cross AC134 x B557 (20) were also reciprocal crosses of parents Laokra 5.5 and Stoneville 857, while third sister group consisted of crosses SA100 x DPL-7340-424 (13) and DPL-7340-424 x SA100 (14), which were reciprocal crosses of parents SA100 and DPL-7340-424. These two groups comprised of all the genotypes of US origin; this could explain the formation of this cluster.
third cluster consisted of both
fourth cluster consisted of the crosses SA100 x Laokra 5.5 (8) and
Laokra 5.5 x SA100 (7) formed the first subgroup and these were reciprocal
crosses of cultivars SA100 and Laokra 5.5, this subgroup was combined
with cultivar (3) B557, whose parent was (
cultivars AC134 (2) and FH87 (4) formed one group and these were the
The fifth group consisted of the only one cultivar Rohi (1) which originated from a cross (D9 x Local exotic) belongs to diploid spp. G. arborium and stands alone from the other cultivars of G. hirsutum spp.
the early 1970, high yielding tetraploid cotton varieties of
A unique feature of the AFLP data is its ability to discriminate all the taxa used in this study making it a very promising marker system in cotton especially since modern cotton cultivars are closely related and have a high level of genetic uniformity (Van Esbroeck et al. 1998). AFLP data clearly differentiated the diploid and tetraploid species from each other (Figure 1).
In conclusion this study showed that with the help of AFLP technique one can easily discriminate closely related taxa in cotton and provides sufficient evidence in the shape of numbers of polymorphic markers for variety identification.
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