| Mutants are widely used for studying genes and their functions. XuZhoul42 fuzzless-lintless line (fl) is a natural mutant of a upland cotton variety XuZhou 142 (wild type, WT). No difference has been observed between the fl and WT line, except that the former bear seeds without hair. In this paper, the fl and WT (regarded as a pair of isogenic lines) were used to search markers related to fiber initiation by using AFLP technique. Main results are as followed. 1. The improvement of extraction method of the cotton genomic DNA It is very difficult to gain the high-quality genomic DNA from cotton for its abundant polysaccharides and phenolic compounds. On the basis of CTA.B method, the extraction procedure was modified in several aspects, including adding PVP1J and -mercaptoethanol in the extraction buffer, inverting the sample microtube rapidly to mix well the sample with the extraction buffer, shortening the time of precipitation, picking Out and re-dissolving the precipitate, centrifuging the DNA solution with high speed to remove the impurity. High purity genomic DNA was extracted from cotton with this improved method. 2. AFLP analysis between the fl and WT line AFLP analysis between the fl and WT line, was conducted with sixty-four primer combinations supplied by AIFLPTM Analysis System Kit. Among 6360clear bands amplified, one differential band (CF1) was detected with the primer combination E-AGG/M-CTA. No stable polymorphism was found with other primer combinations between the two lines. Since the fl and WT line differ only in fiber initiation, the differential band was proposed to relate to fiber initiation. 3. Test of CF1 marker In order to further test the reliability of CF1 relation to fiber initiation, two individuals without seed hair and twenty-three individuals with seed hair were selected from 56 individuals in F2 population, derived from the cross between fl mutant and YuMian No.1, a variety with high lint percentage. CF1 was amplified in all 23 individuals with seed hair, and not in the two individuals without seed hair. Considering the scarcity of individuals without seed hair in F2 population, F3 populations were further used to test. AFLP analysis was performed in 21 individuals with seed hair deriving from 21 F2:3 family respectively and in 17 individuals without seed hair from 7 F2.3 family. The result showed that CF1 was observed in all 21 individuals with seed hair, and not in 17 individuals without seed hair. CF1 was also detected in five upland varieties, YuMian No.1, XuZhoul42, 9202034, T586, SiMian No.3. In sum, CF1 was co-segregated with fiber-bearing and it can be used as a molecular marker related to fiber initiation. 4. Recovery, cloning and sequence analysis of CF1 and Cb Specific fragment CF1 and an adjacent common fragment (Cb) were successfully recovered, cloned and sequenced. CF1 consists of 206 base pairs. BLAST analysis suggested that, CF1 had similarity to some sequence in Arabidopsis thaliana DNA chromosome, Nicotiana tabacum chloroplast genome and a few Homo sapiens clones, and the amino acid sequences deduced by six ORE were similar to phenol hydroxylase alpha subunit, outer surface protein C, NAuH dehydrogenase subunit 1, NADH- ubiquinone oxidoreductase, 2-oxoacid ferredoxin oxidoreduct and hypothetical 14.5KD protein.The common fragment (Cb) has 209 base pairs. It is same as CF1 except for 4 base replacements and a 3-base insertion. CF1 and Cb may be two members in... |
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