首次将PMC-FISH(花粉母细胞荧光原位杂交)技术应用于四倍体栽培棉与二倍体野生棉杂交所形成的三倍体杂种棉F1中,成功的鉴定了目标染色体中的单价体、双价体、多价体,还发现在非目标染色体上有星状和片段状的杂交信号。在以A组棉基因组DNA作为探针的PMC-FISH中,分别对三倍体杂种棉F1及其母本四倍体栽培棉的减数分裂中期I的染色体构型进行了鉴定。结果显示,四个三倍体杂种棉F1的减数分裂时期染色体的构型分别是:陆地棉(AD)1×雷蒙德氏棉(D5)为1IIIaad+1IIaa+4IIad+7IIdd+5Ⅰa+7Ⅰd;海岛棉(AD)2×旱地棉(D4)为1Ⅴaaaad+1IIIadd+2IIad+8IIdd+6Ⅰa+5Ⅰd;陆地棉(AD)1×澳洲棉(C3)为2IIIadd(adc or acc)+1IIaa+9Ⅰa+4IIcc(dc or dd)+14Ⅰd(c);陆地棉(AD)1×南岱华棉(C1-n)为:1IIaa+1IIad(ac)+10Ⅰa+6IIcc(dc or dd)+13Ⅰd(c)。然而,两个四倍体棉染色体的构型都是13IIaa+13IIdd。上述结果还显示,AD×D的两个杂种组合中,二价体多,单价体少,AD×C的两个杂种组合中,二价体少,单价体多;并且AD×D型杂种中A亚组染色体单体的数目比其在AD×C型杂种中的更少。这说明,与C染色体组相比较,D染色体组与四倍体棉种的亲缘关系更近。同时,我们的结果也证明PMC-FISH技术在分析三倍体杂种棉F1的亲缘关系中起着不可取代的作用。
Telomeres form the ends of eukaryotic chromosomes and serve as protective caps that keep chromosomes structure independency and completeness. The first plant telomere DNA was isolated from Arabidopsis thaliana and was shown to have tandemly repeated sequence 5′-TTTAGGG-3′. The Arabidopsis-type telomere has been found in many plants, but several reports indicate that this sequence is absent in some plants. Up to now, no research has been conducted on the telomere of cotton. In this paper, the Arabidopsis-type telomere sequence was amplified and cloned using the primers designed based on the fragment containing telomere sequence in an Arabidopsis bacterial artificial chromosome (BAC). Fluorescence in situ hybridization (FISH) with cotton metaphase chromosomes using the Arabidopsis-type telomere sequence as probes indicated that the signals were located at all chromosome ends of seven diploid and two tetraploid cotton species with different signal intensities among chromosome complements of different cotton species, even between long and short arms of the same chromosome. To identify the signals of FISH, the genome DNA of Xinhai 7, a cultivar of Gossypium barbadense, digested by BAL-31 nuclease was introduced in this study. The result of BAL-31 digestion indicated that the hybridization signals of FISH represent the outermost DNA sequence of each cotton chromosomes. So we first proved that the telomeric repeats of cotton cross-hybridize with that of Arabidopsis. The results of terminal restriction fragment (TRF) showed significant variation in telomere length among cotton species. The telomere length of cultivated cotton was close to 20 kb and was larger than those of wild cotton species whose telomere length ranged from 6 to 20 kb.
[Objective] The experiment aimed to study an efficient method of Nuclei extraction of cotton and provided technical support for constructing large-insert genomic library and sequencing complete genome. [Method] The cotton cotyledons germinated in dark moisture chamber for one week were chopped with a sharp sterile scalpel in a Petri dish which contained ice-cold nucleus isolation buffer (10 mmol/L MgSO4, 5 mmol/L KCl, 0.5 mmol/L HEPES, 1 mg/ml DTT, 0.25% Triton X-100 and 2% PVP40), then the nuclei were collected after selected through 100, 50 and 30 μm nylon meshes and centrifugation. Meanwhile, the tender leaves and cotyledons with different germination time in dark were treated by grinding method and sharp scalpel method. [Result] The chopping with a sharp scalpel method was very simple and rapid, which did not need grind and mercaptoethanol treatment and the successful extraction rate was 100%.[Conclusion] An efficient method of nuclei extraction of cotton with simple, high efficiency, rapid reaction and poison free were established.
