| Cotton（Gossypium spp.）is not only a natural source of textile fiber,but also an important food crop.Cottonseed contains high quality oil and protein,but its comprehensive utilization is restricted greatly because of the gossypol which is toxic to human beings and monogastric animals.Some wild diploid cotton species native to Australia,such as G.bickii,G.australe and G.nelsonii,possess a unique trait,known as the“delayed pigment gland morphogenesis”,that is,no pigment glands are found in dormant seeds until the seeds germinated.This character makes the oil and protein in cottonseed can be fully utilized and the natural insect-resistant trait of the plant can be retained,so that it is possible to achieve comprehensive utilization of cottonseed and improve the benefit of cotton planting.However,wild diploid cotton species have a far relationship with tetraploid cultivars and the low hybrid sterility makes the breeding work difficult.Some scholars pointed out that the synthetic allotetraploid obtained by the hybridization of wild diploid cotton species and diploid cultivars could be used as the intermediate germplasm material to realize the transformation and utilization of some precious traits such as delayed pigment gland morphogenesis.In this study,we crossed G.herbaceum with G.australe and created[A1A1G2G2]allotetraploid by doubling the chromosome with colchicine.The allotetraploid was studied by morphological observation,cytological identification and pollen vigor determination.Meanwhile,we studied the characteristics of gland morphogenesis and gossypol synthesis as well as the expression of related genes and compared them with the[A2A2G1G1]allotetraploid in order to explore the molecular mechanism of delayed formation of pigment gland.The main results were as follows:（1）The[A1A1G2G2]allotetraploid was successfully created by distant hybridization and chromosome doubling.In 2012,we crossed G.herbaceum with G.australe and four seeds were harvested,three of which germinated normally.Finally,two F1 plants survived and both of them were highly sterile.F1 hybrids were then treated with colchicine solution of different concentrations to double the chromosomes.In2016,the diploid hybrids were successfully doubled and we obtained the[A1A1G2G2]allotetraploid.（2）The[A1A1G2G2]allotetraploid was studied by morphological observation.It was found that the characters of the allotetraploid changed significantly after hybridization and polyploidization compared with its parents.50%of the traits invested exhibited typical intermediate phenotype.Meiotic observation showed that the chromosomes of the F1 hybrids were mostly univalent,indicating that the two parents were far related,which was the main reason for F1 hybrid sterility.PMC meiosis of the allotetraploid was normal and the chromosomes could pair together,producing regular tetraspore.The pollen morphology observation and staining identification also showed that the pollen viability of the allotetraploid was high.（3）The density and size of pigment glands were measured.It was showed that the[A1A1G2G2]allotetraploid was glandular,the dormant seeds and the main organs of the mature plants were covered with glands.However,there were no glands in the seeds of the[A2A2G1G1]allotetraploid until 24 hours after germination.Tissue section result confirmed mature gland cavities in the dormant seeds of the[A1A1G2G2]allotetraploid.While in the seeds of the[A2A2G1G1]allotetraploid,a specific ball structure existed,which gradually disintegrated during seed germination and formed pigment gland cavity 20 hours later.The above results showed that the newly created[A1A1G2G2]allotetraploid didn’t retain the trait of delayed pigment gland morphogenesis,indicating that the genetic mechanism of this unique trait is complex.（4）The content of gossypol in the seeds during germination and in the main organs of mature plants was determined.In dormant seeds,seeds with different germination time and main organs of the[A1A1G2G2]allotetraploid,certain amount of gossypol could be detected.However,in the dormant seeds of the[A2A2G1G1]allotetraploid,the gossypol content was very low.These findings indicated that the[A1A1G2G2]allotetraploid didn’t retain the delayed pigment gland morphogenesis,whose dormant seeds and mature plants were glandular and contained gossypol.Meanwhile,the other allotetrploid inherited this unique character from its paternal parent and the dynamic change of gossypol content was similar to that of low gossypol varieties,that is,no gossypol accumulated in dormant seeds due to the absence of glands.（5）Expression profiles of genes related to gland formation and gossypol synthesis during seed germination were analyzed.The results showed that in the[A1A1G2G2]allotetraploid,the expression pattern of GoPGF,a gene related to gland formation,was consistent with that of its maternal parent.The expression level is relatively high in the seeds of 0 h and 4 h after germination.However,in the dormant seeds of the other allotetraploid,the expression level of this gene was very low,which was close to that of G.bickii.As the seeds germinated,the expression level of GoPGF gradually increased.The result of qRT-PCR analysis of genes related to gossypol synthesis revealed that the related genes were highly expressed in the seeds of the[A1A1G2G2]allotetraploid at 24 hours after germination,while in the other allotetraploid,these genes highly expressed in the dormant seeds and during initial stage of germination.The above results illustrated that the expression pattern of genes related to gland formation and gossypol synthesis in the two allotetraploids was different. |
