Fine Mapping And Preliminarily Functional Verification Of A Novel Dwarf Mutant Gene In Upland Cotton

Plant dwarfism is an important agronomic character.The dwarf varieties have the characteristics of strong lodging-resistance,suitable dense plant ing and mechanized managem ent;It is also of great signif icance to reduce the intensity of labor,improving the utilizat ion of fertilizer and increasing the output.Dwarf mutant palys an essential role in dwarf breeding,and it is also a crucial gene resource for the study of plant stem growth and hormone regulation.Cotton has unlim ited growth habits,casuing the plant particular ly tall.At present,most of the country producing cotton all over the world used widely DPC,a chemical inhibitor,to restrict the growth of cotton,which controlled generally the plant height was at around 90-100 cm.Since the success of cultivat ion technology with "dense,dwarf,precocious",the use of chemical inhibitor to regulate cotton plant height has become a chief measure to increase the yield of cotton in Xinjiang.A large number of,however,spraying DPC consumed massive labor and material resources,also causumed serious environmental pollution problems.Cotton,one of the most important economic crops,occupies an important place in the national economy of China and the world.Therefore,it is a critical method of improveing the yield and the efficiency of agricultural production to take full advantage of the natural dwarf sources.Our team found a natural dwarf mutant in LHF10,derived from a Gossypium hirsutum L named Langfang 1 and a Gossypium mustelinum 12 generations of hybrid breeding,in 1998.It reached pure via self pollinat ion during 1998-2005 and named AS98.In this study,a QTL-seq technology was utilized to verify the init ial mapping obtained by previous research.The value of △SNP-index is signif icantly higher than the threshold in the region of 54Mb-55 Mb on the chromosome D12.The interval contains a SSR marker NAU5204,which also exists on the genetic linkage mapping constructed by predecessors,proveing reliably the result of the re-sequencing.That is,the section of 54Mb-55 Mb on chromosome D12 can be regarded as the candidate region associated with the AS98 dwarf gene.We,subsequently,detected the candidate interval by SV(Structure Var iat ion).It is found that there is a CNV(Copy Number Var iat ion)of 14 K large fragment between the dwarf(mutant)and high plant(wild type).To validate the existence of the CNV,we designed the Overlap primer of the CNV.The,ult imately,two terminal primers combinat ion(14k-5F/14k-2R)was used to expand the target fragments in the dwarf plants but failed to expand in the high plants by ordinary PCR.In order to further verify the reliability of the results,we utilized the primer to amplify with genomic DNA as template in 10 dwarf plants and 10 high plants.As expected,10 dwarf plants were all expanded,while the high plants failed to expand.These certify linkage between the CNV and dwarf traits.The research,further,indicated that this CNV contains a gene EXTR-DWARF.Next,we gained the sequence of the gene from the CottonFGD and designed the full length pr imers of the gene by using the Primer Design function of the NCBI.The gene was cloned from the dwarf,semi-dwarf and high plant genomic DNA.The sequencing results indicated that the gene is ident ical in three diverse phenotypic plants.The results of qRT-PCR revealed that the gene expression had a difference significantly in the mutant and the wild type.To further confirm that the gene is a candidate gene to dominate AS98 dwarfing,VIGS(Virus Induced Gene Silencing)test dwarf plant could restore to height of wild type,which also proved that the gene is likely to be related to AS98 plant dwarfing.The material,extremely dwarf,is a main resourc e for studying the mechanism of plant dwarfism in our research.The sites signal region associated with the dwarf was acquired by QTL-seq,and a CNV maker highly linked with the dwarf was identif ied by the analys is of mapping reads from NGS(next generation sequence).The function of EXTR-DWARF gene part of the CNV was related to plant dwarfism by VIGS.There is few reports structural variat ion,discovered by NGS and data analysis technology,casuing phenotypic changes in cotton.The Dwarfing target gene screened in our experiment clarifies the background of the dwarf material and provides a basis for future research.