Localization And Analysis Of Genes Regulating Determinate Inflorescence In Brassica Napu

Brassica napus（AABB,2n=38）is the main cultivated type in China and other countries.The inflorescence of Brassica napus is usually indeterminate,while the determinate inflorescence of Brassica napus has been found in recent years.Previous studies have confirmed that the lines with determinate inflorescence usually had the characteristics of early flowering,early growth period and low plant height.In this study,a newly discovered determinate inflorescence mutant of GD605-2 was used as the experimental materials to study the formation mechanism of determinate inflorescence through microscopic phenotype observation,traditional ILP mapping,BSA-Seq mapping,gene homologous cloning,q RT-PCR analysis,and development and application of associate markers.The results were as follows:1.Using paraffin section technology,the shoot apical meristem of wild-type materials（indeterminate inflorescence）and mutant material（determinate inflorescence）at 3-leaf stage,4-leaf stage,5-leaf stage,6-leaf stage,7-leaf stage and 8-leaf stage,for microscopic phenotype observation.According to the section results,it was inferred that the period of determinate inflorescence variation occurred between the 5-leaf stage and the 6-leaf stage of rape development.2.Using 1,059 pairs of ILP markers to detect the two extreme DNA pools（determinate inflorescence and indeterminate inflorescence）,and the polymorphic markers were used to detect the the mapping population.Finally,two linkage groups containing a total of 17 polymorphic markers on LG01 and LG02 were obtained,in which two determinate candidate genes of Bnadti1 and Bnadti2 were included,respectively.The total length of chromosome LG01 is 77.8 c M,with an average distance among markers of7.07 c M,and the distance from Bnadti1 to Bnap PIP744 and Bnap PIP976 is 0.8 c M and 2.9c M,respectively.In addition,the total length of chromosome LG02 was 41.8 c M,with an average distance among markers of 6.9 c M,and the distance from Bnadti2 to Bnap PIP1870and Bnap PIP859 is 2.9 c M and 1.5 c M,respectively.3.BSA-Seq analysis was performed on two extreme DNA pools（determinate inflorescence and indeterminate inflorescence）and two parents of B.napus.According to the results,sliding window calculation was performed to screen the regions with the highest ED values on chromosomes C02 and C06.In chromosome C02,the candidate QTL region was 3.080 Mb,containing 235 candidate genes.In chromosome C06,the candidate QTL region was 0.280 Mb,containing 47 candidate genes.4.In the ILP mapping,Bnadti1 was located on C02:328,559-68,422 bp,which was coincident with the BSA-Seq mapped region on C02:320,000-3,400,000 bp.To narrow the QTL region,the markers based on the In Del results from BSA-Seq methods were designed and used,and the QTL region was finally narrowed to one 720 kb region（Chr C02:1080000-1800000,720 kb）.Within the narrowed region,only one gene of TFL1 was once reported to be related to the regulation of determinate inflorescence,where one candidate gene of TFL1 was detected,and renamed Bna TFL1.While Bnadti2 was located on C06:24,148,136-33,558,240 bp,which was identified as the same region with the region on C06:27,000,000-27,280,000 from BSA-Seq results.Within the region located Bnadti2,only one candidate gene of AP1 was detected for the regulation of inflorescence development,and renamed Bna AP1 as another candidate gene.5.Using the method of molecular marker-assisted trans-breeding technique,we has successfully transferred the determinate inflorescence trait into 20 breeding materials.6.Use PCR amplification and sequencing technology to clone Bna TFL1 and Bna AP1in the trans-breeding lines.The cloned Bna TFL1 gene has a full length of 1,077 bp.Two amino acid mutations at the 26^th（P→R）and 175^th（A→S）in the trans-breeding were detected,in comparison with the wild plants.Bna TFL1 belongs to hydrophilic,non-secreted and non-transmembrane protein,with a molecular formula of C₉₁₉H₁₄₅₁N₂₆₁O₂₅₉S₅,and the isoelectric point is 9.69.At the same time,the CDS sequence of Bna AP1 was cloned and the total length is 770 bp.Two amino acid mutations at the 122^nd（S→P）and 160^th（A→S）in the trans-breeding were detected,in comparison with the wild plants for Bna AP1 gene.Bna TFL1 belongs to hydrophilic,non-secreted and non-transmembrane protein,with a molecular formula of C₁₃₀₉H₂₀₈₉N₃₉₁O₃₉₉S₁₆₃,and the isoelectric point is 8.25.7.The q RT-PCR analysis of Bna TFL1 and Bna AP1 in different stages and tissues of trans-breeding and wild-type plants were done.The results indicated that Bna TFL1 gene was mainly expressed in the stem of plants,and the expression level in trans-breeding plants was higher than that in the wild-type plants,with the highest expression level at 6-leaf stage.Bna AP1 gene was mainly expressed in the leaves of wild-type plants,and the expression level was the highest at 6 leaf stage.In the trans-breeding plants,Bna AP1 gene was mainly expressed in stem tip,with the highest expression level at 5-leaf stage.In this study,we studied the regulatory genes of GD605-2 with determinate inflorescence through mapping,gene cloning and analysis.The results would lay a foundation for the research of regulation mechanism of rape determinate inflorescence and breeding rape varieties with limited inflorescence.