Fine Mapping Of Hilum Spot In Watermelon Based On Genomic Sequencing Information

Watermelon is an annual vine of cucurbitaceae.With sufficient water and high nutritional value,it is one of the fruits that are often eaten in the summer.The hilum spot of watermelon is the color of the umbilical part that is inconsistent with the surface of the seeds and the trait can be stably inherited.On the one hand,the trait has certain guiding significance in watermelon resources,variety identification and genetic classification.On the other hand,the use of molecular markers to identify markers closely linked to hilum spot can provide ideas and techniques for molecular marker-assisted breeding in watermelon.However,there are no reports on the fine mapping of hilum spot in the current research situation at home and abroad.In this study,the cultivated watermelon line B135?with hilum spot?was used as the male parent and the cultivated watermelon line B132?no hilum spot?as the female parent to obtain F₁ generation B139,B139 and B132 backcrossed to obtain BC₁ population.Morphological observation of the characteristics of 98 individual plants of BC₁ population and analysis of their genetic laws.Based on RAD sequencing technology and QTL-seq technology,preliminary interval localization of hilum spot was completed.Subsequently,the experimental population was screened and verified by molecular markers.The experimental population included 94individual in the BC₁ population that could clearly observe the hilum spot trait,and two randomly selected materials B5-8 and 18CB83,and the physical regions of QTL were gradually reduced.In final region,biosignal analysis was combined to identify candidate genes.Design and develop molecular markers on candidate genes for experimental population to see genotype accuracy.The candidate gene was subjected to qRT-PCR to detect gene expression levels.The molecular markers on the candidate genes were applied to the germplasm resource population of 71 materials for verification.The following experimental results were obtained:?1?F₁ generation B139 had hilum spot.Through statistical analysis of 98individual the trait of hilum spot in BC₁ population,the results showed that BC₁population had hilum spot polymorphism,of which 46 had hilum spot,48 strains without hilum spot,and 4 strains could not be identified because the color of the seed coat was too dark,which was basically consistent with the Mendelian genetic separation ratio of 1:1.From the situation we conclude that the hilum spot of watermelon species are controlled by a major QTL.?2?Based on RAD sequencing and QTL-seq analysis,combined with the phenotype results of BC₁ population,a QTL for hilum spot of watermelon seeds was identified in the chromosome 3,and the LOD value was 3.87,explaining 88.01%of phenotypic variation.The physical interval is 5366042 to 5784364 bp.?3?In the QTL confidence interval,a total of 22 Indel markers were developed,and 11 polymorphic Indel markers were screened by the parents.The initial positioning interval was narrowed down to the molecular markers Zshpr84 and Zshpr87,5.69-5.73Mb,about 40Kb.?4?In the 40Kb interval,the biosignal analysis found that there is only one gene,namely Cla019481.Combined with gene annotation and sequence analysis,Cla019481 was identified as a candidate gene for hilum spot in watermelon.The dCAPS marker Zshpr101 was developed on Cla019481 to be verified in the experimental population.The genotype and phenotype coincidence rate was 100%.?5?The expression level of candidate gene Cla019481 in different developmental stages of having hilum spot and no hilum spot seeds was verified by qRT-PCR.The results showed that the Cla019481 expression level was significantly increased in the seeds have hilum spot at different developmental stages,and there was no significant difference in the seeds of having not hilum spot.It is proved that Cla019481 is the main gene controlling the hilum spot of watermelon.?6?The dCAPS molecular marker Zshpr101 was used to verify the germplasm resource population of 71 materials,and the genotype and phenotype coincidence rate was 69%.