| The plants of the genus Fragaria bloom white flowers.Pink-flowered strawberry is an intergeneric hybrid(Fragaria × Potentilla),which is a new type of perennial herb with both ornamental and edible properties.Its color is the important value that determines the ornamental value of pink-flowered strawberry.The petals show different levels of pink ranging from light pink to red color.The formation of red flower color is not only the result of anthocyanin accumulation but involves many other factors such as co-coloring,vacuolar p H and cell traits.Anthocyanin is the main pigment of pink-flowered strawberry petals.The relationship between the expression of anthocyanin synthesis pathway-related genes and anthocyanin content was analyzed,and some molecular markers related to red flower traits were developed in this paper,which was of great significance to examine the mechanism of flower color formation and molecular breeding in pink-flowered strawberry.In this study,the hybrid progeny of pink-flowered strawberry was used as the test material.The material basis and physical and chemical factors of the flower color formation of pink-flowered strawberry were systematically studied through the observation of petal anatomical structure,the determination of main chromogenic pigments in petals the content of anthocyanin,the p H value of petals and the antioxidant activity.The pink-flowered strawberry cultivar ’Sijihong’showed red petal.The three developmental stages selected with significantly different flower color(‘L’,‘Z’ and ‘D’)were used for transcriptome analysis.The qRT-PCR method was used to identify the transcriptional levels of structural genes and transcriptional regulatory genes involved in the anthocyanin synthesis pathway in different colors of pink-flowered strawberry.At the same time,the transcriptome sequence information combined with SSR marker technology was used to screen the molecular markers related to the flower color traits of pink-flowered strawberry.It lays a theoretical foundation and practical basis for molecular marker-assisted breeding of pink-flowered strawberry.The results were as follows:The color difference meter and color chart were used to compare the different colors of the pink-flowered strawberry hybrid offspring.The results showed that the correlation between redness(a*)and yellowness(b*)is not obvious,while brightness(L*)had a significant negative correlation with redness(a*)and chromaticity(C*).Scanning electron microscopy was used to observe the epidermal cell structure of pink-flowered strawberry petals.It was found that the shape of red,deep pink and pink petal epidermal cells was irregular mosaic,and their cell epidermis tended to be gentle.The shape of white petal and light pink petal epidermis was spherical(oval)convex;The color pigments in the red,deep pink,pink and light pink petals were anthocyanins and flavonoids,and the two pigments werecombined to form a flower color.The color pigments in the white petals were flavonoids,no carotene was found in flower petal;the anatomical structure of the petal was observed by hand slice method,and the longitudinal section of pink-flowered strawberry was of typical structural features,which were divided into upper epidermis,palisade tissue,sponge tissue and table below.In white petals,it contained light yellow flavonoids or flavonols.Red,deep pink,pink and light pink petals contain anthocyanins and flavonoids,and the two pigments were combined to form a flower color.The content of anthocyanin in the petals was determined by p H differential method,and it was found that the anthocyanin content decreased with the saturation reduce of the red color of the petals.Among the five colors,red and deep pink flowers had stronger antioxidant activity.Based on the RNA-seq library,candidate internal reference genes BZIP,TIM,TUB,EF1 a,AP2 and TIP41 with insignificant expression differences were screened.The stability of gene expression of each candidate internal reference gene under different experimental conditions was analyzed by ge Norm,Normfinder and Best Keeper software and compared △Ctmethod.The results showed that the results obtained by different statistical analysis methods were also different.In all samples,the most stability genes used by ge Norm and Best Keeper statistical analysis were AP2 and TIP41;In different colors,the most stability genes were also AP2 and TIP41 by ge Norm,Norm Finder,Best Keeper and △Ct;Among the different ploidy,the most stability genes were AP2 and EF1 a compared by Norm Finder,Best Keeper and △Ct;From the overall analysis results of the four softwares,the highest stability gene was AP2,and followed by TIP41.The optimum number of internal reference genes for accurate quantitative experimental results was determined to be 2.The best combination of required internal parameters was determined to be AP2+TIP41 by the ge Norm pairing coefficient V value.Through RNA-seq,qRT-PCR was used to analyze the gene expression of 20 structural genes and two transcriptional regulatory genes in different flower colors of anthocyanin synthesis pathway of pink-flowered strawberry.And the linear regression equation was established between these gene expressions and total anthocyanin contents.The results showed that the expression of most structural genes was positively correlated with total anthocyanin content,except Fp FLS(R2=0.001),Fp ANR(R2=0.0069),Fp GT1(R2=0.018)and Fp UGT75C1(R2=0.276).And the transcription factor MYB10 in different flower colors was significantly positively correlated with total anthocyanin content,and it positively regulated the anthocyanin synthesis.However,the expression of Fp MYB6 in different colors was negatively correlated with total anthocyanin content,and negatively regulated the anthocyanin accumulation.Based on the SSR in the RNA-seq known genes,the characteristics of genic-SSRsobtained from transcript sequencing of pink-flowered strawberry were analyzed.The SSR locus in the transcript of the pink-flowered strawberry was found to be abundant,and 12,077 unigenes were identified from 139,292 transcripts,and a total of 14,264 SSR loci were found.Among them,1,823 unigenes contained more than one SSR locus,and 758 SSR loci existed in complex forms.Among all repetition types,the SSRlength ranged from 10 to 228 bp with an average length of 16.58 bp.There were different distributions from single base to six base repeats,and the number of each repeat type varies greatly.Of the identified SSRs,176 motif sequence types were searched,and the most abundant of which was a dinucleotide type(GA21.33%).The AGG/CCT motif was the most frequent in the trinucleotide repeat of the coding region.Among the dinucleotide repeats,the AC/GT motif was the most common.Based on deep transcriptome sequencing,a specific marker with four specific markers isolated from the flower traits of pink-flowered strawberry was developed,which was used for the identification of flower color in breeding pink-flowered strawberry.The results were helpful for the early selection of seedlings. |
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