Hui-juan Jia

Development of simple sequence repeat (SSR) markers from a genome survey of Chinese bayberry (Myrica rubra).

BackgroundChinese bayberry (Myrica rubra Sieb. and Zucc.) is a subtropical evergreen tree originating in China. It has been cultivated in southern China for several thousand years, and annual production has reached 1.1 million tons. The taste and high level of health promoting characters identified in the fruit in recent years has stimulated its extension in China and introduction to Australia. A limited number of co-dominant markers have been developed and applied in genetic diversity and identity studies. Here we report, for the first time, a survey of whole genome shotgun data to develop a large number of simple sequence repeat (SSR) markers to analyse the genetic diversity of the common cultivated Chinese bayberry and the relationship with three other Myrica species.ResultsThe whole genome shotgun survey of Chinese bayberry produced 9.01Gb of sequence data, about 26x coverage of the estimated genome size of 323 Mb. The genome sequences were highly heterozygous, but with little duplication. From the initial assembled scaffold covering 255 Mb sequence data, 28,602 SSRs (≥5 repeats) were identified. Dinucleotide was the most common repeat motif with a frequency of 84.73%, followed by 13.78% trinucleotide, 1.34% tetranucleotide, 0.12% pentanucleotide and 0.04% hexanucleotide. From 600 primer pairs, 186 polymorphic SSRs were developed. Of these, 158 were used to screen 29 Chinese bayberry accessions and three other Myrica species: 91.14%, 89.87% and 46.84% SSRs could be used in Myrica adenophora, Myrica nana and Myrica cerifera, respectively. The UPGMA dendrogram tree showed that cultivated Myrica rubra is closely related to Myrica adenophora and Myrica nana, originating in southwest China, and very distantly related to Myrica cerifera, originating in America. These markers can be used in the construction of a linkage map and for genetic diversity studies in Myrica species.ConclusionMyrica rubra has a small genome of about 323 Mb with a high level of heterozygosity. A large number of SSRs were identified, and 158 polymorphic SSR markers developed, 91% of which can be transferred to other Myrica species.

Characterization of aromatic volatile constituents in 11 Asian pear cultivars belonging to different species

Aroma is an important fruit sensory attributes that is influenced by the volatile compounds present in the fruit, and it determines whether a fruit is acceptable to consumers. The volatile compounds in 11 pear cultivars from different species were investigated by headspace solid phase microextraction and gas chromatography-mass spectrometry. In total, 70 volatile compounds, including three alcohols, six aldehydes, fifty-two esters, three acids, three terpenes and three ketones were identified and quantified. Ester was the dominant chemical class of which a total of 52 compounds were detected. Hexanal, hexyl acetate, ethyl hexanoate, ethyl 2-methylbutanoate and ethyl butanoate were the major volatile compounds. Pyrus ussuriensis Maxim showed a higher concentration of volatiles, and higher ester content than in the other Asian pear species studied. Cultivars were clearly grouped into four clusters based on the concentration and number of different volatile compounds present in each cultivar. The clusters of different cultivars could be discriminated from each others using the first two principal components. The volatile profiles of the pears were qualitatively and quantitatively influenced by the cultivar.

Identification of volatile and softening-related genes using digital gene expression profiles in melting peach

To reveal the comprehensive mechanism which is associated with the biosynthesis of volatile compounds and the accompanying texture change, RNA-seq was employed to survey the differentially expressed genes (DEGs), at the transcriptional level, of one cultivar at three stages of ripening and of four melting peach cultivars at harvest stage. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that highly ranked genes are involved in “Ribosome” and “Plant-pathogen interaction,” “Flavonoid biosynthesis,” “Linoleic acid metabolism,” and “Flavone and flavonol biosynthesis” during the fruit-ripening process. The quantitative real-time PCR (qRT-PCR) validation with 15 aroma and softening-related genes showed high correlation with the RNA-seq results. Transcripts of a nonspecific lipid transfer protein (nsLTP, ppa013554) and an ATP-binding cassette transporter (ABC, ppa002351) increased from the early ripening stage to the commercial harvest stage and then declined in the fully ripening stage. The highest transcript abundance was in the aroma-enriched cv. “Hu Jing Mi Lu” (HJ) and lowest in the cv. “Zhong Hua Shou Tao” with slight aroma. Fifty gene families related to the formation of aroma compounds were found. For peach lactone biosynthesis, we inferred that ppa002510 and ppa002282 may be the two important acyl-CoA oxidase genes correlating with the difference in γ-decalactone concentrations among the four cultivars. The expression of one 3-hydroxyacyl-CoA dehydrogenase gene (HCAD, ppa008854) was upregulated during ripening. Thirteen gene families were associated to fruit softening. Four aquaporin (AQP) genes showed cultivar-specificity for HJ, and one of them, ppa009506, reached maximum accumulation of transcripts at harvest stage. The batch of novel genes (nsLTP, ACX, AOC, ABC, HCAD, AQP) found here facilitates understanding of the molecular mechanism of melting peach aroma biosynthesis and fruit softening.

Quantification of Peach Fruit Allergen Lipid Transfer Protein by a Double Monoclonal Antibody-based Sandwich ELISA

Fruit is one of the most commonly reported food allergy sources in China, and peach lipid transfer protein (Pru p 3) has been identified as the major allergen inducing systematic symptoms. Crude allergen extracts and single component allergens have been used in food allergy diagnosis and immunotherapy. Reliable and sensitive analytical methods to quantify Pru p 3 content in fruit will help to identify low-allergenic cultivars among the abundant peach genetic resources in China. In this study, we developed a sensitive sandwich ELISA method to measure Pru p 3 in peach fruit based on two monoclonal antibodies with high sensitivity and linearity. Significant variability was observed in peach cultivars, with a much higher Pru p 3 level in peach peel than in pulp. This method will be very useful to select peach varieties with low Pru p 3 content and as a diagnostic product to detect hidden allergens in processed food.

Development of 107 SSR markers from whole genome shotgun sequences of Chinese bayberry (Myrica rubra) and their application in seedling identification

Chinese bayberry (Myrica rubra Sieb. et Zucc.) is one of the important subtropical fruit crops native to the South of China and Asian countries. In this study, 107 novel simple sequence repeat (SSR) molecular markers, a powerful tool for genetic diversity studies, cultivar identification, and linkage map construction, were developed and characterized from whole genome shotgun sequences. M13 tailing for forward primers was applied as a simple method in different situations. In total, 828 alleles across 45 accessions were detected, with an average of 8 alleles per locus. The number of effective alleles ranged from 1.22 to 10.41 with an average of 4.08. The polymorphic information content (PIC) varied from 0.13 to 0.89, with an average of 0.63. Moreover, these markers could also be amplified in their related species Myrica cerifera (syn. Morella cerifera) and Myrica adenophora. Seventy-eight SSR markers can be used to produce a genetic map of a cross between ‘Biqi’ and ‘Dongkui’. A neighbor-joining (NJ) tree was constructed to assess the genetic relationships among accessions, and the elite accessions ‘Y2010-70’, ‘Y2012-140’, and ‘Y2012-145’, were characterized as potential new genotypes for cultivation.概要研究目的开发多态性高的基因组简单重复序列(SSR)标记, 为杨梅遗传多样性、 遗传图谱构建及品种鉴定相关研究奠定基础。创新要点从杨梅全基因组鸟枪法测序数据中新开发了107 个SSR 标记, 并应用于品种间遗传多样性分析及品系鉴定。研究方法应用生物信息学软件从基因组测序数据中搜索SSR 位点并设计相应引物, 添加通用的M13 尾巴和荧光标识筛选多态性高的SSR 标记。重要结论开发的107 个基因组SSR 标记可以广泛应用于杨梅种质资源和遗传育种研究, 鉴定出三个杨梅新品系。

Changes in volatile organic compound composition during the ripening of ‘Nanguoli’ pears (Pyrus ussuriensis Maxim) harvested at different growing locations

Summary Aroma is an important sensory attribute of fruit that is influenced by the presence of volatile organic compounds (VOCs). Esters were the most prominent volatile compounds in Pyrus ussuriensis fruit, and endowed a ‘fruity’ or ‘sweet’ note to the fruit. We investigated differences in the VOCs in ripening ‘Nanguoli’ pear (P. ussuriensis Maxim) fruit harvested at two different locations [at Anshan, Liaoning Province (ASLN) and at Liaoyuan, Jilin Province (LYJL)] using principal component analysis. Qualitative and quantitative analyses showed that volatile esters were the major VOCs during fruit ripening. The main VOCs were ethyl butanoate, ethyl hexanoate, and hexyl acetate, followed by aldehydes and terpenes, among which the main constituents were hexanal, E-2-hexenal, and αfarnesene. The concentrations of terpenes were higher in fruit grown at ASLN than in fruit grown at LYJL. Ester and terpene concentrations were affected by the stage of ripening, and by location, to some extent. Ripening stage and location affected the production and relative compositions of VOCs in ‘Nanguoli’ pear fruit.

The PpLTP1 Primary Allergen Gene is Highly Conserved in Peach and Has Small Variations in Other Prunus Species

Peach lipid transfer protein (LTP1), Pru p 3.01, is a major allergen causing severe systemic reactions in peach allergic patients in Mediterranean countries and China. Significant expression variability has been reported among peach cultivars at both transcript and protein level. In this study, the allele diversity of the LTP1-encoding gene was assessed in a large set of peach cultivars by direct sequencing of the gene and its upstream region. Evolution of the LTP1-encoding genes in peach (Prunus persica) and three other Prunus species (Prunus kansuensis, Prunus mira, and Prunus davidiana) were also inferred. Sequence analysis revealed that LTP1-encoding genes are highly conserved among peach cultivars and wild peach. Three different allele sequences were obtained from 50 Prunus accessions on the basis of the upstream region of the LTP1-encoding gene and three allele specific markers were derived according to the polymorphic sites. These markers were used to test 316 peach cultivars, most of which clustered within the three main subpopulations of peach, ‘Yu Lu’, ‘Hakuho’ and landraces. The genotypic frequencies and allele frequencies in the oriental peach cultivars in these subpopulations except for ‘Yu Lu’ were found to be in Hardy-Weinberg equilibrium (P < 0.05). The dominant alleles were upLTP1-a in the ‘Yu Lu’ subpopulation and upLTP1-c in the ‘Hakuho’ subpopulation, allele upLTP1-b and allele upLTP1-c had the highest rates in the landraces, and the dominant allele in the three peach wild relatives was allele upLTP1-b. Furthermore, we found many light-responsive elements in the upstream region. Most of the polymorphic sites in Prunus species are located in the intron region. Phylogenetic analysis suggests that Prunus kansuensis KC311794 and Prunus mira KC311791 are related more closely to Prunus persica KC311795 than to Prunus davidiana KC311792 and KC311793. The allele sequences we derived on the basis of this variability were distributed unevenly, indicating the need to study allergenicity in different subpopulations and the association between allele sequences and allergenicity.

Development of 107 SSR markers from whole genome shotgun sequences of Chinese bayberry (Myrica rubra) and their application in seedling identification@@@基于杨梅基因组鸟枪法测序开发的107 个SSR 标记及其在品系鉴定中应用

Chinese bayberry (Myrica rubra Sieb. et Zucc.) is one of the important subtropical fruit crops native to the South of China and Asian countries. In this study, 107 novel simple sequence repeat (SSR) molecular markers, a powerful tool for genetic diversity studies, cultivar identification, and linkage map construction, were developed and characterized from whole genome shotgun sequences. M13 tailing for forward primers was applied as a simple method in different situations. In total, 828 alleles across 45 accessions were detected, with an average of 8 alleles per locus. The number of effective alleles ranged from 1.22 to 10.41 with an average of 4.08. The polymorphic information content (PIC) varied from 0.13 to 0.89, with an average of 0.63. Moreover, these markers could also be amplified in their related species Myrica cerifera (syn. Morella cerifera) and Myrica adenophora. Seventy-eight SSR markers can be used to produce a genetic map of a cross between ‘Biqi’ and ‘Dongkui’. A neighbor-joining (NJ) tree was constructed to assess the genetic relationships among accessions, and the elite accessions ‘Y2010-70’, ‘Y2012-140’, and ‘Y2012-145’, were characterized as potential new genotypes for cultivation.概要研究目的开发多态性高的基因组简单重复序列(SSR)标记, 为杨梅遗传多样性、 遗传图谱构建及品种鉴定相关研究奠定基础。创新要点从杨梅全基因组鸟枪法测序数据中新开发了107 个SSR 标记, 并应用于品种间遗传多样性分析及品系鉴定。研究方法应用生物信息学软件从基因组测序数据中搜索SSR 位点并设计相应引物, 添加通用的M13 尾巴和荧光标识筛选多态性高的SSR 标记。重要结论开发的107 个基因组SSR 标记可以广泛应用于杨梅种质资源和遗传育种研究, 鉴定出三个杨梅新品系。

Development of 107 SSR markers from whole genome shotgun sequences of Chinese bayberry (Myrica rubra

Chinese bayberry (Myrica rubra Sieb. et Zucc.) is one of the important subtropical fruit crops native to the South of China and Asian countries. In this study, 107 novel simple sequence repeat (SSR) molecular markers, a powerful tool for genetic diversity studies, cultivar identification, and linkage map construction, were developed and characterized from whole genome shotgun sequences. M13 tailing for forward primers was applied as a simple method in different situations. In total, 828 alleles across 45 accessions were detected, with an average of 8 alleles per locus. The number of effective alleles ranged from 1.22 to 10.41 with an average of 4.08. The polymorphic information content (PIC) varied from 0.13 to 0.89, with an average of 0.63. Moreover, these markers could also be amplified in their related species Myrica cerifera (syn. Morella cerifera) and Myrica adenophora. Seventy-eight SSR markers can be used to produce a genetic map of a cross between ‘Biqi’ and ‘Dongkui’. A neighbor-joining (NJ) tree was constructed to assess the genetic relationships among accessions, and the elite accessions ‘Y2010-70’, ‘Y2012-140’, and ‘Y2012-145’, were characterized as potential new genotypes for cultivation.概要研究目的开发多态性高的基因组简单重复序列(SSR)标记, 为杨梅遗传多样性、 遗传图谱构建及品种鉴定相关研究奠定基础。创新要点从杨梅全基因组鸟枪法测序数据中新开发了107 个SSR 标记, 并应用于品种间遗传多样性分析及品系鉴定。研究方法应用生物信息学软件从基因组测序数据中搜索SSR 位点并设计相应引物, 添加通用的M13 尾巴和荧光标识筛选多态性高的SSR 标记。重要结论开发的107 个基因组SSR 标记可以广泛应用于杨梅种质资源和遗传育种研究, 鉴定出三个杨梅新品系。