Zhongshan Gao

Genomic characterization of putative allergen genes in peach/almond and their synteny with apple

BackgroundFruits from several species of the Rosaceae family are reported to cause allergic reactions in certain populations. The allergens identified belong to mainly four protein families: pathogenesis related 10 proteins, thaumatin-like proteins, lipid transfer proteins and profilins. These families of putative allergen genes in apple (Mal d 1 to 4) have been mapped on linkage maps and subsequent genetic study on allelic diversity and hypoallergenic traits has been carried out recently. In peach (Prunus persica), these allergen gene families are denoted as Pru p 1 to 4 and for almond (Prunus dulcis)Pru du 1 to 4. Genetic analysis using current molecular tools may be helpful to establish the cause of allergenicity differences observed among different peach cultivars. This study was to characterize putative peach allergen genes for their genomic sequences and linkage map positions, and to compare them with previously characterized homologous genes in apple (Malus domestica).ResultsEight Pru p/du 1 genes were identified, four of which were new. All the Pru p/du 1 genes were mapped in a single bin on the top of linkage group 1 (G1). Five Pru p/du 2 genes were mapped on four different linkage groups, two very similar Pru p/du 2.01 genes (A and B) were on G3, Pru p/du 2.02 on G7,Pru p/du 2.03 on G8 and Pru p/du 2.04 on G1. There were differences in the intron and exon structure in these Pru p/du 2 genes and in their amino acid composition. Three Pru p/du 3 genes (3.01–3.03) containing an intron and a mini exon of 10 nt were mapped in a cluster on G6. Two Pru p/du 4 genes (Pru p/du 4.01 and 4.02) were located on G1 and G7, respectively. The Pru p/du 1 cluster on G1 aligned to the Mal d 1 clusters on LG16; Pru p/du 2.01A and B on G3 to Mal d 2.01A and B on LG9; the Pru p/du 3 cluster on G6 to Mal d 3.01 on LG12; Pru p/du 4.01 on G1 to Mal d 4.03 on LG2; and Pru p/du 4.02 on G7 to Mal d 4.02 on LG2.ConclusionA total of 18 putative peach/almond allergen genes have been mapped on five linkage groups. Their positions confirm the high macro-synteny between peach/almond and apple. The insight gained will help to identify key genes causing differences in allergenicity among different cultivars of peach and other Prunus species.

Whole-Genome Analysis of Diversity and SNP-Major Gene Association in Peach Germplasm.

Peach was domesticated in China more than four millennia ago and from there it spread world-wide. Since the middle of the last century, peach breeding programs have been very dynamic generating hundreds of new commercial varieties, however, in most cases such varieties derive from a limited collection of parental lines (founders). This is one reason for the observed low levels of variability of the commercial gene pool, implying that knowledge of the extent and distribution of genetic variability in peach is critical to allow the choice of adequate parents to confer enhanced productivity, adaptation and quality to improved varieties. With this aim we genotyped 1,580 peach accessions (including a few closely related Prunus species) maintained and phenotyped in five germplasm collections (four European and one Chinese) with the International Peach SNP Consortium 9K SNP peach array. The study of population structure revealed the subdivision of the panel in three main populations, one mainly made up of Occidental varieties from breeding programs (POP1OCB), one of Occidental landraces (POP2OCT) and the third of Oriental accessions (POP3OR). Analysis of linkage disequilibrium (LD) identified differential patterns of genome-wide LD blocks in each of the populations. Phenotypic data for seven monogenic traits were integrated in a genome-wide association study (GWAS). The significantly associated SNPs were always in the regions predicted by linkage analysis, forming haplotypes of markers. These diagnostic haplotypes could be used for marker-assisted selection (MAS) in modern breeding programs.

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.

Assessment of allelic diversity in intron-containing Mal d 1 genes and their association to apple allergenicity

BackgroundMal d 1 is a major apple allergen causing food allergic symptoms of the oral allergy syndrome (OAS) in birch-pollen sensitised patients. The Mal d 1 gene family is known to have at least 7 intron-containing and 11 intronless members that have been mapped in clusters on three linkage groups. In this study, the allelic diversity of the seven intron-containing Mal d 1 genes was assessed among a set of apple cultivars by sequencing or indirectly through pedigree genotyping. Protein variant constitutions were subsequently compared with Skin Prick Test (SPT) responses to study the association of deduced protein variants with allergenicity in a set of 14 cultivars.ResultsFrom the seven intron-containing Mal d 1 genes investigated, Mal d 1.01 and Mal d 1.02 were highly conserved, as nine out of ten cultivars coded for the same protein variant, while only one cultivar coded for a second variant. Mal d 1.04, Mal d 1.05 and Mal d 1.06 A, B and C were more variable, coding for three to six different protein variants. Comparison of Mal d 1 allelic composition between the high-allergenic cultivar Golden Delicious and the low-allergenic cultivars Santana and Priscilla, which are linked in pedigree, showed an association between the protein variants coded by the Mal d 1.04 and -1.06A genes (both located on linkage group 16) with allergenicity. This association was confirmed in 10 other cultivars. In addition, Mal d 1.06A allele dosage effects associated with the degree of allergenicity based on prick to prick testing. Conversely, no associations were observed for the protein variants coded by the Mal d 1.01 (on linkage group 13), -1.02, -1.06B, -1.06C genes (all on linkage group 16), nor by the Mal d 1.05 gene (on linkage group 6).ConclusionProtein variant compositions of Mal d 1.04 and -1.06A and, in case of Mal d 1.06A, allele doses are associated with the differences in allergenicity among fourteen apple cultivars. This information indicates the involvement of qualitative as well as quantitative factors in allergenicity and warrants further research in the relative importance of quantitative and qualitative aspects of Mal d 1 gene expression on allergenicity. Results from this study have implications for medical diagnostics, immunotherapy, clinical research and breeding schemes for new hypo-allergenic cultivars.

Diversity arrays technology (DArT) markers in apple for genetic linkage maps

Diversity Arrays Technology (DArT) provides a high-throughput whole-genome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information. The work presented here details the development and performance of a DArT genotyping array for apple. This is the first paper on DArT in horticultural trees. Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful high-throughput method for obtaining accurate and reproducible marker data, despite the low cost per data point. This method appears to be suitable for aligning the genetic maps of different segregating populations. The standard complexity reduction method, based on the methylation-sensitive PstI restriction enzyme, resulted in a high frequency of markers, although there was 52–54% redundancy due to the repeated sampling of highly similar sequences. Sequencing of the marker clones showed that they are significantly enriched for low-copy, genic regions. The genome coverage using the standard method was 55–76%. For improved genome coverage, an alternative complexity reduction method was examined, which resulted in less redundancy and additional segregating markers. The DArT markers proved to be of high quality and were very suitable for genetic mapping at low cost for the apple, providing moderate genome coverage.

Cultivar Identification and Genetic Diversity of Chinese Bayberry (Myrica rubra) Accessions Based on Fluorescent SSR Markers

AbstractsA collection of 122 Chinese bayberry accessions and one wax myrtle (Myrica cerifera L.) were analyzed with 14 polymorphic simple sequence repeats (SSRs). The average number of alleles per locus was 9.3, and polymorphism information content varied from 0.07 to 0.83, with a mean value of 0.62. The genetic relationships among the 123 accessions were analyzed using the unweighted pair-group method with arithmetic mean (UPGMA). The similarity among all the accessions, based on Dice’s coefficient, varied from 0.78 to 0.99, and 0.74 between the Chinese bayberries and wax myrtle. A set of 122 Chinese bayberries clustered into four groups, with the first group further divided into six subgroups. The accessions originating from the same geographical region were more closely related than those from different regions, although extensive gene flow has taken place. The Mantel test, used to compare similarity matrices calculated from AFLP and SSR data, showed that their combination could provide information on the genetic relationship among the Chinese bayberry accessions. Ten selected SSR markers were able to distinguish most accessions, and multiplex PCR systems were developed. In addition, we found that SSRs developed from Chinese bayberry are transferable to M. cerifera.

The Vf gene for scab resistance in apple is linked to sub-lethal genes

SummaryVf is the most widely used resistance gene in the breeding for scab resistant apple cultivars. Distorted segregation ratios for Vf-resistance have frequently been reported. Here we revealed that sub-lethal genes caused the distorted segregation. The inheritance of Vf was examined in six progenies by testing linked molecular markers. Three progenies showed distorted segregations that could be explained by three sub-lethal genes (sl1, sl2 and sl3), of which sl1, sl2 were closely linked to Vf. The s11 gene was located at about 14 cM from Vf and expressed itself only in the presence of another independently segregating sub-lethal gene sl3. Only the double homozygous recessive genotypes (sl1sl1 sl3sl3) were lethal, which occurred at first as dwarf and poor vigour plants during the first three months after germination. The sl2 gene was also linked to Vf and its lethality was expressed prior to seed germination and also required the homozygous recessive presence of sl3. The map position of sl3 has not yet been identified. The linkage of Vf to sub-lethal genes usually results in a shortage of Vf-resistant progenies. But in some exceptional crosses, it will lead to abundance of resistant seedling.

Evaluation of the Role of IgE Responses to Der p 1 and Der p 2 in Chinese House Dust Mite-Allergic Patients

Background: The role of specific IgE (sIgE) against Der p 1 and Der p 2 in Chinese patients with house dust mite (HDM) allergy has not yet been well investigated. Methods: One hundred patients were enrolled, based on sensitization and doctor-diagnosed allergy to HDM. Questionnaires were administered to document demographic and clinical characteristics. Serum IgE reactivity to Dermatophagoides pteronyssinus (Dp) extract, Der p 1, Der p 2 and Der p 10 was measured by ImmunoCAP. Results: Almost all patients were sensitized to Der p 1 (95%) and Der p 2 (93%), with both allergens together being largely responsible for the total anti-HDM IgE response. No evidence for a significant role of Der p 10 was found. Overall, IgE responses to HDM and its 2 major allergens were higher in children than in adults in this cross-sectional study. With increasing age, IgE responses to Der p 2 become more important. A positive correlation was observed between the reaction of sIgE against Dp, Der p 1 and Der p 2 and the number of organs (including the eyes, nose, lungs and skin) that were affected in patients. Conclusions: In China, Der p 1 and Der p 2 are the dominant allergens in patients with HDM allergy. The relative importance of Der p 1 and Der p 2 changes with age, in favor of Der p 2. Overall, sIgE titers were positively associated with the number of organs affected.

Differential transcript abundance and genotypic variation of four putative allergen-encoding gene families in melting peach

We analysed the temporal and spatial transcript expression of the panel of 18 putative isoallergens from four gene families (Pru p 1–4) in the peach fruit, anther and leaf of two melting cultivars, to gain insight into their expression profiles and to identify the key family members. Genotypic variation of abundantly expressed genes in mature fruit was further screened in nine additional melting cultivars. In the Pru p 1 family, Pru p 1.01 and Pru p 1.06B were predominant and constitutively expressed in all the tissues, with large difference among cultivars observed in mature fruits. Pru p 1.02 was especially abundant only in the leaf. A new member of the Pru p 1 family, Pru p 1.06D, was identified through peach genome mining. In the Pru p 2 family, Pru p 2.01B was predominant in all tissues, whereas Pru p 2.01A was abundant in a few cultivars and undetectable in others. Pru p 3.01 was the most highly expressed member in all tissues except the mesocarp, while the other two members exhibited tissue specificity: Pru p 3.02 was highly expressed in the leaf, and Pru p 3.03 in the anther. Both Pru p 4 isoallergen genes were equally expressed in all tissues of both cultivars. There was high expression variability of Pru p 1 and Pru p 2 members in mature fruits among 11 cultivars, while relative lower for Pru p 3 and Pru p 4. The location, arrangement and features of peach isoallergen genes on the peach genome scaffolds were illustrated.

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.