Chao Gu

Molecular genetics of blood‐fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors

Anthocyanin pigmentation is an important consumer trait in peach (Prunus persica). In this study, the genetic basis of the blood-flesh trait was investigated using the cultivar Dahongpao, which shows high levels of cyanidin-3-glucoside in the mesocarp. Elevation of anthocyanin levels in the flesh was correlated with the expression of an R2R3 MYB transcription factor, PpMYB10.1. However, PpMYB10.1 did not co-segregate with the blood-flesh trait. The blood-flesh trait was mapped to a 200-kb interval on peach linkage group (LG) 5. Within this interval, a gene encoding a NAC domain transcription factor (TF) was found to be highly up-regulated in blood-fleshed peaches when compared with non-red-fleshed peaches. This NAC TF, designated blood (BL), acts as a heterodimer with PpNAC1 which shows high levels of expression in fruit at late developmental stages. We show that the heterodimer of BL and PpNAC1 can activate the transcription of PpMYB10.1, resulting in anthocyanin pigmentation in tobacco. Furthermore, silencing the BL gene reduces anthocyanin pigmentation in blood-fleshed peaches. The transactivation activity of the BL-PpNAC1 heterodimer is repressed by a SQUAMOSA promoter-binding protein-like TF, PpSPL1. Low levels of PpMYB10.1 expression in fruit at early developmental stages is probably attributable to lower levels of expression of PpNAC1 plus the presence of high levels of repressors such as PpSPL1. We present a mechanism whereby BL is the key gene for the blood-flesh trait in peach via its activation of PpMYB10.1 in maturing fruit. Partner TFs such as basic helix-loop-helix proteins and NAC1 are required, as is the removal of transcriptional repressors.

Identification, characterization, and utilization of genome-wide simple sequence repeats to identify a QTL for acidity in apple

BackgroundApple is an economically important fruit crop worldwide. Developing a genetic linkage map is a critical step towards mapping and cloning of genes responsible for important horticultural traits in apple. To facilitate linkage map construction, we surveyed and characterized the distribution and frequency of perfect microsatellites in assembled contig sequences of the apple genome.ResultsA total of 28,538 SSRs have been identified in the apple genome, with an overall density of 40.8 SSRs per Mb. Di-nucleotide repeats are the most frequent microsatellites in the apple genome, accounting for 71.9% of all microsatellites. AT/TA repeats are the most frequent in genomic regions, accounting for 38.3% of all the G-SSRs, while AG/GA dimers prevail in transcribed sequences, and account for 59.4% of all EST-SSRs. A total set of 310 SSRs is selected to amplify eight apple genotypes. Of these, 245 (79.0%) are found to be polymorphic among cultivars and wild species tested. AG/GA motifs in genomic regions have detected more alleles and higher PIC values than AT/TA or AC/CA motifs. Moreover, AG/GA repeats are more variable than any other dimers in apple, and should be preferentially selected for studies, such as genetic diversity and linkage map construction. A total of 54 newly developed apple SSRs have been genetically mapped. Interestingly, clustering of markers with distorted segregation is observed on linkage groups 1, 2, 10, 15, and 16. A QTL responsible for malic acid content of apple fruits is detected on linkage group 8, and accounts for ~13.5% of the observed phenotypic variation.ConclusionsThis study demonstrates that di-nucleotide repeats are prevalent in the apple genome and that AT/TA and AG/GA repeats are the most frequent in genomic and transcribed sequences of apple, respectively. All SSR motifs identified in this study as well as those newly mapped SSRs will serve as valuable resources for pursuing apple genetic studies, aiding the apple breeding community in marker-assisted breeding, and for performing comparative genomic studies in Rosaceae.

Unraveling the mechanism underlying the glycosylation and methylation of anthocyanins in peach

Diversification of anthocyanins in peach is attributed to glycosylation and methylation. Modification of anthocyanin plays an important role in increasing its stability in plants. Here, six anthocyanins were identified in peach (Prunus persica), and their structural diversity is attributed to glycosylation and methylation. Interestingly, peach is quite similar to the wild species Prunus ferganensis but differs from both Prunus davidiana and Prunus kansueasis in terms of anthocyanin composition in flowers. This indicates that peach is probably domesticated from P. ferganensis. Subsequently, genes responsible for both methylation and glycosylation of anthocyanins were identified, and their spatiotemporal expression results in different patterns of anthocyanin accumulation in flowers, leaves, and fruits. Two tandem-duplicated genes encoding flavonoid 3-O-glycosyltransferase (F3GT) in peach, PpUGT78A1 and PpUGT78A2, showed different activity toward anthocyanin, providing an example of divergent evolution of F3GT genes in plants. Two genes encoding anthocyanin O-methyltransferase (AOMT), PpAOMT1 and PpAOMT2, are expressed in leaves and flowers, but only PpAOMT2 is responsible for the O-methylation of anthocyanins at the 3′ position in peach. In addition, our study reveals a novel branch of UGT78 genes in plants that lack the highly conserved intron 2 of the UGT gene family, with a great variation of the amino acid residue at position 22 of the plant secondary product glycosyltransferase box. Our results not only provide insights into the mechanisms underlying anthocyanin glycosylation and methylation in peach but will also aid in future attempts to manipulate flavonoid biosynthesis in peach as well as in other plants.

Identification of S-haplotype-specific S-RNase and SFB alleles in native Chinese apricot (Prunus armeniaca L.).

Summary Chinese apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility (GSI) controlled by a single locus containing at least two linked genes [i.e., the S-RNase gene and the pollen-expressed SFB (or SLF) gene] as do other fruit species in the family, Rosaceae. To elucidate the S-genotypes of 14 native Chinese apricot cultivars, PCR was performed using primers designed from Prunus S-RNase and SFB consensus sequences. After cloning and sequencing the PCR products, the S-genotypes of all 14 apricot cultivars were determined, and eight new S-RNase alleles and nine SFB alleles were identified. The S-RNases shared typical structural features with S-RNases from other Prunus spp. exhibiting GSI. The SFB genes showed similar structural characteristics to SFB genes in other Prunus spp. The intron sequences of the SFB genes revealed sequence and length polymorphisms. The deduced level of amino acid sequence identity for the eight new S-RNase alleles was 66.4 – 100% in P. armeniaca, while the similarity of the SFB alleles was 73.7 – 98.6%. The physical distances between the SFB and S-RNase genes was determined exactly in the S9,S11,S17, and S26-haplotypes, confirming that the S-RNase and SFB genes were linked. The range of distances between the two genes was 299 – 1,061 bp. This study increases our knowledge on the S-genotypes of apricot native to China, and enriches our genomic information on GSI in the Prunus genus.

Molecular cloning and gene expression differences of the anthocyanin biosynthesis-related genes in the red/green skin color mutant of pear (Pyrus communis L.)

To reveal the molecular mechanisms that led to the red/green color mutation of pear between the cultivar ‘Early red Doyenne du Comice’ and its green variant strain, the full-length cDNA of the seven anthocyanin biosynthesis genes (PAL, CHS, CHI, DFR, F3H, ANS, UFGT) was cloned in both cultivars. The accession number has been submitted to National Center for Biotechnology Information (NCBI) as KC460392, KC460393, KC460394, KC460395, KC460396, KC460397, and KC460398, respectively. However, there was no sequence difference between the color mutants, which means that the skin color change was not caused by mutation of any of these genes. Meanwhile, the expression levels of these seven genes were examined by quantitative real-time PCR (qRT-PCR). Results showed that most of the structural genes were up-regulated in the red-skinned cultivar during fruit development, but the CHI and UFGT genes were highly expressed only at an early stage. The expression levels of the transcription factors MYB10, bHLH, and WD40 were also investigated by qRT-PCR, and the MYB10 gene was found to be expressed at significantly higher levels in the red variety than in the green mutant at the early stage, while the expression levels of bHLH and WD40 were higher at a later stage. These data indicate that the expression difference of structural genes in the anthocyanin biosynthesis pathway led to the skin color change of the mutant. However, MYB10, bHLH, and WD40 do not appear to be the key transcription factors that regulate the biosynthesis of anthocyanin and determine the red/green color mutant.

Identification of differentially expressed genes related to coloration in red/green mutant pear (Pyrus communis L.)

Fruit skin color is an important parameter of outer quality and plays an important role in attracting customers. In many plants, it is the result of coordinative regulation of anthocyanin pathway genes. In our study, the differential expression of cDNA library in a pair of pear mutant with red and green color was investigated to find candidate genes which might regulate the anthocyanin biosynthesis and control the coloration of pear. We constructed a cDNA library using the cDNA-amplified fragment length polymorphism approach to analyze the transcriptional differences between the original cultivar “Early red Doyenne du Comice” with high anthocyanin content in the peel and its green color mutant with comparatively low anthocyanin content. Altogether, 47 transcript-derived fragments, putatively involved in anthocyanin biosynthesis, primary metabolism, stress, and defense responses, were identified. The relationships of differentially expressed genes and coloration were investigated by quantitative real-time PCR with fruit skin samples at different developmental stages. A gene putatively involved in anthocyanin biosynthesis was found and named as PyMADS18. Its sequence is similar to genes reported in the literature as regulators of anthocyanin biosynthesis. The expression results indicate that PyMADS18 is likely to be involved in anthocyanin accumulation and regulation of anthocyanin synthesis in early fruit development of pear.

Inactivation of a Gene Encoding Carotenoid Cleavage Dioxygenase (CCD4) Leads to Carotenoid-Based Yellow Coloration of Fruit Flesh and Leaf Midvein in Peach

Yellow fruit flesh color, resulting from the accumulation of carotenoids, is one of the most important commercial traits of peach. Yellow flesh is controlled by a single locus (Y), with white flesh dominant over yellow flesh. In this study, the Y locus was narrowed to a 2.6-cM interval flanked by two markers, SSRy and W2691. SSRy, which is located on the first exon of a gene encoding carotenoid cleavage dioxygenase (CCD4), was cosegregated with the Y locus in two peach F1 populations. RNA-Seq and qRT-PCR analysis revealed transcript level of CCD4 was consistent with carotenoid degradation in peach fruits. All these results suggest that CCD4 is responsible for white and yellow coloration of peach fruit flesh. In fruits of white-fleshed peach, carotenoids are synthesized but subsequently degraded into colorless compounds, leading to the formation of white color. CCD4 is likely to utilize β-carotene as the substrate in peach. Interestingly, CCD4 also controls white and yellow coloration of leaf midveins of peach. Moreover, LCYE was highly expressed in peach leaves, whereas its transcript was not detectable in fruits. This suggests the difference of carotenoid biosynthesis between peach fruits and leaves. Our study not only shows for the first time the pleiotropic effects of CCD4 gene in peach but also provides a morphological marker for easy selection of new peach cultivars with desirable white or yellow flesh colors.

Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis

Red fruits are popular and widely accepted by consumers because of an enhanced appearance and enriched anthocyanins. The molecular mechanism of anthocyanin regulation in red-skinned pear (Pyrus) has been studied, and the genes encoding the biosynthetic steps and several transcription factors (TFs) have been characterized. In this study, a candidate R2R3 MYB TF, PyMYB114, was identified by linkage to the quantitative trait loci (QTL) for red skin color on linkage group 5 in a population of Chinese pear (Pyrus bretschneideri). The function of PyMYB114 was verified by transient transformation in tobacco (Nicotinana tabacum) leaves and strawberry (Fragaria) and pear fruits, resulting in the biosynthesis of anthocyanin. Suppression of PyMYB114 could inhibit anthocyanin biosynthesis in red-skinned pears. The ERF/AP2 TF PyERF3 was found to interact with PyMYB114 and its partner PybHLH3 to co-regulate anthocyanin biosynthesis, as shown by a dual luciferase reporter system and a yeast two-hybrid assay. In addition, the transcript abundance of PyMYB114 and PyMYB10 were correlated, and co-transformation of these two genes into tobacco and strawberry led to enhanced anthocyanin biosynthesis. This interaction network provides insight into the coloration of fruits and the interaction of different TFs to regulate anthocyanin biosynthesis.

Coordinated regulation of anthocyanin biosynthesis through photorespiration and temperature in peach (Prunus persica f. atropurpurea)

The usual red color of young leaves of peach (Prunus persica f. atropurpurea) is due to the accumulation of anthocyanin. Real-time PCR analysis revealed a strong correlation between the expression levels of anthocyanin biosynthetic genes and anthocyanin content in leaves at different developmental stages. The expression profiles of both anthocyanin biosynthetic genes and photorespiratory genes showed significant changes in leaves held in the dark or exposed to heat stress, compared with controls. The expression of anthocyanin biosynthetic genes dramatically decreased in peach red leaves following dark or heat treatments, resulting in a significant decrease of anthocyanin accumulation. However, the photorespiration-related genes GDCH and GOX exhibited increased expression in peach leaves after dark or heat treatment. Moreover, the expression levels of GDCH and GOX in the Arabidopsis chi/f3′h mutant that does not accumulate anthocyanins were higher than in the wild type. Overall, these results support the hypothesis that photorespiration-related genes might be involved in the regulation of anthocyanin biosynthesis. This finding provides a new insight into our understanding of the mechanism underlying the control of anthocyanin biosynthesis in plants.

Self-compatibility of ‘Katy’ apricot ( Prunus armeniaca L.) is associated with pollen-part mutations

Apricot (Prunus armeniaca L.) cultivars originated in China display a typical S-RNase-based gametophytic self-incompatibility (GSI). ‘Katy’, a natural self-compatible cultivar belonging to the European ecotype group, was used as a useful material for breeding new cultivars with high frequency of self-compatibility by hybridizing with Chinese native cultivars. In this work, the pollen-S genes (S-haplotype-specific F-box gene, or SFB gene) of ‘Katy’ were first identified as SFB1 and SFB8, and the S-genotype was determined as S1S8. Genetic analysis of ‘Katy’ progenies under controlled pollination revealed that the stylar S1-RNase and S8-RNase have a normal function in rejecting wild-type pollen with the same S-haplotype, while the pollen grains carrying either the SFB1 or the SFB8 gene are both able to overcome the incompatibility barrier. However, the observed segregation ratios of the S-genotype did not fit the expected ratios under the assumption that the pollen-part mutations are linked to the S-locus. Moreover, alterations in the SFB1 and SFB8 genes and pollen-S duplications were not detected. These results indicated that the breakdown of SI in ‘Katy’ occurred in pollen, and other factors not linked to the S-locus, which caused a loss of pollen S-activity. These findings support a hypothesis that modifying factors other than the S-locus are required for GSI in apricot.