Complete genome sequence of the carrot black rot pathogen Alternaria radicina isolate CBR2.

Black rot, caused by Alternaria radicina, seriously endangers carrots throughout the growing season, affecting both leaves and fleshy roots. In this study, we sequenced and assembled the genome of the A. radicina isolate CBR2. The genome was 34.6 Mb in size and consisted of 6 scaffolds. The sequence information provided in this genome will be used as a reference for further comparative genomics analysis of Alternaria species and will contribute to disease control in carrot production.

First Report of Black Rot of Carrot Caused by Alternaria carotiincultae in China.

China is the world's largest producer and exporter of carrot (Daucus carota L. var. sativa), a well-known and nutritious root vegetable. In the spring seasons of 2021-2023, dark brown lesions were observed on field-grown and cold-stored carrot roots in the Xiamen City, Fujian Province, China. Although just discovered in recent years, the disease has expanded from the initial point to the most planting area in there, and causing over 20% yield loss in the most severely affected fields. This disease symptom is consistent with black rot, a carrot disease found globally and caused by the fungal pathogen Alternaria radicina (Saude et al. 2006). Small pieces of symptomatic roots (3 to 5 mm) from diseased carrot roots were surface disinfested with 75% alcohol for 3 minutes and 10% sodium hypochlorite solution for 8 minutes, and then rinsed in sterile distilled water and cultured on potato dextrose agar (PDA) medium at 28°C with a 12-h dark/light photoperiod. By tissue isolation and single-spore culture, six isolates were obtained from the disease plants in the past three years, and used for morphological and molecular analyses. Unexpectedly, the morphological characteristics of conidia of the six isolates, shape (long and ellipsoid), size (27 to 60 × 17 to 21 µm, n =50), and color (dark olive-brown) were similar to those of A. radicina, but with more transverse septae (Figure 1, Trivedi et al. 2010). Meanwhile, compared to A. radicina, the colony's margin was highly uniform and smooth, without an accumulation of yellow pigment and with fewer dendritic rhizomycin crystals at the bottom of agar media. These characteristics were similar to those of Alternaria carotiincultae, but not to those of A. radicina (Park et al. 2008). Genomic DNA of the six isolates was extracted and further molecular identification was performed. The EF-1α gene was amplified using EF-1/EF-2 primer pairs and sequenced (O'Donnell et al. 1998.). The EF-1α gene sequences of the six isolates were compared using DNAMAN 5.2.9 software. They were found to be 100% identical, and the sequences has been deposited in GenBank under accession number OR449062. BLAST analysis of the amplicon revealed 100% nucleotide sequence identity with the A. carotiincultae strain YZU 151039 (GenBank accession No. MK279390), while 2bp difference between the sequences of A. radicina strains present in GenBank. Pathogenicity tests of the isolate were carried on the unwounded carrots (5 roots each, with three replications). These tap roots were surface disinfected with 75% alcohol and disinfected by immersion in 0.75% sodium hypochlorite solution for 10 minutes, then immediately rinsed with sterile water and dried with sterile filter paper. Mycelial plugs (6 mm diameter) were taken from the margin of a vigorously growing colony and inoculated into the sterilized carrot roots. Another group of sterilised tap roots inoculated with sterile agar plugs were used as negative control. All the roots were incubated in the dark at 25°C with 80% humidity. After 2 days, colonies were observed on the surface of the roots that had been inoculated with the mycelial plugs. After 7 days, the inoculated tap roots showed symptoms of black rot with dark brown sunken lesions as the diseased plants in the field. However, no disease was observed on the control roots. Following the previous method, a strain was re-isolated from the inoculated carrot roots and again identified as A. carotiincultae, thus fulfilling Koch's postulates, and confirming that A. carotiincultae is the pathogen causing dark brown lesions of carrots. To our knowledge, this is the first report of A. carotiincultae causing carrot black rot in China. Attention should be paid to the damage caused by this pathogen during the production and storage stages of carrots, and strategies should be developed to prevent its spread.

Draft Genome Sequence of Carrot Alternaria Leaf Blight Pathogen Alternaria dauci.

The fungal genus Alternaria, which causes a variety of crop diseases, is widely distributed in the world. Alternaria leaf blight, caused by Alternaria dauci, is one of the most common and destructive diseases in carrot. The infection of A. dauci leads to dramatic decay on both foliage and taproot in severe cases, which results in significant yield losses. In this study, we sequenced and assembled the genome of A. dauci isolate CALB1, which isolated from the major carrot producing areas of China. A total of 65 contigs were assembled, and the estimated genome size was 34.9 Mb. The draft genome of A. dauci can be used for comparative genomic analysis of Alternaria species and provide genetic information for further research on plant-pathogen interactions.

Genome-wide identification and characterization profile of phosphatidy ethanolamine-binding protein family genes in carrot.

Members of the family of Phosphatidy Ethanolamine-Binding Protein (PEBP) have been shown to be key regulators of the transition of plants from vegetative to reproductive phases. Here, a total of 12 PEBP proteins were identified in the carrot (Daucus carota L.) genome and classified into FT-like (4), TFL1-like (6), and MFT-like 2) subfamilies, that had different lengths (110-267 aa) and were distributed unevenly across seven chromosomes. Moreover, 13 and 31 PEBP proteins were identified in other two Apiaceae species, celery (Apium graveolens L.) and coriander (Coriandrum sativum L.). The phylogenetic and evolutionary results of these PEBP family proteins were obtained based on the protein sequences. In the three Apiaceae species, purifying selection was the main evolutionary force, and WGD, segmental duplication, and dispersed duplication have played key roles in the PEBP family expansion. The expression analysis showed that carrot PEBP genes exhibited relatively broad expression patterns across various tissues. In the period of bolting to flowering, the carrot FT-like subfamily genes were upregulated as positive regulators, and TFL1-like subfamily genes remained at lower expression levels as inhibitors. More interestingly, the members of carrot FT-like genes had different temporal-spatial expression characteristics, suggesting that they have different regulatory functions in the carrot reproductive phase. In summary, this study contributes to our understanding of the PEBP family proteins and provides a foundation for exploring the mechanism of carrot bolting and flowering for the breeding of cultivars with bolting resistance.

Detection of Chromosomal Segments Introgressed from Wild Species of Carrot into Cultivars: Quantitative Trait Loci Mapping for Morphological Features in Backcross Inbred Lines.

Cultivated carrot is thought to have been domesticated from a wild species, and various phenotypes developed through human domestication and selection over the past several centuries. Little is known about the genomic contribution of wild species to the phenotypes of present-day cultivars, although several studies have focused on identifying genetic loci that contribute to the morphology of storage roots. A backcross inbred line (BIL) population derived from a cross between the wild species Daucus carota ssp. carota "Songzi" and the orange cultivar "Amsterdam forcing" was developed. The morphological features in the BIL population became more diverse after several generations of selfing BC2F1 plants. Only few lines retained features of wild parent. Genomic resequencing of the two parental lines and the BILs resulted in 3,223,651 single nucleotide polymorphisms (SNPs), and 13,445 bin markers were generated using a sliding window approach. We constructed a genetic map with 2027 bins containing 154,776 SNPs; the total genetic distance was 1436.43 cM and the average interval between the bins was 0.71 cm. Five stable QTLs related to root length, root shoulder width, dry material content of root, and ratio of root shoulder width to root middle width were consistently detected on chromosome 2 in both years and explained 23.4-66.9% of the phenotypic variance. The effects of introgressed genomic segments from the wild species on the storage root are reported and will enable the identification of functional genes that control root morphological traits in carrot.

The Response of COL and FT Homologues to Photoperiodic Regulation in Carrot (Daucus carota L.).

Carrot (Daucus carota L.) is a biennial plant requiring vernalization to induce flowering, but long days can promote its premature bolting and flowering. The basic genetic network controlling the flowering time has been constructed for carrot, but there is limited information on the molecular mechanisms underlying the photoperiodic flowering response. The published carrot genome could provide an effective tool for systematically retrieving the key integrator genes of GIGANTEA (GI), CONSTANS-LIKE (COL), FLOWERING LOCUS T (FT), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) homologues in the photoperiod pathway. In this study, the bolting time of wild species "Songzi" (Ws) could be regulated by different photoperiods, but the orange cultivar "Amsterdam forcing" (Af) displayed no bolting phenomenon. According to the carrot genome and previous de novo transcriptome, 1 DcGI, 15 DcCOLs, 2 DcFTs, and 3 DcSOC1s were identified in the photoperiod pathway. The circadian rhythm peaks of DcGI, DcCOL2, DcCOL5a, and DcCOL13b could be delayed under long days (LDs). The peak value of DcCOL2 in Af (12.9) was significantly higher than that in Ws (6.8) under short day (SD) conditions, and was reduced under LD conditions (5.0). The peak values of DcCOL5a in Ws were constantly higher than those in Af under the photoperiod treatments. The expression levels of DcFT1 in Ws (463.0) were significantly upregulated under LD conditions compared with those in Af (1.4). These responses of DcCOL2, DcCOL5a, and DcFT1 might be related to the different bolting responses of Ws and Af. This study could provide valuable insights into understanding the key integrator genes in the carrot photoperiod pathway.

Differentially Expressed Genes between Carrot Petaloid Cytoplasmic Male Sterile and Maintainer during Floral Development.

Petaloid cytoplasmic male sterility (CMS) is a maternally inherited loss of male fertility due to the complete conversion of stamens into petal-like organs, and CMS lines have been widely utilized in carrot breeding. Petaloid CMS is an ideal model not only for studying the mitochondrial-nuclear interaction but also for discovering genes that are essential for floral organ development. To investigate the comprehensive mechanism of CMS and homeotic organ alternation during carrot flower development, we conducted transcriptome analysis between the petaloid CMS line (P2S) and its maintainer line (P2M) at four flower developmental stages (T1-T4). A total of 2838 genes were found to be differentially expressed, among which 1495 genes were significantly downregulated and 1343 genes were significantly upregulated in the CMS line. Functional analysis showed that most of the differentially expressed genes (DEGs) were involved in protein processing in the endoplasmic reticulum, plant hormone signal transduction, and biosynthesis. A total of 16 MADS-box genes were grouped into class A, B, C, and E, but not class D, genes. Several key genes associated with oxidative phosphorylation showed continuously low expression from stage T2 in P2S, and the expression of DcPI and DcAG-like genes also greatly decreased at stage T2 in P2S. This indicated that energy deficiency might inhibit the expression of B- and C-class MADS-box genes resulting in the conversion of stamens into petals. Stamen petaloidy may act as an intrinsic stress, upregulating the expression of heat shock protein (HSP) genes and MADS-box genes at stages T3 and T4 in P2S, which results in some fertile revertants. This study will provide a better understanding of carrot petaloid CMS and floral development as a basis for further research.

The dual role of phytoene synthase genes in carotenogenesis in carrot roots and leaves.

Carrot (Daucus carota L.) is an important food crop and is useful for studying carotenogenesis due to the quantity and diversity of carotenoids in its roots. Phytoene synthase catalyzes the first committed step in the carotenoid biosynthesis pathway, and its overexpression is the main driving force in the orange phenotype. At present, we lack fundamental knowledge of the role of these genes and their effects on carotenoid accumulation in leaves. In the present study, three backcross inbred lines (BC2S4) with different colored roots derived from a cross between the orange inbred line (Af) and related wild species were used to investigate the role of the duplicated DcPSY genes in root carotenogenesis. Promoter analysis showed that DcPSY genes have diverged substantially in their regulatory sequences after gene duplication. Expression levels of DcPSY1 and DcPSY2 were generally positively correlated with carotenoid content during root development. In mature leaves, total carotenoid content was higher than that in the roots, DcPSY1 expression increased extremely higher than DcPSY2 expression compared with roots, and DcPSY1 was more sensitive than DcPSY2 during leaf de-etiolation under sunlight. These results suggest that DcPSY1 seems to make an important contribution to carotenoid accumulation in the leaves and is important for photosynthesis and photoprotection, but they are not the determining factors of root color. This expands our understanding of the regulation of carotenoid biosynthesis in carrot.

[QTL mapping for contents of main carotenes and lycopene in carrot (Daucus carota L.)].

An F2 population derived from two carrot inbred lines, P50006 and HCM A.C. with high carotene accumulation, was developed and used to map and analyze quantitative trait locus (QTL) associated with the accumulation of alpha and beta-carotene, total carotene and lycopene. Broad-sense heritabilities of these traits were 0.75, 0.50, 0.31, and 0.93, respectively. A genetic map with 91 SRAP (Sequence-related amplified polymorphism) markers was developed, which spanned 502.9 cM in 9 linkage groups with a mean marker interval of 5.5 cM. Mixed-model-based composite interval mapping was performed to analyze QTL and epistasis effects. One major QTL each for beta-carotene, total carotene and lycopene accumulation were detected which can explain 12.79%, 12.87%, and 14.61% of total phenotypic variations, respectively. Additive genetic variance was primarily responsible for genetic variability in all three major QTL. In addition, a pair of epistasis QTL for beta-carotene and lycopene accumulation was detected, which were able to explain 15.1% and 6.5% of total phenotypic variation, respectively. The dominant x additive and dominant x dominant interaction variance were primary epistasis effect for beta-carotene and lycopene. These SRAP markers linked to QTL could be used in selection or QTL pyramiding for high carotene and lycopene content in carrot breeding.