The Y2 locus conditions α- and β-carotene accumulation in the xylem (core) of carrot roots. The dominant allele suppresses carotene, but not xanthophyll accumulation, resulting in yellow-cored roots. Individuals homozygous for the recessive allele are rich in carotenes and are therefore orange-cored. Increased consumer interest in high carotene produce requires improved understanding of carotene biosynthesis and color development and more-efficient breeding techniques. We examined 103 F2 individuals generated from inbred populations differing in core carotene content. Bulked segregant analysis identified AFLP bands putatively linked to Y2. Linkage was confirmed for some bands by mapping. Linked bands were excised from gels, re-amplified, cloned into pGEM vectors, and sequenced. Cloned fragments and sequence information were used to characterize larger genomic regions to identify codominant markers. Currently we are developing codominant, PCR-based markers that can be used to rapidly genotype individuals in breeding programs, to characterize wild, feral, and cultivated populations for diversity and evolution studies, and to examine the role of Y2 in carotene accumulation.
The β-fructofuranosidases (invertases) cleave sucrose and related sugars into fructose and glucose. The enzyme is present in most plant tissues in multiple forms. Carrots contain an insoluble β-fructofuranosidase, which is ionically bound to the cell wall and soluble β-fructofuranosidases. The Rs locus in carrot conditions the accumulation of sucrose, fructose, and glucose. The inbred B493 is homozygous for the recessive allele (rs/rs) and accumulates high levels of sucrose, while most carrots accumulate glucose and fructose (Rs/Rs). Increased consumer interest in sweeter carrots require greater understanding of sucrose metabolism and its biochemistry. We established gene transformation systems for carrots using Agrobacterium-mediated and particle bombardment-mediated methods. Intact soluble invertase cDNA was synthesized from B493 and other carrots as measured by RT-PCR. The cDNA fragment was cloned into pBI121 and into a sequencing vector. B493 callus was transformed by Agrobacterium containing the pBI121 vector with invertase driven by the 35S promoter. Invertase expression was compared in rs/rs and Rs/Rs transformed carrots (with invertase overexpression) and non-transformed carrots. These results improve our understanding of the role of soluble invertase in sucrose metabolism of carrot.
When stored at temperatures less than 10 °C, tubers of all cultivated potatoes exhibit cold-induced sweetening (CIS) during which starch degrades to sucrose, glucose, and fructose. Upon frying at high temperatures, the reducing sugars (Fru, Glu) interact with free amino acids via the non-enzymatic Maillard reaction to form dark-colored chips that are unacceptable to consumers. In addition, scientists recently discovered that the toxic chemical acrylamide is also produced during frying. Although storage at warmer temperatures reverses CIS and circumvents dark chip production, the probability of storage loss due to shrinkage and disease increases. Wild Solanum species form the backbone of many potato-breeding programs. In this study, we evaluated 36 different plant introductions (PI) including 20 different species, grown in Madison and Rhinelander, Wis., to identify germplasm resistant to CIS for genetic analysis. After storage for 2–3 months at 4 °C, tuber sugar and amino acid content were analyzed via HPLC and slices were fried to determine chip color. Sugar and chipping data support previous research indicating CIS resistance in S. okadae, S. raphanifolium, and S. phujera. Interestingly, some germplasm selections with high reducing sugar content produced light-colored chips, indicating exceptions to the typical correlation between reducing sugar content and chip color. Genetic bases to these exceptions are under evaluation.
Markers were placed on linkage groups, ordered, and merged for two unrelated F2 populations of carrot (Daucus carota L.). Included were 277 and 242 dominant Amplified fragment-length polymorphism (AFLP) markers and 10 and eight codominant markers assigned to the nine linkage groups of Brasilia × HCM and B493 × QAL F2 populations, respectively. The merged linkage groups were based on two codominant markers and 28 conserved dominant AFLP markers (based upon sequence and size) shared by both populations. The average marker spacing was 4.8 to 5.5 cM in the four parental coupling phase maps. The average marker spacing in the six merged linkage groups was 3.75 cM with maximum gaps among linkage groups ranging from 8.0 to 19.8 cM. Gaps of a similar size were observed with the linkage coupling phase maps of the parents, indicating that linkage group integration did not double the bias which comes with repulsion phase mapping. Three out of nine linkage groups of carrot were not merged due to the absence of common markers. The six merged linkage groups incorporated similar numbers of AFLP fragments from the four parents, further indicating no significant increase in bias expected with repulsion phase linkage. While other studies have merged linkage maps with shared AFLPs of similar size, this is the first report to use shared AFLPs with highly conserved sequence to merge linkage maps in carrot. The genome coverage in this study is suitable to apply quantitative trait locus analysis and to construct a cross-validated consensus map of carrot, which is an important step toward an integrated map of carrot.
Garlic (Allium sativum L.) is an asexually propagated crop that displays much morphological diversity. Studies which have assessed garlic diversity with isozymes and randomly amplified polymorphic DNA (RAPD) markers generally agreed with the morphological observations but sometimes failed to discriminate clones. To discriminate among closely related garlic clones in more detail, we introduced amplified fragment-length polymorphism (AFLPs) to evaluate the genetic diversity and phenetic relatedness of 45 garlic clones and three A. longicuspis clones and we compared AFLP results with RAPD markers and isozymes. Three AFLP primer combinations generated a total of 183 polymorphic fragments. Although similarities between the clusters were low (≥0.30), some clones within the clusters were very similar (>0.95) with AFLP analysis. Sixteen clones represented only six different banding patterns, within which they shared 100% polymorphic AFLPs and RAPD markers, and likely are duplicates. In agreement with the results of other investigators, A. longicuspis and A. sativum clones were clustered together with no clear separation, suggesting these species are not genetically or specifically distinct. The topology of AFLP, RAPD, and isozyme dendrograms were similar, but RAPD and isozyme dendrograms reflected less and much less polymorphism, respectively. Comparison of unweighted pair group method with arithmetic averaging (UPGMA) dendrograms of AFLP, RAPD, and isozyme cluster analyses using the Mantel test indicated a correlation of 0.96, 0.55, and 0.57 between AFLP and RAPD, AFLP and isozyme, and RAPD and isozyme, respectively. Polymorphic AFLPs are abundant in garlic and demonstrated genetic diversity among closely related clones which could not be differentiated with RAPD markers and isozymes. Therefore, AFLP is an additional tool for fingerprinting and detailed assessment of genetic relationships in garlic.
Five cycles of phenotypic recurrent selection for total dissolved solids and sugar type (reducing vs. nonreducing) were performed on four carrot (Daucus carota L.) populations of common background. The populations contained high or low percentage of total dissolved solids (HTDS and LTDS, respectively) with high or low levels of reducing sugar (HRS and LRS, respectively). Effective selection for total dissolved solids (TDS) and sugar type was indicated by significant gains over five cycles of selection. TDS decreased in LTDS/HRS and LTDS/LRS populations by 21.9% and 15.9%, respectively. Corresponding increases of 22.4% and 28.2% were observed in HTDS/HRS and HTDS/LRS populations. Mean reducing sugar levels in HRS roots after five cycles of selection were limited to 2.0% of root fresh weight; sucrose was the primary storage carbohydrate. Reducing sugars were not detected in LRS roots. Mean total sugar levels in the HTDS and LTDS populations were 7.1% and 3.1% of root fresh weight, respectively. Realized heritability estimates ranged from 0.40 to 0.45 for the four populations. The onset of flowering was markedly delayed in plants of the two HTDS populations after five cycles of selection.
Allium plants possess organosulfur compounds and carbohydrates that provide unique flavor and health-enhancing properties. In previous studies of onion F3 families, significant phenotypic and genetic correlations have been reported between pungency, in vitro antiplatelet activity (IVAA), and soluble solids content (SSC); although in other studies SSC and pungency have not always been correlated. In this study we analyzed SSC, pungency, garlic-induced in vitro antiplatelet activity and the content of three predominant thiosulfinates in bulbs from two garlic families obtained from unrelated self-pollinated plants. A strong positive correlation was observed between pungency and IVAA for both sample sets, indicating that it will be difficult to develop garlic populations with low pungency and high IVAA. Allicin was the most abundant thiosulfinate and its content was positively correlated with pungency and IVAA (r= 0.70 and 0.74, respectively). The thiosulfinates AllS(O)SPropenyl and AllS(O)SMe were also positively correlated with pungency and IVAA. When compared with IVAA, AllS(O)SMe had higher r values than AllS(O)SPropenyl (0.88 and 0.50, respectively). These differences could reflect differential platelet anti-aggregatory properties of different thiosulfinates. SSC was not correlated with IVAA, pungency, or thiosulfinates content, suggesting that soluble solids in garlic can be independently selected.
Production of a visible flower stalk, or bolting, has been used as a major trait to categorize garlic (Allium sativum L.) clones. Analysis of mitochondrial genome variation with polymerase chain reaction (PCR) revealed differences between bolting and nonbolting clones of garlic. Screening 333 garlic accessions from diverse geographic origins revealed a 1403-bp mitochondrial DNA marker associated with bolting that the authors call “Bolt Marker” (BltM). Bolt Marker did not amplify in any of the 131 nonbolting clones, whereas amplification of this marker was observed in 127 of 130 (97.7%) garlic clones that bolted completely in Wisconsin. Seventy-two garlic clones bolted incompletely (clones in which some but not all of the plants bolted), and this marker was not amplified in 69 (95.8%) of these clones. Because of the significant association of BltM with bolting, this PCR-based marker can be used to discriminate complete-bolting garlic clones reliably from nonbolting and incomplete-bolting ones. Sequence characterization of this marker revealed that BltM is a chimera involving both mitochondrial and chloroplast DNA. The DNA sequences including and flanking both the 5′ and 3′ ends of this marker are consistent with an ≈4.8-kbp chloroplast DNA fragment having been inserted into the mitochondrial genome downstream from the mitochondrial cox3 gene. Sequence alignment of the chloroplast genes in this chimeric region with the homologous sequences in GenBank indicate the presence of deletions, insertions, and single nucleotide polymorphisms in the coding sequences, resulting in putative, incomplete open reading frames or frame shift mutations. Hence, the authors speculate that this insertion may have occurred long ago in the evolution of garlic.
Carotenoids are isoprenoid compounds synthesized in plants that serve as photoprotectants essential for photosynthesis and provide plant tissues with red, orange, and yellow pigmentation. These compounds are important in human health, because they serve as both vitamin A precursors as well as having antioxidant properties. Carrot (Daucus carota ssp. sativus) provides an important source of carotenoids in the human diet, providing up to 30% of provitamin A in the United States. Although essential to human health, very little is currently understood about the accumulation of carotenoids in carrot. To better understand the molecular mechanism for carotenoid accumulation in carrot, we used reverse-transcription quantitative polymerase chain reaction (PCR) to evaluate the expression of nine genes in the carotenoid biosynthetic pathway in storage root tissue. No significant difference was found among white, yellow, orange, and dark orange carrot roots in seven of the nine genes evaluated. However, increased phytoene synthase 1 (PSY1) and phytoene synthase 2 (PSY2) expression was observed in orange and dark orange carrot roots compared with yellow and white carrots. Increased PSY1 and PSY2 expression was not observed in the leaf tissue of these genotypes, indicating a different mechanism for carotenoid accumulation in the leaf tissue of carrot. This study is the first to demonstrate that naturally occurring mutations that dramatically increase carotenoid accumulation in orange carrot are associated with increased PSY1 and PSY2 expression and it provides insights into the mechanism underlying the biosynthesis of these important photoprotectants and nutrients.