A Rosaceae Family-Level Approach To Identify Loci Influencing Soluble Solids Content in Blackberry for DNA-Informed Breeding.
ABSTRACT: A Rosaceae family-level candidate gene approach was used to identify genes associated with sugar content in blackberry (Rubus subgenus Rubus). Three regions conserved among apple (Malus × domestica), peach (Prunus persica), and alpine strawberry (Fragaria vesca) were identified that contained previously detected sweetness-related quantitative trait loci (QTL) in at least two of the crops. Sugar related genes from these conserved regions and 789 sugar-associated apple genes were used to identify 279 Rubus candidate transcripts. A Hyb-Seq approach was used in conjunction with PacBio sequencing to generate haplotype level sequence information of sugar-related genes for 40 cultivars with high and low soluble solids content from the University of Arkansas and USDA blackberry breeding programs. Polymorphisms were identified relative to the 'Hillquist' blackberry (R. argutus) and ORUS 4115-3 black raspberry (R. occidentalis) genomes and tested for their association with soluble solids content (SSC). A total of 173 alleles were identified that were significantly (? = 0.05) associated with SSC. KASP genotyping was conducted for 92 of these alleles on a validation set of blackberries from each breeding program and 48 markers were identified that were significantly associated with SSC. One QTL, qSSC-Ruh-ch1.1, identified in both breeding programs accounted for an increase of 1.5 °Brix and the polymorphisms were detected in the intron space of a sucrose synthase gene. This discovery represents the first environmentally stable sweetness QTL identified in blackberry. The approach demonstrated in this study can be used to develop breeding tools for other crops that have not yet benefited directly from the genomics revolution.
Project description:Sweetness is one of the main drivers of consumer preference, and thus is given high priority in apple breeding programmes. Due to the complexity of sweetness evaluation, soluble solid content (SSC) is commonly used as an estimation of this trait. Nevertheless, it has been demonstrated that SSC and sweet taste are poorly correlated. Though individual sugar content may vary greatly between and within apple cultivars, no previous study has tried to investigate the relationship between the amount of individual sugars, or ratios of these, and apple sweetness. In this work, we quantified the major sugars (sucrose, glucose, fructose, xylose) and sorbitol and explored their influence on perceived sweetness in apple; we also related this to malic acid content, SSC and volatile compounds. Our data confirmed that the correlation between sweetness and SSC is weak. We found that sorbitol content correlates (similarly to SSC) with perceived sweetness better than any other single sugar or total sugar content. The single sugars show no differentiable importance in determining apple sweetness. Our predictive model based on partial least squares regression shows that after sorbitol and SSC, the most important contribution to apple sweetness is provided by several volatile compounds, mainly esters and farnesene.
Project description:BACKGROUND:Sugar content is an important determinant of fruit sweetness, but details on the complex molecular mechanism underlying fruit sugar accumulation remain scarce. Here, we report the role of sucrose transporter (SUT) family in regulating fruit sugar accumulation in apple. RESULTS:Gene-tagged markers were developed to conduct candidate gene-based association study, and an SUT4 member MdSUT4.1 was found to be significantly associated with fruit sugar accumulation. MdSUT4.1 encodes a tonoplast localized protein and its expression level had a negative correlation with fruit sugar content. Overexpression of MdSUT4.1 in strawberry and apple callus had an overall negative impact on sugar accumulation, suggesting that it functions to remobilize sugar out of the vacuole. In addition, MdSUT4.1 is located on chromosomal region harboring a previously reported QTL for sugar content, suggesting that it is a candidate gene for fruit sugar accumulation in apple. CONCLUSIONS:MdSUT4.1 is involved in the regulation of fruit sugar accumulation in apple. This study is not only helpful for understanding the complex mechanism of fruit sugar accumulation, but it also provides molecular tools for genetic improvement of fruit quality in breeding programs of apple.
Project description:Sugar content is the major determinant of both fruit quality and consumer acceptance in melon (Cucumis melo L), and is a primary target for crop improvement. Near-isogenic lines (NILs) derived from the intraspecific cross between a "Piel de Sapo" (PS) type and the exotic cultivar "Songwhan Charmi" (SC), and several populations generated from the cross of PS × Ames 24294 ("Trigonus"), a wild melon, were used to identify QTL related to sugar and organic acid composition. Seventy-eight QTL were detected across several locations and different years, with three important clusters related to sugar content located on chromosomes 4, 5, and 7. Two PS × SC NILs (SC5-1 and SC5-2) sharing a common genomic interval of 1.7 Mb at the top of chromosome 5 contained QTL reducing soluble solids content (SSC) and sucrose content by an average of 29 and 68%, respectively. This cluster collocated with QTL affecting sugar content identified in other studies in lines developed from the PS × SC cross and supported the presence of a stable consensus locus involved in sugar accumulation that we named SUCQSC5.1. QTL reducing soluble solids and sucrose content identified in the "Trigonus" mapping populations, as well as QTL identified in previous studies from other ssp. agrestis sources, collocated with SUCQSC5.1, suggesting that they may be allelic and implying a role in domestication. In subNILs derived from the PS × SC5-1 cross, SUCQSC5.1 reduced SSC and sucrose content by an average of 18 and 34%, respectively, and was fine-mapped to a 56.1 kb interval containing four genes. Expression analysis of the candidate genes in mature fruit showed differences between the subNILs with PS alleles that were "high" sugar and SC alleles of "low" sugar phenotypes for MELO3C014519, encoding a putative BEL1-like homeodomain protein. Sequence differences in the gene predicted to affect protein function were restricted to SC and other ssp. agrestis cultivar groups. These results provide the basis for further investigation of genes affecting sugar accumulation in melon.
Project description:According to estimates of various taxonomists, the genus Rubus L. (Rosaceae Juss.) consists of 12-16 subgenera comprising ~750 species. The two largest subgenera are Idaeobatus (Focke) Focke, which includes raspberries, and the type subgenus Rubus (=Eubatus Focke), which contains blackberry species. Representatives of the genus Rubus have high nutritional and economic values, as well as medicinal properties. Breeding programs are aimed at broadening genetic diversity and creating new varieties of raspberries and blackberries that are resistant to biotic and abiotic stressors and have high fruit quality. Modern breeding and genetic programs increasingly use the achievements of molecular genetics and genomics. This paper reviews the literature data on the application of molecular markers in fundamental and applied research aimed at studying the genetic diversity of cultivated and wild species of the genus Rubus. The review describes the main types of molecular markers (RFLP, RAPD, SCoT, SSR, ISSR, AFLP, SCAR, SSCP) and their application for studying the species of the genus Rubus. The results of the work on the use of DNA markers for solving different tasks are presented, including: studying the phylogenetic relationships of species, clarifying controversial issues of taxonomy, analyzing interspecific and intraspecific diversity, genotyping and pedigree analysis of raspberry and blackberry varieties, studying somaclonal variation and others. The most important applied result is the development of molecular genetic maps for raspberry and blackberry species, on which numerous genes and QTLs conferring various valuable traits have been mapped. At the same time, the number of markers that are promising for effective molecular screening is still insufficient.
Project description:BACKGROUND: The recent development of novel repeat-fruiting types of blackberry (Rubus L.) cultivars, combined with a long history of morphological marker-assisted selection for thornlessness by blackberry breeders, has given rise to increased interest in using molecular markers to facilitate blackberry breeding. Yet no genetic maps, molecular markers, or even sequences exist specifically for cultivated blackberry. The purpose of this study is to begin development of these tools by generating and annotating the first blackberry expressed sequence tag (EST) library, designing primers from the ESTs to amplify regions containing simple sequence repeats (SSR), and testing the usefulness of a subset of the EST-SSRs with two blackberry cultivars. RESULTS: A cDNA library of 18,432 clones was generated from expanding leaf tissue of the cultivar Merton Thornless, a progenitor of many thornless commercial cultivars. Among the most abundantly expressed of the 3,000 genes annotated were those involved with energy, cell structure, and defense. From individual sequences containing SSRs, 673 primer pairs were designed. Of a randomly chosen set of 33 primer pairs tested with two blackberry cultivars, 10 detected an average of 1.9 polymorphic PCR products. CONCLUSION: This rate predicts that this library may yield as many as 940 SSR primer pairs detecting 1,786 polymorphisms. This may be sufficient to generate a genetic map that can be used to associate molecular markers with phenotypic traits, making possible molecular marker-assisted breeding to compliment existing morphological marker-assisted breeding in blackberry.
Project description:Many important apple (Malus × domestica Borkh.) fruit quality traits are regulated by multiple genes, and more information about quantitative trait loci (QTLs) for these traits is required for marker-assisted selection. In this study, we constructed genetic linkage maps of the Japanese apple cultivars 'Orin' and 'Akane' using F1 seedlings derived from a cross between these cultivars. The 'Orin' map consisted of 251 loci covering 17 linkage groups (LGs; total length 1095.3 cM), and the 'Akane' map consisted of 291 loci covering 18 LGs (total length 1098.2 cM). We performed QTL analysis for 16 important traits, and found that four QTLs related to harvest time explained about 70% of genetic variation, and these will be useful for marker-assisted selection. The QTL for early harvest time in LG15 was located very close to the QTL for preharvest fruit drop. The QTL for skin color depth was located around the position of MYB1 in LG9, which suggested that alleles harbored by 'Akane' are regulating red color depth with different degrees of effect. We also analyzed soluble solids and sugar component contents, and found that a QTL for soluble solids content in LG16 could be explained by the amount of sorbitol and fructose.
Project description:The Panel on Plant Health of EFSA conducted a pest categorisation of 17 viruses of Rubus L. that were previously classified as either non-EU or of undetermined standing in a previous opinion. These infectious agents belong to different genera and are heterogeneous in their biology. Blackberry virus X, blackberry virus Z and wineberry latent virus were not categorised because of lack of information while grapevine red blotch virus was excluded because it does not infect Rubus. All 17 viruses are efficiently transmitted by vegetative propagation, with plants for planting representing the major pathway for entry and spread. For some viruses, additional pathway(s) are Rubus seeds, pollen and/or vector(s). Most of the viruses categorised here infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Cherry rasp leaf virus, raspberry latent virus, raspberry leaf curl virus, strawberry necrotic shock virus, tobacco ringspot virus and tomato ringspot virus meet all the criteria to qualify as potential Union quarantine pests (QPs). With the exception of impact in the EU territory, on which the Panel was unable to conclude, blackberry chlorotic ringspot virus, blackberry leaf mottle-associated virus, blackberry vein banding-associated virus, blackberry virus E, blackberry virus F, blackberry virus S, blackberry virus Y and blackberry yellow vein-associated virus satisfy all the other criteria to be considered as potential QPs. Black raspberry cryptic virus, blackberry calico virus and Rubus canadensis virus 1 do not meet the criterion of having a potential negative impact in the EU. For several viruses, the categorisation is associated with high uncertainties, mainly because of the absence of data on biology, distribution and impact. Since the opinion addresses non-EU viruses, they do not meet the criteria to qualify as potential Union regulated non-quarantine pests.
Project description:Sugar content is related to fruit sweetness, and the complex mechanisms underlying fruit sugar accumulation still remain elusive. Here, we report a peach PpTST1 gene encoding tonoplast sugar transporter that is located in the quantitative trait loci (QTL) interval on Chr5 controlling fruit sucrose content. One derived Cleaved Amplified Polymorphic Sequence (dCAPS) marker was developed based on a nonsynonymous G/T variant in the third exon of PpTST1. Genotyping of peach cultivars with the dCAPS marker revealed a significant difference in fruit sugar content among genotypes. PpTST1 is located in the tonoplast, and substitution of glutamine by histidine caused by the G/T variation has no impact on subcellular location. The expression profile of PpTST1 exhibited a consistency with the sugar accumulation pattern, and its transient silencing significantly inhibited sugar accumulation in peach fruits. All of these results demonstrated the role of PpTST1 in regulating sugar accumulation in peach fruit. In addition, cis-elements for binding of MYB and WRKY transcript factors were found in the promoter sequence of PpTST1, suggesting a gene regulatory network of fruit sugar accumulation. Our results are not only helpful for understanding the mechanisms underlying fruit sugar accumulation, but will also be useful for the genetic improvement of fruit sweetness in peach breeding programs.
Project description:Most Rubus species have a biennial cycle of flowering and fruiting with an intervening period of winter dormancy, in common with many perennial fruit crops. Annual-fruiting (AF) varieties of raspberry (Rubus idaeus and Rubus occidentalis L.) and blackberry (Rubus subgenus Rubus) are able to flower and fruit in one growing season, without the intervening dormant period normally required in biennial-fruiting (BF) varieties. We used a red raspberry (R. idaeus) population segregating for AF obtained from a cross between NC493 and 'Chilliwack' to identify genetic factors controlling AF. Genotyping by sequencing (GBS) was used to generate saturated linkage maps in both parents. Trait mapping in this population indicated that AF is controlled by two newly identified loci (RiAF3 and RiAF4) located on Rubus linkage groups (LGs) 3 and 4. The location of these loci was analyzed using single-nucleotide polymorphism (SNP) markers on independent red raspberry and blackberry populations segregating for the AF trait. This confirmed that AF in Rubus is regulated by loci on LG 3 and 4, in addition to a previously reported locus on LG 7. Comparative RNAseq analysis at the time of floral bud differentiation in an AF and a BF variety revealed candidate genes potentially regulating the trait.
Project description:The cultivated strawberry (Fragaria×ananassa) is consumed worldwide for its flavor and nutritional benefits. Genetic analysis of commercially important traits in strawberry are important for the development of breeding methods and tools for this species. Although several quantitative trait loci (QTL) have been previously detected for fruit quality and flowering traits using low-density genetic maps, clarity on the sub-genomic locations of these QTLs was missing. Recent discoveries in allo-octoploid strawberry genomics led to the development of the IStraw90 single-nucleotide polymorphism (SNP) array, enabling high-density genetic maps and finer resolution QTL analysis. In this study, breeder-specified traits were evaluated in the Eastern (Michigan) and Western (Oregon) United States for a common set of breeding populations during 2 years. Several QTLs were validated for soluble solids content (SSC), fruit weight (FWT), pH and titratable acidity (TA) using a pedigree-based QTL analysis approach. For fruit quality, a QTL for SSC on linkage group (LG) 6A, a QTL for FWT on LG 2BII, a QTL for pH on LG 4CII and two QTLs for TA on LGs 2A and 5B were detected. In addition, a large-effect QTL for flowering was detected at the distal end of LG 4A, coinciding with the FaPFRU locus. Marker haplotype analysis in the FaPFRU region indicated that the homozygous recessive genotype was highly predictive of seasonal flowering. SNP probes in the FaPFRU region may help facilitate marker-assisted selection for this trait.