Molecular Mapping of Putative Genomic Regions Controlling Fruit and Seed Morphology of Watermelon.

The genetic regulatory basis of qualitative and quantitative phenotypes of watermelon is being investigated in different types of molecular and genetic breeding studies around the world. In this study, biparental F2 mapping populations were developed over two experimental years, and the collected datasets of fruit and seed traits exhibited highly significant correlations. Whole-genome resequencing of comparative parental lines was performed and detected single nucleotide polymorphism (SNP) loci were converted into cleaved amplified polymorphic sequence (CAPS) markers. The screened polymorphic markers were genotyped in segregating populations and two genetic linkage maps were constructed, which covered a total of 2834.28 and 2721.45 centimorgan (cM) genetic lengths, respectively. A total of 22 quantitative trait loci (QTLs) for seven phenotypic traits were mapped; among them, five stable and major-effect QTLs (PC-8-1, SL-9-1, SWi-9-1, SSi-9-1, and SW-6-1) and four minor-effect QTLs (PC-2-1 and PC-2-2; PT-2-1 and PT-2-2; SL-6-1 and SSi-6-2; and SWi-6-1 and SWi-6-2) were observed with 3.77-38.98% PVE. The adjacent QTL markers showed a good fit marker-trait association, and a significant allele-specific contribution was also noticed for genetic inheritance of traits. Further, a total of four candidate genes (Cla97C09G179150, Cla97C09G179350, Cla97C09G180040, and Cla97C09G180100) were spotted in the stable colocalized QTLs of seed size linked traits (SL-9-1 and SWi-9-1) that showed non-synonymous type mutations. The gene expression trends indicated that the seed morphology had been formed in the early developmental stage and showed the genetic regulation of seed shape formation. Hence, we think that our identified QTLs and genes would provide powerful genetic insights for marker-assisted breeding aimed at improving the quality traits of watermelon.

Development and Application of a Cleaved Amplified Polymorphic Sequence Marker (Phyto) Linked to the Pc5.1 Locus Conferring Resistance to Phytophthora capsici in Pepper (Capsicum annuum L.).

Phytophthora capsici causes destructive disease in several crop species, including pepper (Capsicum annuum L.). Resistance in this species is physiologically and genetically complex due to many P. capsici virulence phenotypes and different QTLs and R genes among the identified resistance sources. Several primer pairs were designed to follow an SNP (G/A) within the CA_011264 locus linked to the Pc5.1 locus. All primer pairs were designed on DNA sequences derived from CaDMR1, a homoserine kinase (HSK), which is a gene candidate responsible for the major QTL on chromosome P5 for resistance to P. capsici. A panel of 69 pepper genotypes from the Southern Seed germplasm collection was used to screen the primer pairs designed. Of these, two primers (Phyto_for_2 and Phyto_rev_2) surrounding the SNP proved successful in discriminating susceptible and resistant genotypes when combined with a restriction enzyme (BtgI). This new marker (called Phyto) worked as expected in all genotypes tested, proving to be an excellent candidate for marker-assisted selection in breeding programs aimed at introgressing the resistant locus into pure lines.

Next-Generation Sequencing (NGS) Identified Species-Specific SSR and SNP Markers, Allow the Unequivocal Identification of Strawberry Tree (Arbutus unedo L.) Germplasm Accessions and Contribute to Assess Their Genetic Relationships.

The strawberry tree (Arbutus unedo L.), an evergreen bush to small tree of the Ericaceae family, is a main component of the natural flora of the Mediterranean basin that also grows profusely through the Iberian Peninsula, southwestern France, and Ireland. The small edible red fruits are usually used to produce preserves, jams, and liquors, as the Portuguese "aguardente de medronho". The leaves and fruits have been used for a long time in traditional medicine, and their bioactive compounds are presently the subject of intense research. A strawberry tree germplasm collection was recently established by the company Corte Velada (Odiáxere, Portugal). A set of 50 germplasm accessions was selected for a breeding program. A next-generation sequencing project was performed, resulting in the establishment of the first strawberry tree genome assembly and further identification of 500 SSR and 500 SNP loci. Individual molecular fingerprints for the unequivocal identification of the selected 50 accessions were established based on 71 markers alleles amplified by 4 SSR and 9 SNP markers. The same species-specific markers alleles combined with 61 random amplified markers amplified by 5 RAPD and 5 ISSR primers were used to assess the genetic variability and genetic relationships among the selected accessions.

Assessment of Wild Rocket (Diplotaxis tenuifolia (L.) DC.) Germplasm Accessions by NGS Identified SSR and SNP Markers.

Rocket is the common designation for two baby-leaf salad crops of the Brassicaceae family: Eruca sativa (L.) Cav., usually referred to as annual garden rocket, and Diplotaxis tenuifolia (L.) DC. commonly named to as perennial wild rocket. E. sativa is used for human consumption since antiquity. However, the growing consumer preference for D. tenuifolia is being accompanied by the fast increase in its production area and commercialization of new cultivars. Nevertheless, the worldwide number of wild rocket accessions maintained in germplasm collections is very reduced, the solution for which situation the project "REMIRucula" intends to contribute, establishing a germplasm collection at the INIAV, Oeiras, Portugal. Herein, we report on the establishment via next generation sequencing (NGS) of the first genome assembly of D. tenuifolia and the identification of specific single sequence repeat (SSR) and single nucleotide polymorphisms (SNP) loci for the establishment of specific DNA-markers for this species. A representative set of 87 D. tenuifolia and 3 E. sativa accessions were assessed by 5 SSR and 9 SNP-CAPS markers, allowing a drastic discrimination between both species and the establishment of unequivocal molecular fingerprints for the analyzed accessions. The non-discrimination within six pairs and one trio of D. tenuifolia accessions is discussed.

Fine Mapping the Soybean Mosaic Virus Resistance Gene in Soybean Cultivar Heinong 84 and Development of CAPS Markers for Rapid Identification.

Heinong 84 is one of the major soybean varieties growing in Northeast China, and is resistant to the infection of all strains of soybean mosaic virus (SMV) in the region including the most prevalent strain, N3. However, the resistance gene(s) in Heinong 84 and the resistant mechanism are still elusive. In this study, genetic and next-generation sequencing (NGS)-based bulk segregation analysis (BSA) were performed to map the resistance gene using a segregation population from the cross of Heinong 84 and a susceptible cultivar to strain N3, Zhonghuang 13. Results show that the resistance of Heinong 84 is controlled by a dominant gene on chromosome 13. Further analyses suggest that the resistance gene in Heinong 84 is probably an allele of Rsv1. Finally, two pairs of single-nucleotide-polymorphism (SNP)-based primers that are tightly cosegregated with the resistance gene were designed for rapidly identifying resistant progenies in breeding via the cleaved amplified polymorphic sequence (CAPS) assay.

Genetic Mapping and QTL Analysis of Stigma Color in Melon (Cucumis melo L.).

Melon is an important Cucurbitaceae crop. Field observations had shown that the green stigmas of melon are more attractive to pollinators than yellow stigmas. In this study, F2 and F2:3 populations obtained by crossing MR-1 (green stigma) and M4-7 (yellow stigma) were used for genetic analysis and mapping. A genetic map of 1,802.49 cm was constructed with 116 cleaved amplified polymorphism sequence (CAPS) markers. Two stable quantitative trait loci (QTLs) linked to the trait of stigma color were identified on chromosomes 2 (SC2.1) and 8 (SC8.1), respectively. An expanded F2 population was used to narrow down the confidence regions of SC2.1 and SC8.1. As a result, SC2.1 was further mapped to a 3.6 cm region between CAPS markers S2M3 and S2B1-3, explaining 9.40% phenotypic variation. SC8.1 was mapped to a 3.7-cm region between CAPS markers S8E7 and S8H-1, explaining 25.92% phenotypic variation. This study broadens our understanding of the mechanisms of stigma color regulation and will be of benefit to the breeding of melon.

Identification of genome compositions in allopolyploid species of the genus Elymus (Poaceae: Triticeae) in the Asian part of Russia by CAPS analysis.

The genus Elymus L., together with wheat, rye, and barley, belongs to the tribe Triticeae. Apart from its economic value, this tribe is characterized by abundance of polyploid taxa formed in the course of remote hybridization. Single-copy nuclear genes are convenient markers for identification of source genomes incorporated into polyploids. In the present work, a CAPS-marker is developed to distinguish basic St, H, and Y genomes comprising polyploid genomes of Asiatic species of the genus Elymus. The test is based on electrophoretic analysis of restriction patterns of a PCR-amplified fragment of the gene coding for beta-amylase. There are about 50 Elymus species in Russia, and most of them are supposed to possess one of three haplome combinations, StH, StY and StHY. Boreal StH-genomic species endemic for Russia are the least studied. On the basis of nucleotide sequences from public databases, TaqI restrictase was selected, as it produced patterns of restriction fragments specific for St, H, and Y haplomes easily recognizable in agarose gel. A sample of 68 accessions belonging to 32 species was analyzed. In 15 species, the earlier known genomic constitutions were confirmed, but in E. kamoji this assay failed to reveal the presence of H genome. This unusual H genome was suggested to originate from a different Hordeum species. In 16 species, genomic constitutions were identified for the first time. Fifteen accessions from Asian Russia possessed the genomic constitution StStHH, and E. amurensis, phylogenetically close to the StY-genomic species E. ciliaris, had the genomic constitution StStYY. It is inferred that the center of species diversity of the StH-genomic group is shifted to the north as compared to the center of origin of StY-genomic species, confined to China. Key words: Elymus; taxonomy; allopolyploids; genome constitution; CAPS markers.

Fine Mapping of Lycopene Content and Flesh Color Related Gene and Development of Molecular Marker-Assisted Selection for Flesh Color in Watermelon (Citrullus lanatus).

Lycopene content and flesh color are important traits determined by a network of carotenoid metabolic pathways in watermelon. Based on our previous study of genetic inheritance and initial mapping using F2 populations of LSW-177 (red flesh) × cream of Saskatchewan (pale yellow flesh), red flesh color was controlled by one recessive gene regulating red and pale yellow pigmentation, and a candidate region related to lycopene content was detected spanning a 392,077-bp region on chromosome 4. To obtain a more precise result for further study, three genetic populations and a natural panel of 81 watermelon accessions with different flesh colors were used in this research. Herein, we narrowed the preliminary mapping region to 41,233 bp with the linkage map generated from F2 populations of LSW-177 (red flesh) × cream of Saskatchewan (pale yellow flesh) with 1,202 individuals. Two candidate genes, Cla005011 and Cla005012, were found in the fine mapping region; therein Cla005011 was a key locus annotated as a lycopene ß-cyclase gene. Phylogenetic tree analysis showed that Cla005011 was the closest relative gene in gourd. LSW-177 × PI 186490 (white flesh) and another BC1 population derived from garden female (red flesh) × PI 186490 were generated to verify the accuracy of the red flesh candidate gene region. By analyzing the expression levels of candidate genes in different developmental stages of different color watermelon varieties, Cla005011 for the expression differences was not the main reason for the flesh color variation between COS and LSW-177. This indicated that the LCYB gene might regulate fruit color changes at the protein level. A new marker-assisted selection system to identify red and yellow flesh colors in watermelon was developed with flesh color-specific CAPS markers and tested in 81 watermelon accessions.

Development of AhMITE1 markers through genome-wide analysis in peanut (Arachis hypogaea L.).

OBJECTIVE: In peanut, the DNA polymorphism is very low despite enormous phenotypic variations. This limits the use of genomics-assisted breeding to enhance peanut productivity. This study aimed to develop and validate new AhMITE1 and cleaved amplified polymorphic sequences (CAPS) markers. RESULTS: In total, 2957 new AhMITE1 markers were developed in addition to identifying 465 already reported markers from the whole genome re-sequencing data (WGRS) of 33 diverse genotypes of peanut. The B sub-genome (1620) showed more number of markers than the A sub-genome (1337). Distribution also varied among the chromosomes of both the sub-genomes. Further, 52.6% of the markers were from genic regions; where 31.0% were from intronic regions and 5.2% were from exonic regions. Of the 343 randomly selected markers, 82.2% showed amplification validation, with up to 35.5% polymorphism. From the SNPs on the A03, B01, B02 and B03 chromosomes, 11,730 snip-SNPs (potential CAPS sites) were identified, and 500 CAPS markers were developed from chromosome A03. Of these markers, 30.0% showed validation and high polymorphism. This study demonstrated the potential of the WGRS data to develop AhMITE1 and CAPS markers, which showed high level of validation and polymorphism. These marker resources will be useful for various genetic studies and mapping in peanut.

Massive Analysis of cDNA Ends (MACE) for transcript-based marker design in pea (Pisum sativum L.).

Aimed at gene-based markers design, we generated and analyzed transcriptome sequencing datasets for six pea (Pisum sativum L.) genetic lines that have not previously been massively genotyped. Five cDNA libraries obtained from nodules or nodulated roots of genetic lines Finale, Frisson, Sparkle, Sprint-2 and NGB1238 were sequenced using a versatile 3'-RNA-seq protocol called MACE (Massive Analysis of cDNA Ends). MACE delivers a single next-generation sequence from the 3'-end of each individual cDNA molecule that precisely quantifies the respective transcripts. Since the contig generated from the 3'-end of the cDNA by assembling all sequences encompasses the highly polymorphic 3'-untranslated region (3'-UTR), MACE efficiently detects single nucleotide variants (SNVs). Mapping MACE reads to the reference nodule transcriptome assembly of the pea line SGE (Transcriptome Shotgun Assembly GDTM00000000.1) resulted in characterization of over 34,000 polymorphic sites in more than 9700 contigs. Several of these SNVs were located within recognition sequences of restriction endonucleases which allowed the design of co-dominant CAPS markers for the particular transcript. Cleaned reads of sequenced libraries are available from European Nucleotide Archive (http://www.ebi.ac.uk/) under accessions PRJEB18101, PRJEB18102, PRJEB18103, PRJEB18104, PRJEB17691.

Development of STS and CAPS markers for variety identification and genetic diversity analysis of tea germplasm in Taiwan.

BACKGROUND: Tea (Camellia sinensis) is an important economic crop in Taiwan. Particularly, two major commercial types of tea (Paochong tea and Oolong tea) which are produced in Taiwan are famous around the world, and they must be manufactured with specific cultivars. Nevertheless, many elite cultivars have been illegally introduced to foreign countries. Because of the lower cost, large amount of "Taiwan-type tea" are produced and imported to Taiwan, causing a dramatic damage in the tea industry. It is very urgent to develop the stable, fast and reliable DNA markers for fingerprinting tea cultivars in Taiwan and protecting intellectual property rights for breeders. Furthermore, genetic diversity and phylogenetic relationship evaluations of tea germplasm in Taiwan are imperative for parental selection in the cross-breeding program and avoidance of genetic vulnerability. RESULTS: Two STS and 37 CAPS markers derived from cytoplasmic genome and ESTs of tea have been developed in this study providing a useful tool for distinguishing all investigated germplasm. For identifying 12 prevailing tea cultivars in Taiwan, five core markers, including each one of mitochondria and chloroplast, and three nuclear markers, were developed. Based on principal coordinate analysis and cluster analysis, 55 tea germplasm in Taiwan were divided into three groups: sinensis type (C. sinensis var. sinensis), assamica type (C. sinensis var. assamica) and Taiwan wild species (C. formosensis). The result of genetic diversity analysis revealed that both sinensis (0.44) and assamica (0.41) types had higher genetic diversity than wild species (0.25). The close genetic distance between the first (Chin-Shin-Oolong) and the third (Shy-Jih-Chuen) prevailing cultivars was found, and many recently released varieties are the descents of Chin-Shin-Oolong. This implies the potential risk of genetic vulnerability for tea cultivation in Taiwan. CONCLUSIONS: We have successfully developed a tool for tea germplasm discrimination and genetic diversity analysis, as well as a set of core markers for effective identification of prevailing cultivars in Taiwan. According to the results of phylogenetic analysis on prevailing tea cultivars, it is necessary to broaden genetic diversity from wild species or plant introduction in future breeding programs.