Development Of Functional Markers Research Articles

De Novo Assembly and Characterization of Developing Spikes Transcriptome of Finger Millet (Eleusine coracana): a Minor Crop Having Nutraceutical Properties

Finger millet (Eleusine coracana), a minor crop, is a rich source of micronutrients especially calcium and protein. In the present study, developing spikes transcriptome of two finger millet genotypes differing in grain calcium content was sequenced using Illumina HiSeq 2000 Genome analyser. A total of 72.44 and 64.84 million reads of high and low grain calcium genotype were assembled using Trinity into a non-redundant set of 120,130 and 109,218 transcripts (≥200 bp). The average lengths of transcripts were 921 and 789 bp and N50 lengths of 1,450 and 1,191 bp with coverage of 60,911.1 and 59,969.9 FPKM, respectively. At the protein level, 74.32 and 75.39 % finger millet transcripts showed significant similarity with the proteins of rice followed by sorghum. A total of 44,255 (36.8 %) and 43,780 (40 %) transcripts were assigned to 106 gene ontology (GO) terms for three main categories: biological function, cellular component and molecular function. Further, functional characterization of transcripts led to the identification of nutritionally important genes encoding calcium transporters and seed storage proteins. In addition, simple sequence repeat (SSR) motifs in the transcripts were also identified. The transcriptome data generated here provides a rich resource for discovery of genes expressed throughout the spike development and development of functional molecular markers.

De novo assembly and characterization of the fruit transcriptome of Chinese jujube (Ziziphus jujuba Mill.) Using 454 pyrosequencing and the development of novel tri-nucleotide SSR markers.

Chinese jujube (Ziziphus jujuba Mill.) is an economically important deciduous tree that has high therapeutic value and health benefits. However, a lack of sequence data and molecular markers have constrained genetic and breeding studies for better fruit quality and other traits in Chinese jujube. In this study, two combined cDNA libraries of ‘Dongzao’ fruit representing the early and late stages of fruit development were constructed and sequenced on the 454 GS FLX Titanium platform. In total, 1,124,197 reads were generated and then de novo assembled into 97,479 unigenes. A total of 52,938 unigenes were homologous to genes in the NCBI non-redundant sequence database. A total of 33,123 unigenes were assigned to one or more Gene Ontology terms, and 16,693 unigenes were classified into 319 Kyoto Encyclopedia of Genes and Genomes pathways. The results showed that the Smirnoff-Wheeler pathway was the main pathway for the biosynthesis of ascorbic acid in Chinese jujube. The number of differentially expressed genes between the two stages of fruit development was 1,764, among which 974 and 790 genes were up-regulated and down-regulated, respectively. Furthermore, 9,893 sequences were identified containing SSRs. 93 primer pairs designed from the sequences with a tri-nucleotide repeat showed successful PCR amplification and could be validated in Chinese jujube accessions and Z. mauritiana Lam and Z. acidojujuba as well, of which 71 primer pairs were polymorphic. The obtained transcriptome provides a most comprehensive resource currently available for gene discovery and the development of functional markers in Z. jujuba. The newly developed microsatellite markers could be used in applications such as genetic linkage analysis and association studies, diversity analysis, and marker-assisted selection in Chinese jujube and related species.

Identification of candidate genes for rice grain aroma by combining QTL mapping and transcriptome profiling approaches

The present report is in continuation to our earlier reports on the identification and fine mapping of three aroma QTLs in basmati rice using a bi-parental mapping population derived from a cross between Pusa 1121, a basmati rice variety, and Pusa 1342, a non-aromatic rice variety. We used a combination of genetic mapping and transcriptome profiling to narrow down the number of differentially expressed genes in rice to identify potential candidate genes for rice grain aroma. Highly aromatic and non-aromatic recombinant inbred lines (RILs) were identified through sensory analysis of mature milled grains. RILs with similar phenotypes were bulked together using bulk segregant analysis approach which drastically reduced the number of differentially expressed genes from 4016 to 1344. The transcriptome profiles generated were analyzed through Affymetrix rice genome array containing probe sets designed from all the predicted rice gene sequences. Microarray-based transcriptome profiling revealed one down-regulated gene co-located in QTL region aro3.1 on chromosome 3, eight genes co-located in the aro4.1 region on chromosome 4 and the badh2 gene on chromosome 8 to be differentially expressed in the aromatic parent and aromatic bulk. These genes are the most suitable candidates for future validation and development of new molecular functional markers to facilitate marker assisted breeding.

Gene mining of sulfur-containing amino acid metabolic enzymes in soybean

The genes of sulfur-containing amino acid synthetases in soybean are essential for the synthesis of sulfur-containing amino acids. Gene mining of these enzymes is the basis for the molecular assistant breeding of high sulfur-containing amino acids in soybean. In this study, using software BioMercator2.1, 113 genes of sulfur-containing amino acid enzymes and 33 QTLs controlling the sulfur-containing amino acids content were mapped onto Consensus Map 4.0, which was integrated by genetic and physical maps of soybean. Sixteen candidate genes associated to the synthesis of sulfur-containing amino acids were screened based on the synteny between gene loci and QTLs, and the effect values of QTLs. Through a bioinformatic analysis of the copy number, SNP information, and expression profile of candidate genes, 12 related enzyme genes were identified and mapped on 8 linkage groups, such as D1a, M, A2, K, and G. The genes corresponding to QTL regions can explain 6%?38.5% genetic variation of sulfur-containing amino acids, and among them, the indirect effect values of 9 genes were more than 10%. These 12 genes were involved in sulfur-containing amino acid metabolism and were highly expressed in the cotyledons and flowers, showing an abundance of SNPs. These genes can be used as candidate genes for the development of functional markers, and it will lay a foundation for molecular design breeding in soybean.

Identifying a source of a bacterial blight resistance gene xa5 in rice variety ‘IR62266’ and development of a functional marker ‘PAxa5’, the easy agarose based detection

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important diseases in rice worldwide causing tremendous damage to rice yield in many countries. The use of resistant rice varieties is the most economical and effective method to manage and control this disease. IR62266, a rice variety developed by IRRI , was identified as a good genetic resource for broad spectrum BB resistance in Thailand. The genetic factor determining BB resistance was studied in the F 2:3 population developed from a cross between IR62266 and KDML105 using three Thai Xoo isolates , TXO1 , TXO2 , and TXO5. A total of 190 F 2:3 individuals were challenged with TXO1 , TXO2 , and TXO5 and lesion lengths were measured. The resistant reactions of the F 2:3 population showed the ratio of 3 susceptible : 1 resistant, thereby indicating a single recessive gene controlled BB resistance in IR62266. Genetic analysis indicated that RM122 was located on the short arm of chromosome 5 and was the tightly linked to the resistance gene accounting for 66.03% of phenotypic variance . The RM122 was located very close to the cloned xa5 gene, a broad spectrum BB resistance gene . . Therefore, the PAxa5 was developed as a new functional SNP marker and tested in the F 2:3 individuals. PAxa5 clearly differentiated the resistant and susceptible reactions in the population. PAxa5 was then validated in a wide range of germplasms and was found to clearly identify xa5 . PAxa5 is currently implemented in the marker-assisted breeding program to facilitate the selection for the xa5 gene

De novo transcriptome assembly of Vitis flexuosa grapevines inoculated with Elsinoe ampelina

In this study, the transcriptome of Vitis flexuosa leaves inoculated with Elsinoe ampelina was analysed to identify useful genes and elucidate their function and differential expression patterns through assembly and annotation gene ontology of data from sequencing short reads on the Illumina platform. We assembled ~121 million high-quality trimmed reads using Velvet and Oases with optimal parameters into a non-redundant set of 70,899 transcripts ( ≥ 200 bp in length). The transcripts exhibited an average length of 1138 bp and a N50 length of 1695 bp, with the largest contig length being 9623 bp. Functional categorization revealed the conservation of genes involved in various molecular functions, including protein binding (21.1%) and oxidoreductase activity (11.7%), in V. flexuosa. The V. flexuosa transcript set generated in this study will serve as a resource for gene discovery and development of functional molecular markers.

Quantitative Trait Loci and Candidate Genes Associated with Fatty Acid Content of Watermelon Seed

Seed oil percentage (SOP) and fatty acid composition of watermelon (Citrullus lanatus) seeds are important traits in Africa, the Middle East, and Asia where the seeds provide a significant source of nutrition and income. Oil yield from watermelon seed exceeds 50% (w/w) and is high in unsaturated fatty acids, a profile comparable to that of sunflower (Helianthus annuus) and soybean (Glycine max) oil. As a result of novel non-food uses of plant-derived oils, there is an increasing need for more sources of vegetable oil. To improve the nutritive value of watermelon seed and position watermelon as a potential oil crop, it is critical to understand the genetic factors associated with SOP and fatty acid composition. Although the fatty acid composition of watermelon seed is well documented, the underlying genetic basis has not yet been studied. Therefore, the current study aimed to elucidate the quality of watermelon seed oil and identify genomic regions and candidate genes associated with fatty acid composition. Seed from an F2 population developed from a cross between an egusi type (PI 560023), known for its high SOP, and Strain II (PI 279261) was phenotyped for palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2). Significant (P < 0.05) correlations were found between palmitic and oleic acid (0.24), palmitic and linoleic acid (–0.37), stearic and linoleic acid (–0.21), and oleic and linoleic acid (–0.92). A total of eight quantitative trait loci (QTL) were associated with fatty acid composition with a QTL for oleic and linoleic acid colocalizing on chromosome (Chr) 6. Eighty genes involved in fatty biosynthesis including those modulating the ratio of saturated and unsaturated fatty acids were identified from the functionally annotated genes on the watermelon draft genome. Several fatty acid biosynthesis genes were found within and in close proximity to the QTL identified in this study. A gene (Cla013264) homolog to fatty acid elongase (FAE) was found within the 1.5-likelihood-odds (LOD) interval of the QTL for palmitic acid (R2 = 7.6%) on Chr 2, whereas Cla008157, a homolog to omega-3-fatty acid desaturase and Cla008263, a homolog to FAE, were identified within the 1.5-LOD interval of the QTL for palmitic acid (R2 = 24.7%) on Chr 3. In addition, the QTL for palmitic acid on Chr 3 was located ≈0.60 Mbp from Cla002633, a gene homolog to fatty acyl- [acyl carrier protein (ACP)] thioesterase B. A gene (Cla009335) homolog to ACP was found within the flanking markers of the QTL for oleic acid (R2 = 17.9%) and linoleic acid (R2 = 21.5%) on Chr 6, whereas Cla010780, a gene homolog to acyl-ACP desaturase was located within the QTL for stearic acid (R2 = 10.2%) on Chr 7. On Chr 8, another gene (Cla013862) homolog to acyl-ACP desaturase was found within the 1.5-LOD interval of the QTL for oleic acid (R2 = 13.5%). The genes identified in this study are possible candidates for the development of functional markers for application in marker-assisted selection for fatty acid composition in watermelon seed. To the best of our knowledge, this is the first study that aimed to elucidate genetic control of the fatty acid composition of watermelon seed.

Stacking of five favorable alleles for amylase content, fragrance and disease resistance into elite lines in rice (Oryza sativa) by using four HRM-based markers and a linked gel-based marker

Marker-assisted selection (MAS) for qualitative traits such as grain quality and resistance to certain diseases has proven to be highly effective. Multiple genes responsible for various quality components and disease resistances can be simultaneously stacked to boost the performance and to lengthen the commercial lifespan of high-yield varieties. Grain quality genes (fgr and Wx) and three disease resistance genes (Pita, Pik and Xa23) have been well characterized and used in MAS breeding. However, stacking all of them together into a single variety has not been reported. We reported here the stacking of the five genes into elite lines in rice. We achieved this through the development of functional markers from causal mutations at fgr, Wx and Pita, a gene-targeted marker at Pik and the use of a linked marker for Xa23. We employed and optimized the high-resolution melting (HRM) analysis method for use as the genotyping platform of fgr, Wx, Pik and Pita. By combining high-throughput DNA isolation, multiplex and nested-PCR methods, we showed that HRM could serve as cost-effective, highly automated, moderate-throughput and reliable non-gel genotyping platform for a small-scale MAS program.

Leaf-, panel- and latex-expressed sequenced tags from the rubber tree (Hevea brasiliensis) under cold-stressed and suboptimal growing conditions: the development of gene-targeted functional markers for stress response.

Hevea brasiliensis is a native species of the Amazon Basin of South America and the primary source of natural rubber worldwide. Due to the occurrence of South American Leaf Blight disease in this area, rubber plantations have been extended to suboptimal regions. Rubber tree breeding is time-consuming and expensive, but molecular markers can serve as a tool for early evaluation, thus reducing time and costs. In this work, we constructed six different cDNA libraries with the aim of developing gene-targeted molecular markers for the rubber tree. A total of 8,263 reads were assembled, generating 5,025 unigenes that were analyzed; 912 expressed sequence tags (ESTs) represented new transcripts, and two sequences were highly up-regulated by cold stress. These unigenes were scanned for microsatellite (SSR) regions and single nucleotide polymorphisms (SNPs). In total, 169 novel EST-SSR markers were developed; 138 loci were polymorphic in the rubber tree, and 98 % presented transferability to six other Hevea species. Locus duplication was observed in H. brasiliensis and other species. Additionally, 43 SNP markers in 13 sequences that showed similarity to proteins involved in stress response, latex biosynthesis and developmental processes were characterized. cDNA libraries are a rich source of SSR and SNP markers and enable the identification of new transcripts. The new markers developed here will be a valuable resource for linkage mapping, QTL identification and other studies in the rubber tree and can also be used to evaluate the genetic variability of other Hevea species, which are valuable assets in rubber tree breeding.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-014-0095-2) contains supplementary material, which is available to authorized users.

Genetic and physical mapping of the QTLAR3 controlling blight resistance in chickpea (Cicer arietinum L)

Physical and genetic maps of chickpea a QTL related to Ascochyta blight resistance and located in LG2 (QTLAR3) have been constructed. Single-copy markers based on candidate genes located in the Ca2 pseudomolecule were for the first time obtained and found to be useful for refining the QTL position. The location of the QTLAR3 peak was linked to an ethylene insensitive 3-like gene (Ein3). The Ein3 gene explained the highest percentage of the total phenotypic variation for resistance to blight (44.3 %) with a confidence interval of 16.3 cM. This genomic region was predicted to be at the Ca2 physical position 32–33 Mb, comprising 42 genes. Candidate genes located in this region include Ein3, Avr9/Cf9 and Argonaute 4, directly involved in disease resistance mechanisms. However, there are other genes outside the confidence interval that may play a role in the blight resistance pathway. The information reported in this paper will facilitate the development of functional markers to be used in the screening of germplasm collections or breeding materials, improving the efficiency and effectiveness of conventional breeding methods.

An efficient and cost-effective approach for genic microsatellite marker-based large-scale trait association mapping: identification of candidate genes for seed weight in chickpea

The large-scale validation and high-throughput genotyping of numerous informative genic microsatellite markers are required for association mapping to identify candidate genes for complex quantitative traits in chickpea. However, the screening and genotyping of such informative markers in individual genotypes/whole association panels for trait association mapping involves massive costs in terms of resources, time and labour due to low genetic polymorphism in chickpea. We have developed an alternative time-saving and cost-effective pool-based trait association mapping approach by combining pooled DNA analysis (with 616 genic microsatellite markers) and individual genotype (large structured association panel) genotyping. Using this approach we have identified seven seed weight-associated transcription factor gene-derived microsatellite markers (with minor allele frequency >15 %) in desi and kabuli chickpea. Strong marker allele effects of these five transcription factors with increasing seed weight in the contrasting desi and kabuli genotypes were evident. Bi-parental linkage mapping using 241 of the informative gene-based microsatellite markers resulted in the identification and mapping of nine such markers linked with three major quantitative trait loci (explaining a total phenotypic variance of 23.5–34.7 %) on chromosomes 1 (CaqSW1.1: 73.5–74.5 cM and CaqSW1.2: 79.3–81.3 cM) and 2 (CaqSW2.1: 65.7–67.5 cM) controlling 100-seed weight in chickpea. The integration of pool-based trait association mapping with differential expression profiling, traditional bi-parental linkage mapping and high-resolution microsatellite-single nucleotide polymorphism marker-based haplotyping/linkage disequilibrium mapping delineated four transcription factor genes (DUF3594, bZIP, DUF1635 and SBP) controlling seed weight in desi and kabuli chickpea. The strategies implemented in our study can be used in large-scale trait association mapping for the rapid identification of candidate genes and in the development of functional markers for traits of agricultural importance in crop species including chickpea.

Quantitative trait loci controlling aluminum tolerance in soybean: candidate gene and single nucleotide polymorphism marker discovery

Aluminum (Al) toxicity is an important abiotic stress that affects soybean production in acidic soils throughout the world. Development of Al-tolerant cultivars is an efficient and environmentally friendly solution to the problem. A previous report identified quantitative trait loci (QTL) for Al tolerance inherited from PI 416937, using restriction fragment length polymorphism markers, in a population of Young × PI 416937. The population was genotyped with 162 simple sequence repeats to enhance the power of QTL detection and enable the selection of candidate genes for functional marker development. Two QTL that explained 54 % of the phenotypic variation in root extension under Al stress conditions (HIAL) were refined on chromosomes (chr) Gm08 and Gm16. Three QTL located on chr Gm08, Gm16 and Gm19 explained 59 % of the phenotypic variation in root extension as a percent of control (PC). Two major QTL, designated qAL_HIAL_08 and qAL_PC_08, controlling HIAL and PC, respectively, were mapped to the same genomic region on chr Gm08 and inherited their favorable allele from PI 416937. These QTL explained 45 and 41 % of phenotypic variation in HIAL and PC, respectively. Six homologues for citrate synthase (CS) genes were found in the soybean genome sequence at chr Gm02, Gm08, Gm14, Gm15, and Gm18. Sixteen single nucleotide polymorphisms (SNPs) were identified in the CS homologue on chr Gm08. A SimpleProbe assay of Glyma08g42400-SNP was developed for the major QTL on chr Gm08. The SNPs identified from this region could be used for marker-assisted selection of Al tolerance.

Allelic variation of a soluble acid invertase gene (SAI-1) and development of a functional marker in sweet sorghum [Sorghum bicolor (L.) Moench

Sweet sorghum [Sorghum bicolor (L.) Moench] accumulates large amounts of sugars in the stem parenchyma and can be used to manufacture sucrose and alcohol. Soluble acid invertase (SAI) plays an important role in sucrose accumulation in the sweet sorghum stem. Characterization of SAI genes and development of functional markers are important for marker-assisted selection in breeding programs. Here, the full-length genomic DNA sequence of a SAI gene (SAI-1) was cloned by in silico cloning and experimentally validated. The SAI-1 gene comprises three exons and two introns, totaling 4,966 bp, with an open reading frame of 2,025 bp encoding a polypeptide of 674 amino acids. The protein encoded by SAI-1 (molecular weight 73.4 kDa) contains a glycosyl hydrolase family 32 conserved domain, which catalyzes the hydrolysis of sucrose. Four allelic variants at the SAI-1 locus, SAI-1a, SAI-1b, SAI-1c and SAI-1d (GenBank accession nos. JX535515, JX535516, JX535517 and JX535518), were identified in 165 sweet sorghum cultivars. A co-dominant marker, SBX1, was developed to discriminate these variants. The mean Brix value from cultivars with a 141-bp (SAI-1c) PCR product was higher than in cultivars with a 136-bp (SAI-1b) product, and the mean Brix value from cultivars with a 136-bp (SAI-1b) product was significantly higher than those with a 132-bp (SAI-1a) product. The results showed that a domain of ATTGA repeat sequences in the second intron might be an important regulatory site, and that the functional marker SBX1 was closely related to the Brix value, and could therefore be used in sweet sorghum breeding programs.

Discovery of a new fragrance allele and development of functional markers for identifying diverse fragrant genotypes in rice

Discovery of new fragrance alleles provides important genetic resources for breeding fragrant rice. In this study, a hybrid complementation test demonstrated the association of a new fragrance allele without mutation in the coding region with flavor formation in a fragrant rice variety Nankai 138. The new allele (badh2-p-5′UTR) has a 3-bp deletion in the 5′ untranslated region and an 8-bp insertion in the promoter (−1,314 site upstream from the initiation codon). Surprisingly, we found that there is also an 8-bp insertion in the promoter of the badh2-E7 allele. We developed a new sequence tagged site functional marker to identify the badh2-p-5′UTR and badh2-E7 alleles according to the 8-bp insertion in their promoters. A cleaved amplified polymorphic sequence (AluI) functional marker targeting a common base substitution in the intron 2 of three badh2 alleles, viz. badh2-p-5′UTR, badh2-E7 and badh2-E2, was developed to identify diverse genotypes for fragrance in rice. Based on the results of sequence alignments among the three badh2 alleles, we suggest that the badh2-E7 and badh2-p-5′UTR alleles may have the same genetic origin. In addition, the genetic distance between the badh2-E7 and badh2-p-5′UTR alleles may be closer than that between the badh2-E2 and the badh2-p-5′UTR alleles, or between the badh2-E2 and the badh2-E7 alleles.

Haplotype, molecular marker and phenotype effects associated with mineral nutrient and grain size traits of TaGS1a in wheat

Glutamine synthetase (GS) is a key enzyme in the formation of the amino acid glutamine during N assimilation. The characterization of GS genes and the development of functional markers are important for marker-assisted selection (MAS) in wheat breeding programs. In the present study, the full-length genomic DNA (gDNA) sequence of TaGS1a was obtained from 60 wheat varieties. TaGS1a comprises 3415bp and has eleven exons and ten introns. Nine single nucleotide polymorphisms (SNPs) and two insertions and deletions of DNA segments (InDels) were detected in introns, resulting two haplotypes: Hap 1 and Hap 2. A cleaved amplified polymorphic sequence (CAPS) marker was developed to distinguish the two haplotypes. The TaGS1a-CAPS marker was located on chromosome 6D using Chinese Spring nullisomic–tetrasomic lines, and mapped at 2.5 cM from the SSR marker barc1121b in a RIL population. The completely corresponding results between quantitative trait locus (QTL) analysis and association analysis suggested that the TaGS1a gene had functions for grain size traits, including thousand grain weight (TGW), grain width (GW), grain height (GH), GL/GW ratio (GLW), factor form density (FFD), grain area (GA) and grain volume (GV) during maturity stage, and for the ratio of root/shoot for dry weight and nitrogen, phosphorus, potassium contents (RSDW, RSNC, RSPC and RSKC) during seedling stage.

Optimization of De Novo Short Read Assembly of Seabuckthorn (Hippophae rhamnoides L.) Transcriptome

Seabuckthorn ( Hippophae rhamnoides L.) is known for its medicinal, nutritional and environmental importance since ancient times. However, very limited efforts have been made to characterize the genome and transcriptome of this wonder plant. Here, we report the use of next generation massive parallel sequencing technology (Illumina platform) and de novo assembly to gain a comprehensive view of the seabuckthorn transcriptome. We assembled 86,253,874 high quality short reads using six assembly tools. At our hand, assembly of non-redundant short reads following a two-step procedure was found to be the best considering various assembly quality parameters. Initially, ABySS tool was used following an additive k-mer approach. The assembled transcripts were subsequently subjected to TGICL suite. Finally, de novo short read assembly yielded 88,297 transcripts (> 100 bp), representing about 53 Mb of seabuckthorn transcriptome. The average length of transcripts was 610 bp, N50 length 1198 BP and 91% of the short reads uniquely mapped back to seabuckthorn transcriptome. A total of 41,340 (46.8%) transcripts showed significant similarity with sequences present in nr protein databases of NCBI (E-value < 1E-06). We also screened the assembled transcripts for the presence of transcription factors and simple sequence repeats. Our strategy involving the use of short read assembler (ABySS) followed by TGICL will be useful for the researchers working with a non-model organism’s transcriptome in terms of saving time and reducing complexity in data management. The seabuckthorn transcriptome data generated here provide a valuable resource for gene discovery and development of functional molecular markers.

Development of user-friendly functional molecular markers for VvDXS gene conferring muscat flavor in grapevine

High fruit and wine quality combined with good climatic adaptation and disease resistance are essential objectives of grape breeding. While several molecular markers are available for pyramiding resistance to fungal pathogens, molecular tools for predicting fruit composition are still scarce. Muscat flavor, caused by the accumulation of monoterpenoids in the berry, is an important target trait for breeding, sought after in both table grapes and wine. Four missense mutations in the VvDXS gene in grape germplasm have been shown to be tightly linked to muscat flavor. Here we present highly reproducible and breeder-friendly functional markers for each of the targeted polymorphisms developed by using either the multiplexed minisequencing SNaPshot™ method, the high-resolution melting (HRM) assay or the cleaved amplified polymorphic sequence system. A total of 242 grapevine accessions were analyzed to optimize these different genotyping methods and to provide allele-specific markers for accurate selection of muscat flavor at early stages of grape breeding programs. The HRM and the minisequencing SNaPshot multiplex assays allow for high-throughput automated screening and are suitable for large-scale breeding programs and germplasm characterization.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-013-9929-6) contains supplementary material, which is available to authorized users.

Identification of cold-responsive genes in energycane for their use in genetic diversity analysis and future functional marker development

Breeding for cold tolerance in sugarcane will allow its cultivation as a dedicated biomass crop in cold environments. Development of functional markers to facilitate marker-assisted breeding requires identification of cold stress tolerance genes. Using suppression subtractive hybridization, 465 cold-responsive genes were isolated from the cold-tolerant energycane Ho02-144. Predicted gene interactions network indicated several associated pathways that may coordinately regulate cold tolerance responses in energycane. Expression analysis of a select set of genes, representing signaling and transcription factors, genes involved in polyamine and antioxidant biosynthesis, protein degradation and in the repair of damaged proteins in the cytosol, showed their time-dependent regulation under cold-stress. Comparative expression profiles of these genes between Ho02-144 and a cold-sensitive clone (L79-1002) showed that almost all genes were induced immediately upon imposition of cold stress and maintained their expression in Ho02-144 whereas they were either downregulated or their upregulation was very low in L79-1002. Simple sequence repeat markers derived from 260 cold-responsive genes showed allelic diversity among the cold-sensitive commercial hybrids that were distinct from the Saccharum spontaneum clones. Future efforts will target sequence polymorphism information of these genes in our ongoing QTL and association mapping studies to identify functional markers associated with cold tolerance in sugar/energycane.

Desarrollo de marcadores funcionales asociados a caracteres fenotípicos para identificación varietal en soja

La organización de los sistemas agrícolas requiere la verificación de la identidad y pureza genética de los cultivares. El aumento en el número de cultivares de soja a ser evaluados y su estrecha base genética dificulta su identificación mediante evaluación de descriptores fenotípicos. La Unión Internacional para la Protección de las Obtenciones Vegetales (UPOV) ha reconocido la utilidad de marcadores moleculares asociados con caracteres fenotípicos descriptivos. Con el objetivo de desarrollar ese tipo de marcadores, se seleccionaron in silico seis SSR génicos o genómicos (Sat286, Satt229, GmPrx1, GMES1173, Satt571 y GmHi), más dos marcadores previamente reportados (GmF35H y SoyF3H). Todos fueron evaluados en 35 cultivares de soja. Los SSR seleccionados para peroxidasa en testa y color de hilo (GmPrx1 y GmHi) fueron monomórficos. El contenido de información polimórfica (PIC) promedio del conjunto de marcadores polimórficos fue 0,48, con una media de 3,12 alelos por locus. GmF35H discriminó las variedades de acuerdo al color de flor (blanca y violeta). El análisis discriminante determinó alto porcentaje de clasificación correcta para el carácter hábito de crecimiento (95,8%) y color de pubescencia (80,6%), utilizando Sat286 y SoyF3H respectivamente. Los valores de clasificación para color de vaina (74,2%) y tamaño de folíolo (73,5%) fueron intermedios, utilizando GMES1173 y Satt571 respectivamente. El marcador Satt229 no resultó discriminante para ciclo a floración (50%) y maduración (42,8%). Los marcadores moleculares seleccionados dentro o cercanos a secuencias de interés pueden ser integrados a un sistema de identificación genética de variedades de soja como complemento a los descriptores fenotípicos clásicos.

Development of Functional Markers for Detection of Inactive DFR-A Alleles Responsible for Failure of Anthocyanin Production in Onions (Allium cepa L.)

Inactivation of the gene coding for dihydroflavonol 4-reductase (DFR) is responsible for the color difference between red and yellow onions (Allium cepa L.). Two inactive DFR-A alleles, DFR-<TEX>$A^{PS}$</TEX> and DFR-<TEX>$A^{DEL}$</TEX>, were identified in our previous study. A functional marker was developed on the basis of the premature stop codon that inactivated the DFR-<TEX>$A^{PS}$</TEX> allele. A derived cleaved amplified polymorphic sequences (dCAPS) primer was designed to detect the single nucleotide polymorphism, an A/T transition, which produced the premature stop codon. Digested PCR products clearly distinguished the homozygous and heterozygous red <TEX>$F_2$</TEX> individuals. Meanwhile, to develop a molecular marker for detection of the DFR-<TEX>$A^{DEL}$</TEX> allele in which entire DFR-A gene was deleted, genome walking was performed and approximately 3 kb 5' and 3' flanking sequences of the DFR-<TEX>$A^R$</TEX> coding region were obtained. PCR amplification using multiple primers binding to the extended flanking regions showed that more of the extended region of the DFR-A gene was deleted in the DFR-<TEX>$A^{DEL}$</TEX> allele. A dominant simple PCR marker was developed to identify the DFR-<TEX>$A^{DEL}$</TEX> allele using the dissimilar 3' flanking sequences of the DFR-A gene and homologous DFR-B pseudogene. Distribution of the DFR-<TEX>$A^{PS}$</TEX> and DFR-<TEX>$A^{DEL}$</TEX> alleles in yellow onion cultivars bred in Korea and Japan was surveyed using molecular makers developed in this study. Results showed predominant existence of the DFR-<TEX>$A^{PS}$</TEX> allele in yellow onion cultivars.