Specific Length Amplified Fragment Research Articles

An aldehyde dehydrogenase gene, GhALDH7B4_A06, positively regulates fiber strength in upland cotton (Gossypium hirsutum L.).

High fiber strength (FS) premium cotton has significant market demand. Consequently, enhancing FS is a major objective in breeding quality cotton. However, there is a notable lack of known functionally applicable genes that can be targeted for breeding. To address this issue, our study used specific length-amplified fragment sequencing combined with bulk segregant analysis to study FS trait in an F2 population. Subsequently, we integrated these results with previous quantitative trait locus mapping results regarding fiber quality, which used simple sequence repeat markers in F2, F2:3, and recombinant inbred line populations. We identified a stable quantitative trait locus qFSA06 associated with FS located on chromosome A06 (90.74-90.83 Mb). Within this interval, we cloned a gene, GhALDH7B4_A06, which harbored a critical mutation site in coding sequences that is distinct in the two parents of the tested cotton line. In the paternal parent Ji228, the gene is normal and referred to as GhALDH7B4_A06O; however, there is a nonsense mutation in the maternal parent Ji567 that results in premature termination of protein translation, and this gene is designated as truncated GhALDH7B4_A06S. Validation using recombinant inbred lines and gene expression analysis revealed that this mutation site is correlated with cotton FS. Virus-induced gene silencing of GhALDH7B4 in cotton caused significant decreases in FS and fiber micronaire. Conversely, GhALDH7B4_A06O overexpression in Arabidopsis boosted cell wall component contents in the stem. The findings of our study provide a candidate gene for improving cotton fiber quality through molecular breeding.

A High-Resolution Linkage Map Construction and QTL Analysis for Morphological Traits in Anthurium (Anthurium andraeanum Linden).

Anthurium andraeanum Linden is a prominent ornamental plant belonging to the family Araceae and is cultivated worldwide. The morphology characteristics are crucial because they significantly impact ornamental values, commercial properties, and the efficiency of space utilization in production. However, only a few related investigations have been conducted in anthurium to date. In this study, an F1 genetic segregation population containing 160 progenies was generated through hybridization between potted and cut anthurium varieties. Fifteen morphological traits were assessed and revealed substantial levels of genetic variation and widespread positive correlation. Based on specific length amplified fragment (SLAF) sequencing technology, 8171 single nucleotide polymorphism (SNP) markers were developed, and the high-density linkage map of 2202.27 cM in length distributed on 15 linkage groups was constructed successfully, with an average distance of 0.30 cM. Using the inclusive composite interval mapping (ICIM) method, 59 QTLs related to 15 key morphological traits were successfully identified, which explained phenotypic variance (PVE) ranging from 6.21% to 17.74%. Thirty-three of those associated with 13 traits were designated as major QTLs with PVE > 10%. These findings offer valuable insights into the genetic basis of quantitative traits and are beneficial for molecular marker-assisted selection (MAS) in anthurium breeding.

Construction of a high-resolution genetic map and identification of single nucleotide polymorphism markers relevant to flower stalk height in onion.

Onion (Allium cepa L., 2n=16) is an economically and nutritionally important vegetable crop worldwide. Construction of a high-resolution genetic map and map-based gene mining in onion have lagged behind other vegetable crops such as tomato and pepper. In this study, we constructed a high-resolution genetic map of onion using 321 F2 individuals from a cross between two double haploid lines DH-1×DH-17 and employing specific length amplified fragment (SLAF)-seq technology. The genetic map containing 10,584 polymorphic SLAFs with 21,250 single nucleotide polymorphism (SNP) markers and 8 linkage groups was developed for onion, which spanned 928.32 cM, with an average distance of 0.09 cM between adjacent markers. Using this map, we carried out QTL mapping of Ms locus related to the male-fertile trait and reproduced previous mapping results, which proved that this map was of good quality. Then, four QTLs (located on LG2, LG5, and LG8) were detected for flower stalk height, explaining 26.60% of the phenotypic variance. Among them, we proposed that 20 SLAF markers (in three QTLs) of flower stalk height trait were effective favorable allelic variant markers associated with heterosis. Overall, the genetic map was structured using SLAF-seq based on DH lines, and it is the highest-quality and highest-resolution linkage map of onion to date. It lays a foundation for the fine mapping and candidate gene identification of flower stalk height, and provides new insights into the developmental genetic mechanisms in onion breeding.

Primary Mapping and Analysis of the CmARM14 Candidate Gene for Mature Fruit Abscission in Melon

Mature fruit abscission (MFA) is an important trait in terms of both harvest and yield. MFA can affect the production and economic value of melon fruit. An F3 population segregating for a single gene and derived from a cross between line M2-10, which shows no mature fruit abscission (None MFA), and the MFA line ZT00091 was used to map candidate genes. Specific length amplified fragment (SLAF) sequencing, in conjunction with bulked-segregant analysis (BSA), was used to map loci governing the natural fruit abscission of plants composing the F3-57 family. A candidate locus, mfa10.1, located on chromosome 10 between genomic positions 73,229 and 818,251, was obtained. An insertion-deletion (InDel) marker and 46 recombinant individuals were used to narrow the candidate region to within 35 kb at the genomic position of 650,203 to 685,250; this region included six candidate genes. qRT–PCR gene expression and gene sequence data showed that the CmARM14 gene, which encodes a RING-type E3 ubiquitin transferase (MELO3C012406), was a candidate for melon MFA. Subcellular localization observations revealed that the CmARM14 fusion protein was localized to the golgi apparatus. Taken together, these results provide a molecular basis for melon breeding.

Identification of major QTLs and candidate genes for pod and seed weight per plant in soybean

Pod and seed weight per plant (PSW) is one of the most important components of soybean yield. We analysed 147 recombinant inbred lines (RILs) of soybean. Using a combination of simple sequence repeat (SSR) and specific-length amplified fragment (SLAF) high-density maps, we applied composite interval mapping (CIM) and multiple interval mapping (MIM) to map quantitative trait loci (QTLs) for PSW across multiple years (2006–10 and 2013). We mapped 24 QTLs for PSW, with 10 QTLs identified by SSR genetic map and 14 by SLAF genetic map. Five consensus QTLs were integrated, and they were validated by a chromosome segment substitution line (CSSL) population. Furthermore, the functions of all genes located in consensus QTL intervals were predicted; nine candidate genes function directly or indirectly in regulating seed development as well as seed size and weight. Our results lay a foundation for the cloning of candidate genes related to PSW and marker-assisted breeding in soybean.

Genome-wide assessment of population structure and genetic diversity of Chinese Lou onion using specific length amplified fragment (SLAF) sequencing.

Lou onion (Allium fistulosum L. var. viviparum) is an abundant source of flavonols which provides additional health benefits to diseases. Genome-wide specific length amplified fragment (SLAF) sequencing method is a rapidly developed deep sequencing technologies used for selection and identification of genetic loci or markers. This study aimed to elucidate the genetic diversity of 122 onion accessions in China using the SLAF-seq method. A set of 122 onion accessions including 107 A.fistulosum L. var. viviparum Makino, 3 A.fistulosum L. var. gigantum Makino, 3 A.mongolicum Regel and 9 A.cepa L. accessions (3 whites, 3 reds and 3 yellows) from different regions in China were enrolled. Genomic DNA was isolated from young leaves and prepared for the SLAF-seq, which generated a total of 1,387.55 M reads and 162,321 high quality SNPs (integrity >0.5 and MAF >0.05). These SNPs were used for the construction of neighbor-joining phylogenetic tree, in which 10 A.fistulosum L. var. viviparum Makino accessions from Yinchuan (Ningxia province) and Datong (Qinghai province) had close genetic relationship. The 3 A.cepa L. clusters (red, white and yellow) had close genetic relationship especially with the 97 A.fistulosum L. var. viviparum Makino accessions. Population structure analysis suggested entire population could be clustered into 3 groups, while principal component analysis (PCA) showed there were 4 genetic groups. We confirmed the SLAF-seq approach was effective in genetic diversity analysis in red onion accessions. The key findings would provide a reference to the Lou onion germplasm in China.

QTL mapping and transcriptome analysis identify candidate genes regulating pericarp thickness in sweet corn

BackgroundIn recent years, the planting area of sweet corn in China has expanded rapidly. Some new varieties with high yields and good adaptabilities have emerged. However, the improvement of edible quality traits, especially through the development of varieties with thin pericarp thickness, has not been achieved to date. Pericarp thickness is a complex trait that is the key factor determining the edible quality of sweet corn. Genetic mapping combined with transcriptome analysis was used to identify candidate genes controlling pericarp thickness.ResultsTo identify novel quantitative trait loci (QTLs) for pericarp thickness, a sweet corn BC4F3 population of 148 lines was developed using the two sweet corn lines M03 (recurrent parent) and M08 (donor parent). Additionally, a high-density genetic linkage map containing 3876 specific length amplified fragment (SLAF) tags was constructed and used for mapping QTLs for pericarp thickness. Interestingly, 14 QTLs for pericarp thickness were detected, and one stable QTL (qPT10–5) was detected across multiple years, which explained 7.78–35.38% of the phenotypic variation located on chromosome 10 (144,631,242-145,532,401). Forty-two candidate genes were found within the target region of qPT10–5. Moreover, of these 42 genes, five genes (GRMZM2G143402, GRMZM2G143389, GRMZM2G143352, GRMZM6G287947, and AC234202.1_FG004) were differentially expressed between the two parents, as revealed by transcriptome analysis. According to the gene annotation information, three genes might be considered candidates for pericarp thickness. GRMZM2G143352 and GRMZM2G143402 have been annotated as AUX/IAA transcription factor and ZIM transcription factor, respectively, while GRMZM2G143389 has been annotated as FATTY ACID EXPORT 2, chloroplastic.ConclusionsThis study identified a major QTL and candidate genes that could accelerate breeding for the thin pericarp thickness variety of sweet corn, and these results established the basis for map-based cloning and further functional research.

Construction of a SNP-Based Genetic Map Using SLAF-Seq and QTL Analysis of Morphological Traits in Eggplant.

Eggplant (Solanum melongena; 2n = 24) is an economically important fruit crop of the family Solanaceae that was domesticated in India and Southeast Asia. Construction of a high-resolution genetic map and map-based gene mining in eggplant have lagged behind other crops within the family such as tomato and potato. In this study, we conducted high-throughput single nucleotide polymorphism (SNP) discovery in the eggplant genome using specific length amplified fragment (SLAF) sequencing and constructed a high-density genetic map for the quantitative trait locus (QTL) analysis of multiple traits. An interspecific F2 population of 121 individuals was developed from the cross between cultivated eggplant “1836” and the wild relative S. linnaeanum “1809.” Genomic DNA extracted from parental lines and the F2 population was subjected to high-throughput SLAF sequencing. A total of 111.74 Gb of data and 487.53 million pair-end reads were generated. A high-resolution genetic map containing 2,122 SNP markers and 12 linkage groups was developed for eggplant, which spanned 1530.75 cM, with an average distance of 0.72 cM between adjacent markers. A total of 19 QTLs were detected for stem height and fruit and leaf morphology traits of eggplant, explaining 4.08–55.23% of the phenotypic variance. These QTLs were distributed on nine linkage groups (LGs), but not on LG2, 4, and 9. The number of SNPs ranged from 2 to 11 within each QTL, and the genetic interval varied from 0.15 to 10.53 cM. Overall, the results establish a foundation for the fine mapping of complex QTLs, candidate gene identification, and marker-assisted selection of favorable alleles in eggplant breeding.

Construction of a High-Density Genetic Map and Analysis of Seed-Related Traits Using Specific Length Amplified Fragment Sequencing for Cucurbita maxima.

Seed traits are agronomically important for Cucurbita breeding, but the genes controlling seed size, seed weight and seed number have not been mapped in Cucurbita maxima (C. maxima). In this study, 100 F2 individual derived from two parental lines, “2013-12” and “9-6”, were applied to construct a 3,376.87-cM genetic map containing 20 linkage groups (LGs) with an average genetic distance of 0.47 cM using a total of 8,406 specific length amplified fragment (SLAF) markers in C. maxima. Ten quantitative trait loci (QTLs) of seed width (SW), seed length (SL) and hundred-seed weight (HSW) were identified using the composite interval mapping (CIM) method. The QTLs affecting SW, SL and HSW explained a maximum of 38.6%, 28.9% and 17.2% of the phenotypic variation and were detected in LG6, LG6 and LG17, respectively. To validate these results, an additional 150 F2 individuals were used for QTL mapping of SW and SL with cleaved amplified polymorphic sequence (CAPS) markers. We found that two major QTLs, SL6-1 and SW6-1, could be detected in both SLAF-seq and CAPS markers in an overlapped region. Based on gene annotation and non-synonymous single-nucleotide polymorphisms (SNPs) in the major SWand SL-associated regions, we found that two genes encoding a VQ motif and an E3 ubiquitin-protein ligase may be candidate genes influencing SL, while an F-box and leucinerich repeat (LRR) domain-containing protein is the potential regulator for SW in C. maxima. This study provides the first high-density linkage map of C. maxima using SNPs developed by SLAF-seq technology, which is a powerful tool for associated mapping of important agronomic traits, map-based gene cloning and marker-assisted selection (MAS)-based breeding in C. maxima.

A high-density genetic map constructed using specific length amplified fragment (SLAF) sequencing and QTL mapping of seed-related traits in sesame (Sesamum indicum L.)

BackgroundSesame (Sesamum indicum L., 2n = 2x = 26) is an important oilseed crop with high oil content but small seed size. To reveal the genetic loci of the quantitative seed-related traits, we constructed a high-density single nucleotide polymorphism (SNP) linkage map of an F2 population by using specific length amplified fragment (SLAF) technique and determined the quantitative trait loci (QTLs) of seed-related traits for sesame based on the phenotypes of F3 progeny.ResultsThe genetic map comprised 2159 SNP markers distributed on 13 linkage groups (LGs) and was 2128.51 cM in length, with an average distance of 0.99 cM between adjacent markers. QTL mapping revealed 19 major-effect QTLs with the phenotypic effect (R2) more than 10%, i.e., eight QTLs for seed coat color, nine QTLs for seed size, and two QTLs for 1000-seed weight (TSW), using composite interval mapping method. Particularly, LG04 and LG11 contained collocated QTL regions for the seed coat color and seed size traits, respectively, based on their close or identical locations. In total, 155 candidate genes for seed coat color, 22 for seed size traits, and 54 for TSW were screened and analyzed.ConclusionsThis report presents the first QTL mapping of seed-related traits in sesame using an F2 population. The results reveal the location of specific markers associated with seed-related traits in sesame and provide the basis for further seed quality traits research.

Construction of the first high-density genetic linkage map of pikeperch (Sander lucioperca) using specific length amplified fragment (SLAF) sequencing and QTL analysis of growth-related traits

High-density genetic linkage maps play an important role in genome assembly, quantitative trait locus (QTL) mapping, and marker-assisted selection. Pikeperch (Sander lucioperca) is a popular inland aquaculture species in Europe and Asia, but the genetic improvement of this species is still in its infancy. In this study, genomic DNA extracted from two pikeperch parents and 150 F1 offspring was subjected to high-throughput sequencing and specific length amplified fragment (SLAF) library construction. In total, 436.58 M reads (87.28 G data) were acquired, with average sequencing depths of 100.16-fold for the parents and 19.51-fold for each F1 offspring. A total of 237,687 high-quality SLAFs were detected, 34,774 of which were polymorphic and used for linkage map construction. The final merged linkage map consisted of 8159 SLAFs in 24 linkage groups (LGs) and spanned 3421.81 cM, with an average inter-marker distance of 0.46 cM. Based on this high-density genetic linkage map, 21 QTLs were identified for eight growth-related traits: total length, body weight, body length, head length, body height, body thickness, body girth, and slaughter weight. Three QTLs for each trait, including slaughter weight, body girth, body weight, and body thickness, were detected on LG6 or LG16. Moreover, five QTLs for body length were detected on LG6, LG9, and LG18. QTLs for total length, body height, and head length were detected on LG6. The linkage map and QTLs for growth-related traits identified in this study should be useful for improving pikeperch marker-assisted selection in the future.

SLAF-based high-density genetic map construction and QTL mapping for major economic traits in sea urchin Strongylocentrotus intermedius

Sea urchin (Strongylocentrotus intermedius) has long been a model species for developmental and evolutionary research, but only a few studies have focused on gene mapping. Here, we reported a high-density genetic map containing 4,387 polymorphism specific-length amplified fragment (SLAF) markers spanning 21 linkage groups (LG) for sea urchin. Based on this genetic map and phenotyping data for eight economic traits, 33 potentially significant QTLs were detected on ten different LGs with explanations ranging from 9.90% to 46.30%, partly including 10 QTLs for test diameter, six QTLs for body weight and eight QTLs for Aristotle’s lantern weight. Moreover, we found a QTL enrichment LG, LG15, gathering QTLs for test diameter, body weight, gonad weight, light orange-yellow color difference (≥E1) and light yellow color difference (≥E2). Among all QTLs, we genotyped four QTLs for test diameter, Aristotle’s lantern weight and body weight using High Resolution Melting (HRM) technology. Finally, we used the verified SNP marker (detected using SLAF sequencing) to explore their marker-assisted selection (MAS) breeding application potential and found that SNP-29 associated tightly with body weight and that heterozygous genotype was a dominant genotype, indicating that SNP-29 was a promising marker for MAS.

WITHDRAWN: Genome-wide association analysis of inguinal/scrotal hernia in pigs using specific length amplified fragment (SLAF) sequencing

// Danyang Zou 1, * , Bo Zuo 1, 2, * , Mingfei Zhu 1 , Lei Rong 1 , Huimin Mao 3 , Hongdan Niu 1 , Ling Wang 1 and Rong Zheng 1 1 Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei, P.R. China 2 The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, P.R. China 3 The Tianpeng Group, Jiangshan, Zhejiang, 324111, P.R. China * These authors share co-first authorship Correspondence to: Rong Zheng, email: zhengrong@mail.hzau.edu.cn Keywords: GWAS; SLAF-seq; inguinal/scrotal hernia; pig; farmCPU Received: October 06, 2017 Accepted: October 27, 2017 Epub: December 22, 2017 ABSTRACT Inguinal and scrotal hernias are the most frequent congenital disorders in pigs, and they may cause severe economic loss in the pig breeding industry. Genetic factors play a significant role in the susceptibility to hernias, but the genetic mechanisms of inguinal/scrotal hernia are poorly understood. In this study, we performed a genome-wide association study (GWAS) with specific-locus amplified fragment sequencing (SLAF-seq) on 120 (59 cases and 61 controls) full and half sib pigs to identify genetic loci underlying variations in inguinal/scrotal hernias. A total of 218,460 high-quality SNPs were generated for further statistical analysis with case-controls and the farmCPU model. Based on these two methods, a total of 59 SNPs were significantly ( P < 0.01) associated with inguinal/scrotal hernia. Finally, 14 novel candidate genes implicated in the defect were identified, and 5 genes ( CPNE5 , DEGS1 , PLCG2 , PRKCE and NUAK1 ) were related to cell apoptosis, which is one of the pivotal pathogenesis factors of inguinal/scrotal hernia. In addition, 4 of them were shown strong associations with the hernia were confirmed in 270 samples ( P < 0.05). This finding provides new evidence that genes related to cell apoptosis may be associated with inguinal/scrotal hernias.

Construction of a high-density genetic map for grape using specific length amplified fragment (SLAF) sequencing.

Genetic maps are important tools in plant genomics and breeding. We report a large-scale discovery of single nucleotide polymorphisms (SNPs) using the specific length amplified fragment sequencing (SLAF-seq) technique for the construction of high-density genetic maps for two elite wine grape cultivars, ‘Chardonnay’ and ‘Beibinghong’, and their 130 F1 plants. A total of 372.53 M paired-end reads were obtained after preprocessing. The average sequencing depth was 33.81 for ‘Chardonnay’ (the female parent), 48.20 for ‘Beibinghong’ (the male parent), and 12.66 for the F1 offspring. We detected 202,349 high-quality SLAFs of which 144,972 were polymorphic; 10,042 SNPs were used to construct a genetic map that spanned 1,969.95 cM, with an average genetic distance of 0.23 cM between adjacent markers. This genetic map contains the largest molecular marker number of the grape maps so far reported. We thus demonstrate that SLAF-seq is a promising strategy for the construction of high-density genetic maps; the map that we report here is a good potential resource for QTL mapping of genes linked to major economic and agronomic traits, map-based cloning, and marker-assisted selection of grape.

Uncovering SNP and indel variations of tetraploid cottons by SLAF-seq

BackgroundCotton (Gossypium spp.), as the world’s most utilized textile fibre source, is an important, economically valuable crop worldwide. Understanding the genomic variation of tetraploid cotton species is important for exploitation of the excellent characteristics of wild cotton and for improving the diversity of cotton in breeding. However, the discovery of DNA polymorphisms in tetraploid cotton genomes has lagged behind other important crops.ResultsA total of 111,795,823 reads, 467,735 specific length amplified fragment (SLAF) tags and 139,176 high-quality DNA polymorphisms were identified using specific length amplified fragment sequencing (SLAF-seq), including 132,880 SNPs and 6,296 InDels between the reference genome (TM-1) and the five tetraploid cotton species. Intriguingly, gene ontology (GO) enrichment analysis revealed that a number of significant terms were related to reproduction in G. barbadense acc. 3–79. Based on the new data sets, we reconstructed phylogenetic trees that showed a high concordance to the phylogeny of diploid and polyploid cottons. A large amount of interspecific genetic variations were identified, and some of them were validated by the single-strand conformation polymorphism (SSCP) method, which will be applied in introgression genetics and breeding with G. hirsutum cv. Emian22 as the receptor and the other species as donors.ConclusionsUsing SLAF-seq, a large number of DNA polymorphisms were identified. The comprehensive analysis of DNA polymorphisms provided invaluable insights into the different tetraploid cotton species. More importantly, the identification of numerous interspecific genetic variations provides the basis and is very practical for future introgression breeding. The results presented herein provide a valuable genomic resource for new insights into the genetics and breeding of cotton.

Genome-wide assessment of population structure and genetic diversity and development of a core germplasm set for sweet potato based on specific length amplified fragment (SLAF) sequencing.

Sweet potato, Ipomoea batatas (L.) Lam., is an important food crop that is cultivated worldwide. However, no genome-wide assessment of the genetic diversity of sweet potato has been reported to date. In the present study, the population structure and genetic diversity of 197 sweet potato accessions most of which were from China were assessed using 62,363 SNPs. A model-based structure analysis divided the accessions into three groups: group 1, group 2 and group 3. The genetic relationships among the accessions were evaluated using a phylogenetic tree, which clustered all the accessions into three major groups. A principal component analysis (PCA) showed that the accessions were distributed according to their population structure. The mean genetic distance among accessions ranged from 0.290 for group 1 to 0.311 for group 3, and the mean polymorphic information content (PIC) ranged from 0.232 for group 1 to 0.251 for group 3. The mean minor allele frequency (MAF) ranged from 0.207 for group 1 to 0.222 for group 3. Analysis of molecular variance (AMOVA) showed that the maximum diversity was within accessions (89.569%). Using CoreHunter software, a core set of 39 accessions was obtained, which accounted for approximately 19.8% of the total collection. The core germplasm set of sweet potato developed will be a valuable resource for future sweet potato improvement strategies.

A High-Density Genetic Linkage Map for Cucumber (Cucumis sativus L.): Based on Specific Length Amplified Fragment (SLAF) Sequencing and QTL Analysis of Fruit Traits in Cucumber.

High-density genetic linkage map plays an important role in genome assembly and quantitative trait loci (QTL) fine mapping. Since the coming of next-generation sequencing, makes the structure of high-density linkage maps much more convenient and practical, which simplifies SNP discovery and high-throughput genotyping. In this research, a high-density linkage map of cucumber was structured using specific length amplified fragment sequencing, using 153 F2 populations of S1000 × S1002. The high-density genetic map composed 3,057 SLAFs, including 4,475 SNP markers on seven chromosomes, and spanned 1061.19 cM. The average genetic distance is 0.35 cM. Based on this high-density genome map, QTL analysis was performed on two cucumber fruit traits, fruit length and fruit diameter. There are 15 QTLs for the two fruit traits were detected.

Mining candidate genes associated with powdery mildew resistance in cucumber via super-BSA by specific length amplified fragment (SLAF) sequencing.

BackgroundPowdery mildew (PM) is the most common fungal disease of cucumber and other cucurbit crops, while breeding the PM-resistant materials is the effective way to defense this disease, and the recent development of modern genetics and genomics make us aware of that studying the resistance genes is the essential way to breed the PM high-resistance plant. With the ever increasing throughput of next-generation sequencing (NGS), the development of specific length amplified fragment sequencing (SLAF-seq) as a high-resolution strategy for large-scale de novo SNP discovery is gradually applied for functional gene mining. Here we combined the bulked segregant analysis (BSA) with SLAF-seq to identify candidate genes associated with PM resistance in cucumber.MethodsA segregating population comprising 251 F2 individuals was developed using H136 (female parent) as susceptible parent and BK2 (male parent) as resistance donor. After PMR test, total genomic DNA was prepared from each plant. Systemic genomic analysis of the GC content, repeat sequence, etc. was carried out by prediction software SLAF_Predict to establish condition to ensure the uniformity and density of the molecular markers. After samples were gel purified, SLAFs were generated at Biomarker Technologies Corporation in Beijing. Based on SLAF tags and the PMR test result, the hot region were annotated.ResultsA total of 73,100 high-quality SLAF tags with an average depth of 99.11× were sequenced. Among these, 5,355 polymorphic tags were identified with a polymorphism rate of 7.34 %, including 7.09 % SNPs and other polymorphism types. Finally, 140 associated SLAFs were identified, and two main Hot Regions were detected on chromosome 1 and 6, which contained five genes invovled in defense response, toxin metabolism, cell stress response, and injury response in cucumber.ConclusionsAssociated markers identified by super-BSA in this study, could not only speed up the study of the PMR genes, but also provide a feasible solution for breeding the marker-assisted PMR cucumber. Moreover, this study could also be extended to any other species with reference genome.

Construction of a high-density genetic map and mapping of a sex-linked locus for the brown alga Undaria pinnatifida (Phaeophyceae) based on large scale marker development by specific length amplified fragment (SLAF) sequencing.

BackgroundUndaria pinnatifida is an important economic brown alga in East Asian countries. However, its genetic and genomic information is very scarce, which hinders further research in this species. A high-density genetic map is a basic tool for fundamental and applied research such as discovery of functional genes and mapping of quantitative trait loci (QTL). In this study the recently developed specific length amplified fragment sequencing (SLAF-seq) technology was employed to construct a high-density genetic linkage map and locate a sex determining locus for U. pinnatifida.ResultsA total of 28.06 Gb data including 140.31 M pair-end reads was obtained. After linkage analysis 4626 SLAF markers were mapped onto the genetic map. After adding the sex linked simple sequence repeat (SSR) marker [GenBank:AY738602.1], the final genetic map was 1816.28 cM long, consisting of 30 linkage groups with an average distance of 0.39 cM between adjacent markers. The length of LGs ranged from 20.12 to 106.95 cM. A major sex associated QTL was mapped to LG22 within a window starting at 29.01 cM and ending at 68.81 cM with a total of 68 SLAF markers. The SSR marker and five SLAF markers (Marker6556, 19020, 43089, 60771 and 26359) were identified as tightly sex-linked markers, as indicated by the absence of recombination between them and the sex phenotype. These markers were located at the position of 59.50 cM, which was supposed to be the sex determining region.ConclusionsA high-density genetic linkage map was constructed using SLAF-seq technique and F1 gametophyte population for the first time in the economically important brown alga U. pinnatifida. For the first time, a major sex associated QTL suggesting a sex determining region was mapped to a single LG. This map will facilitate the further fundamental and applied research such as QTL mapping and map-based gene clone in U. pinnatifida and provide a reference for studies in other kelp species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2184-y) contains supplementary material, which is available to authorized users.

An SNP-based saturated genetic map and QTL analysis of fruit-related traits in cucumber using specific-length amplified fragment (SLAF) sequencing.

BackgroundCucumber, Cucumis sativus L., is an economically important vegetable crop which is processed or consumed fresh worldwide. However, the narrow genetic base in cucumber makes it difficult for constructing high-density genetic maps. The development of massively parallel genotyping methods and next-generation sequencing (NGS) technologies provides an excellent opportunity for developing single nucleotide polymorphisms (SNPs) for linkage map construction and QTL analysis of horticultural traits. Specific-length amplified fragment sequencing (SLAF-seq) is a recent marker development technology that allows large-scale SNP discovery and genotyping at a reasonable cost. In this study, we constructed a high-density SNP map for cucumber using SLAF-seq and detected fruit-related QTLs.ResultsAn F2 population of 148 individuals was developed from an intra-varietal cross between CC3 and NC76. Genomic DNAs extracted from two parents and 148 F2 individuals were subjected to high-throughput sequencing and SLAF library construction. A total of 10.76 Gb raw data and 75,024,043 pair-end reads were generated to develop 52,684 high-quality SLAFs, out of which 5,044 were polymorphic. 4,817 SLAFs were encoded and grouped into different segregation patterns. A high-resolution genetic map containing 1,800 SNPs was constructed for cucumber spanning 890.79 cM. The average distance between adjacent markers was 0.50 cM. 183 scaffolds were anchored to the SNP-based genetic map covering 46% (168.9 Mb) of the cucumber genome (367 Mb). Nine QTLs for fruit length and weight were detected, a QTL designated fl3.2 explained 44.60% of the phenotypic variance. Alignment of the SNP markers to draft genome scaffolds revealed two mis-assembled scaffolds that were validated by fluorescence in situ hybridization (FISH).ConclusionsWe report herein the development of evenly dispersed SNPs across cucumber genome, and for the first time an SNP-based saturated linkage map. This 1,800-locus map would likely facilitate genetic mapping of complex QTL loci controlling fruit yield, and the orientation of draft genome scaffolds.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1158) contains supplementary material, which is available to authorized users.