Specific-locus Amplified Fragment Sequencing Research Articles

Differentiation and Temperature Adaptation of Pampus Echinogaster Based on Genome-Wide SNPs

Climate change and overfishing have led to the degradation of the quality and stock of Pampus echinogaster. Exploring the genetic structure and local adaptive evolutionary mechanisms of P. echinogaster is crucial for the management of this species. This population genomic study of nine geographical populations of P. echinogaster in China was conducted by specific-locus amplified fragment sequencing (SLAF-seq). A total of 935,215 SLAF tags were obtained, and the average sequencing depth of the SLAF tags was 20.80×. After filtering, a total of 46,187 high-consistency genome-wide single nucleotide polymorphisms (SNPs) were detected. Based on all SNPs, the population size of the nine P. echinogaster geographical populations was large. The Shantou population had the lowest genetic diversity, and the Tianjin population had the highest. Moreover, the population genetic structure based on all SNPs revealed significant gene exchange and no significant genetic differentiation among the nine P. echinogaster geographical populations, probably because of the population expansion after the last glacial period, the lack of balance between migration and genetic drift, and the long-distance diffusion of eggs and larvae. We suspect that variation of material metabolism, ion transfer, breeding, stress response, and inflammatory reactions-related genes were critical for adaptation to spatially heterogeneous temperature selection in natural P. echinogaster populations.

Phylogeographical Pattern and Population Evolution History of Indigenous Elymus sibiricus L. on Qinghai-Tibetan Plateau.

Elymus sibiricus L. is a perennial allotetraploid belonging to Triticeae of Poaceae, Elymus L., as the type species of genus Elymus L. The existing geographical distribution pattern and genetic spatial structure of E. sibiricus on Qinghai–Tibetan Plateau (QTP) are not yet clear. In this study, population genetic structure and demography history of 216 individuals from 44 E. sibiricus populations on QTP were studied used specific-locus amplified fragment sequencing (SLAF-seq). The result of genetic diversity showed that there was no single genetic diversity center was observed across all E. sibiricus populations. The results of genetic variation showed that 44 populations were clearly divided into the following three groups: Qinghai Plateau (Group I), South Tibet (Group II), and Hengduan Mountains (Group III). From the three analyses of AMOVA, Mantel test and Treemix, strong genetic differentiation across all populations and low genetic differentiation among populations within three groups. Molecular dating indicated that E. sibiricus diverged at 16.08 Ma (during the early Miocene) can be linked to the Himalayan Motion stage of QTP uplift. It is speculated that the reasons affecting the current phylogeographical pattern are as follows: (1) The environmental changes due to the uplift of the QTP; (2) The geographic distance between the populations (Groups I and III are close in geographic distance, and gene flow are frequent); (3) Geographical barriers (the Tanggula and Bayangela Mountains between Groups I and II). This study provides new evidence and historical perspective to the future exploration of the evolution and geographic distribution pattern of Elymus L.

Genome-Wide Association Analysis and Gene Mining of Resistance to China Race 1 of Frogeye Leaf Spot in Soybean.

Soybean frogeye leaf spot (FLS) is a worldwide fungal disease. Its higher occurrence frequency and wider distribution range always led to severe yield losses of soybean, therefore, breeding new cultivars with FLS resistance has been an important breeding goal for soybean breeders. In this study, an association panel of 183 representative soybean accessions was used to evaluate their resistance to FLS race 1, and to identify quantitative trait nucleotides (QTNs) and candidate genes based on genome-wide association study (GWAS) and high-throughput single-nucleotide polymorphisms (SNPs). A total of 23,156 high-quality SNPs were developed using the specific locus-amplified fragment sequencing (SLAF-seq) approach. Finally, 13 novel association signals associated with FLS race 1 resistance were identified by the compressed mixed linear model (CMLM). In addition, 119 candidate genes were found within the 200-kb flanking genomic region of these 13 peak SNPs. Based on the gene-based association analysis, haplotype analysis, expression pattern analysis, and virus-induced gene silencing (VIGS) systems, four genes (Glyma.05G121100, Glyma.17G228300, Glyma.19G006900, and Glyma.19G008700) were preliminarily proved to play an important role in the soybean resistance to FLS race 1.

Genetic Diversity and Population Differentiation of Chinese Lizard Gudgeon (Saurogobio dabryi) in the Upper Yangtze River

Dam construction on the upper Yangtze River has dramatically altered riverine ecosystems and caused habitat fragmentation of fishes, which might influence the genetic structure of fish populations. In this study, we examined the possible genetic effects of dam construction on Chinese lizard gudgeon (Saurogobio dabryi) populations in the upper Yangtze River, China. Seven populations were sampled, and genetic structure was analyzed using single nucleotide polymorphism (SNP) markers through the specific locus amplified fragment sequencing (SLAF-seq) method. The numbers of SNPs were lower in the upstream populations than in the downstream populations. Genetic similarity was increased from downstream to upstream. The upstream populations of S. dabryi might be more vulnerable to genetic drift than those downstream. Structure analysis indicated three distinct genetic groups of S. dabryi in the upper Yangtze River, among which the genetic differentiation values (Fst) were at a high level. The genetic differentiation of S. dabryi exhibited a close correlation with spatial distance. We did not detect a significant correlation between isolation time and genetic differentiation, suggesting that impacts of dams on the genetic structure of S. dabryi can be relatively minimal on a short time scale. The results quantify the genetic diversity and population structure patterns of S. dabryi after habitat fragmentation caused by dams, which will provide a reference for resource protection and management of this species in the upper Yangtze River.

Combined GWAS and Transcriptome Analyses Provide New Insights Into the Response Mechanisms of Sunflower Against Drought Stress.

Sunflower is one of the most important oil crops in the world, and drought stress can severely limit its production and quality. To understand the underlying mechanism of drought tolerance, and identify candidate genes for drought tolerance breeding, we conducted a combined genome-wide association studies (GWAS) and RNA-seq analysis. A total of 226 sunflower inbred lines were collected from different regions of China and other countries. Eight phenotypic traits were evaluated under control and drought stress conditions. Genotyping was performed using a Specific-Locus Amplified Fragment Sequencing (SLAF-seq) approach. A total of 934.08 M paired-end reads were generated, with an average Q30 of 91.97%. Based on the 243,291 polymorphic SLAF tags, a total of 94,162 high-quality SNPs were identified. Subsequent analysis of linkage disequilibrium (LD) and population structure in the 226 accessions was carried out based on the 94,162 high-quality SNPs. The average LD decay across the genome was 20 kb. Admixture analysis indicated that the entire population most likely originated from 11 ancestors. GWAS was performed using three methods (MLM, FarmCPU, and BLINK) simultaneously. A total of 80 SNPs showed significant associations with the 8 traits (p < 1.062 × 10−6). Next, a total of 118 candidate genes were found. To obtain more reliable candidate genes, RNA-seq analysis was subsequently performed. An inbred line with the highest drought tolerance was selected according to phenotypic traits. RNA was extracted from leaves at 0, 7, and 14 days of drought treatment. A total of 18,922 differentially expressed genes were obtained. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed up-regulated genes were mainly enriched in the branched-chain amino acid catabolic process, while the down-regulated genes were mainly enriched in the photosynthesis-related process. Six DEGs were randomly selected from all DEGs for validation; these genes showed similar patterns in RNA-seq and RT-qPCR analysis, with a correlation coefficient of 0.8167. Through the integration of the genome-wide association study and the RNA-sequencing, 14 candidate genes were identified. Four of them (LOC110885273, LOC110872899, LOC110891369, LOC110920644) were abscisic acid related protein kinases and transcription factors. These genes may play an important role in sunflower drought response and will be used for further study. Our findings provide new insights into the response mechanisms of sunflowers against drought stress and contribute to further genetic breeding.

Cytogenetic identification and molecular marker development of a novel wheat-Thinopyrum ponticum translocation line with powdery mildew resistance.

A new wheat-Thinopyrum ponticum translocation line with excellent powdery mildew resistance was produced, and alien-specific PCR markers and FISH probes were developed by SLAF-seq. Powdery mildew is one of the most threatening diseases in wheat production. Thinopyrum ponticum (Podp.) Barkworth and D. R. Dewey, as a wild relative, has been used for wheat genetic improvement for the better part of a century. In view of the good powdery mildew resistance of Th. ponticum, we have been working to transfer the resistance genes from Th. ponticum to wheat by creating translocation lines. In this study, a new wheat-Th. ponticum translocation line with excellent resistance and agronomic performance was developed and through seedling disease evaluation, gene postulation and diagnostic marker analysis proved to carry a novel Pm gene derived from Th. ponticum. Cytogenetic analysis revealed that a small alien segment was translocated to the terminal of chromosome 1D to form new translocation TTh-1DS·1DL chromosome. The translocation breakpoint was determined to lie in 21.5Mb region of chromosome 1D by using Wheat660K SNP array analysis. Based on specific-locus amplified fragment sequencing (SLAF-seq) technology, eight molecular markers and one repetitive sequence probe were developed, which were specific for Th. ponticum. Fortunately, the probe could be used in distinguishing six alien chromosome pairs in partial amphiploid Xiaoyan 7430 by fluorescence in situ hybridization (FISH). Furthermore, a Thinopyrum-specific oligonucleotide probe was designed depending on the sequence information of the FISH probe. The novel translocation line could be used in wheat disease resistance breeding, and these specific markers and probes will enable wheat breeders to rapidly trace the alien genome with the novel Pm gene(s).

Identification of fruit firmness QTL ff2.1 by SLAF-BSA and QTL mapping in melon

Fruit firmness is an important target of melon breeding, as it is associated with shelf life and economic value; however, the precise mechanism that determines fruit firmness during fruit ripening remains elusive. In the present study, one hundred forty-four F2 plants and F2–3 families derived from the high-firmness melon line M2–10 and the low-firmness melon line ZT091 were used to identify major quantitative trait loci by specific-locus amplified fragment sequencing with bulked segregant analysis. Simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) markers based on the resequencing of parental lines were also used to narrow the associated region to identify candidate genes. Two regions associated with fruit firmness were investigated, including a 4.87 Mb region on chr. 2 and a 28.7 Mb region on chr. 5 of the melon genome. SSR and CAPS markers were used to construct a genetic map of the associated regions: QTL ff5.1 was located between CmSSR13509 and CmSSR13423 and explained 38.44% of the observed variation, with an LOD threshold of 17.44; ff2.1 was located between CmSSR07709 and SNP22228 and explained 28.14% of the variation, with an LOD threshold of 3.8, and this region included 106 Kb and 10 candidate genes. Quantitative real-time PCR was performed to investigated the investigate candidate gene expression at 15, 20 and 25 days after pollination in the parental lines, and significant expression levels were detected for most of the genes, including four genes of unknown function and MELO3C017519, MELO3C017520, MELO3C017522, MELO3C029506, and MELO3C029520. These results revealed a new QTL, ff2.1, for melon fruit firmness-related gene identification.

Integration of GWAS and transcriptome analyses to identify SNPs and candidate genes for aluminum tolerance in rapeseed (Brassica napus L.)

BackgroundThe exchangeable aluminum (Al), released from the acid soils, is another addition to the environmental stress factors in the form of Al toxicity stress. Al stress affects the normal crop development and reduces the overall yield of rapeseed (Brassica napus L.). The response mechanism of plants to Al toxicity is complicated and difficult to understand with few QTL related studies in rapeseed under Al toxicity stress.ResultUsing 200,510 SNPs developed by SLAF-seq (specific-locus amplified fragment sequencing) technology, we carried out the genome-wide association analysis (GWAS) in a population of 254 inbred lines of B. napus with large genetic variation and Al-tolerance differences. There were 43 SNPs significantly associated with eight Al-tolerance traits in the seedling stage were detected on 14 chromosomes, and 777 candidate genes were screened at the flanking 100 kb region of these SNPs. Moreover, RNA-seq detected 8291 and 5341 DEGs (the differentially expressed gene) in the Al -tolerant line (ATL) and -sensitive line (ASL), respectively. Based on integration of GWAS and RNA-seq analysis, 64 candidate genes from GWAS analysis differentially expressed at least once in 6 h vs 0 h or 24 h vs 0 h conditions in ATL or ASL. Moreover, four out of sixty-four candidate genes (BnaA03g30320D, BnaA10g11500D, BnaC03g38360D and BnaC06g30030D) were differentially expressed in both 6 h and 24 h compared to 0 h (control) conditions in both lines. The proposed model based on the candidate genes excavated in this study highlighted that Al stress disturb the oxidation-redox balance, causing abnormal synthesis and repair of cell wall and ABA signal transduction, ultimately resulting in inhibition of root elongation.ConclusionsThe integration of GWAS and transcriptome analysis provide an effective strategy to explore the SNPs and candidate genes, which has a potential to develop molecular markers for breeding Al tolerant rapeseed varieties along with theoretical basis of molecular mechanisms for Al toxicity response of Brassica napus plants.

Establishment and application of an SNP molecular identification system for grape cultivars

We aimed to develop a set of single nucleotide polymorphism (SNP) markers that can be used to distinguish the main cultivated grape (Vitis L.) cultivars in China and provide technical support for domestic grape cultivar protection, cultivar registration, and market rights protection. A total of 517 high-quality loci were screened from 4 241 729 SNPs obtained by sequencing 304 grape accessions using specific locus amplified fragment sequencing, of which 442 were successfully designed as Kompetitive Allele Specific PCR (KASP) markers. A set of 27 markers that completely distinguishes 304 sequenced grape accessions was determined by using the program, and 26 effective markers were screened based on 23 representative grape cultivars. Finally, a total of 46 out of 48 KASP markers, including 22 markers selected by the research group in the early stage, were re-screened based on 348 grape accessions. Population structure, principal component, and cluster analyses all showed that the 348 grape accessions were best divided into two populations. In addition, cluster analysis subdivided them into six subpopulations. According to genetic distance, V. labrusca, V. davidii, V. heyneana, and V. amurensis were far from V. vinifera, while V. vinifera×V. labrusca and V. amurensis×V. vinifera were somewhere in between these two groups. Furthermore, a core set of 25 KASP markers could distinguish 95.69% of the 348 grape accessions, and the other 21 markers were used as extended markers. Therefore, SNP molecular markers based on KASP typing technology provide a new way for mapping DNA fingerprints in grape cultivars. With high efficiency and accuracy and low cost, this technology is more competitive than other current identification methods. It also has excellent application prospects in the grape distinctness, uniformity, and stability (DUS) test, as well as in promoting market rights protection in the near future.

Molecular cytogenetics and development of St-chromosome-specific molecular markers of novel stripe rust resistant wheat\u2013Thinopyrum intermedium and wheat\u2013Thinopyrum ponticum substitution lines

BackgroundOwing to their excellent resistance to abiotic and biotic stress, Thinopyrum intermedium (2n = 6x = 42, JJJsJsStSt) and Th. ponticum (2n = 10x = 70) are both widely utilized in wheat germplasm innovation programs. Disomic substitution lines (DSLs) carrying one pair of alien chromosomes are valuable bridge materials for transmission of novel genes, fluorescence in situ hybridization (FISH) karyotype construction and specific molecular marker development.ResultsSix wheat–Thinopyrum DSLs derived from crosses between Abbondanza nullisomic lines (2n = 40) and two octoploid Trititrigia lines (2n = 8x = 56), were characterized by sequential FISH–genome in situ hybridization (GISH), multicolor GISH (mc-GISH), and an analysis of the wheat 15 K SNP array combined with molecular marker selection. ES-9 (DS2St (2A)) and ES-10 (DS3St (3D)) are wheat–Th. ponticum DSLs, while ES-23 (DS2St (2A)), ES-24 (DS3St (3D)), ES-25(DS2St (2B)), and ES-26 (DS2St (2D)) are wheat–Th. intermedium DSLs. ES-9, ES-23, ES-25 and ES-26 conferred high thousand-kernel weight and stripe rust resistance at adult stages, while ES-10 and ES-24 were highly resistant to stripe rust at all stages. Furthermore, cytological analysis showed that the alien chromosomes belonging to the same homoeologous group (2 or 3) derived from different donors carried the same FISH karyotype and could form a bivalent. Based on specific-locus amplified fragment sequencing (SLAF-seq), two 2St-chromosome-specific markers (PTH-005 and PTH-013) and two 3St-chromosome-specific markers (PTH-113 and PTH-135) were developed.ConclusionsThe six wheat–Thinopyrum DSLs conferring stripe rust resistance can be used as bridging parents for transmission of valuable resistance genes. The utility of PTH-113 and PTH-135 in a BC1F2 population showed that the newly developed markers could be useful tools for efficient identification of St chromosomes in a common wheat background.

Identification and DNA Marker Development for a Wheat-Leymus mollis 2Ns (2D) Disomic Chromosome Substitution.

Leymus mollis (2n = 4x = 28, NsNsXmXm), a wild relative of common wheat (Triticum aestivum L.), carries numerous loci which could potentially be used in wheat improvement. In this study, line 17DM48 was isolated from the progeny of a wheat and L. mollis hybrid. This line has 42 chromosomes forming 21 bivalents at meiotic metaphase I. Genomic in situ hybridization (GISH) demonstrated the presence of a pair chromosomes from the Ns genome of L. mollis. This pair substituted for wheat chromosome 2D, as shown by fluorescence in situ hybridization (FISH), DNA marker analysis, and hybridization to wheat 55K SNP array. Therefore, 17DM48 is a wheat-L. mollis 2Ns (2D) disomic substitution line. It shows longer spike and a high level of stripe rust resistance. Using specific-locus amplified fragment sequencing (SLAF-seq), 13 DNA markers were developed to identify and trace chromosome 2Ns of L. mollis in wheat background. This line provides a potential bridge germplasm for genetic improvement of wheat stripe rust resistance.

Genome Wide Association Study Identifies Candidate Genes Related to the Earlywood Tracheid Properties in Picea crassifolia Kom.

Picea crassifolia Kom. is one of the timber and ecological conifers in China and its wood tracheid traits directly affect wood formation and adaptability under harsh environment. Molecular studies on P. crassifolia remain inadequate because relatively few genes have been associated with these traits. To identify markers and candidate genes that can potentially be used for genetic improvement of wood tracheid traits, we examined 106 clones of P. crassifolia, and investigated phenotypic data for 14 wood tracheid traits before specific-locus amplified fragment sequencing (SLAF-seq) was employed to perform a genome wide association study (GWAS). Subsequently, the results were used to screen single nucleotide polymorphism (SNP) loci and candidate genes that exhibited a significant correlation with the studied traits. We developed 4,058,883 SLAF-tags and 12,275,765 SNP loci, and our analyses identified a total of 96 SNP loci that showed significant correlations with three earlywood tracheid traits using a mixed linear model (MLM). Next, candidate genes were screened in the 100 kb zone (50 kb upstream, 50 kb downstream) of each of the SNP loci, whereby 67 candidate genes were obtained in earlywood tracheid traits, including 34 genes of known function and 33 genes of unknown function. We provide the most significant SNP for each trait-locus combination and candidate genes occurring within the GWAS hits. These resources provide a foundation for the development of markers that could be used in wood traits improvement and candidate genes for the development of earlywood tracheid in P. crassifolia.

Construction of a High-Density Genetic Map and Identification of Quantitative Trait Loci Linked to Fruit Quality Traits in Apricots Using Specific-Locus Amplified Fragment Sequencing.

Improving fruit quality is one of the main tasks in modern commercial apricot breeding. Because of the lack of high-density linkage maps and fine mapping, it is difficult to obtain molecular markers that can assist in breeding for quantitative inheritance of fruit quality traits. In this study, specific-locus amplified fragment sequencing was used to genotype 169 seedlings of F1 apricot (Prunus armeniaca L.) progenies derived from crossing “Chuanzhihong” (H) with “Saimaiti” (S). After aligning to the Prunus armeniaca reference genome and filtering out low-quality variants, 6,012 high-quality single nucleotide polymorphisms were obtained and employed to construct a genetic map for each parent. The genetic linkage maps showed eight linkage groups of apricot, covering a distance of 809.6 cM in “H” and 1076.4 cM in “S”. The average distance between markers in “H” and “S” was 0.62 and 0.95 cM, respectively. To map quantitative trait loci (QTLs) for fruit quality, we investigated fruit quality traits, including fruit weight (FW), fruit height (FH), fruit lateral width (FL), fruit ventral width (FV), soluble solids content (SSC), and fruit firmness (FF) for all seedlings genotyped in 2018 and 2019. Eleven and nine QTLs linked to fruit quality traits were anchored on the “H” and “S” maps, respectively, and 1,138 putative candidate genes for 16 most significant regions on the corresponding chromosome were identified based on gene annotation. Among them, fruit size contained 648 genes in 11 intervals on the reference genome, SSC contained 372 genes in 3 intervals, and FF contained 117 genes in 2 intervals. Our findings uncovered the genetic basis of apricot fruit quality, and provided candidate genes for further molecular genetic studies on fruit quality and QTL targets for future marker-assisted selection of apricot quality improvement breeding.

QTL mapping for growth-related traits by constructing the first genetic linkage map in Simao pine

BackgroundSimao pine is one of the primary economic tree species for resin and timber production in southwest China. The exploitation and utilization of Simao pine are constrained by the relatively lacking of genetic information. Construction a fine genetic linkage map and detecting quantitative trait locis (QTLs) for growth-related traits is a prerequisite section of Simao Pine's molecular breeding program.ResultsIn our study, a high-resolution Simao pine genetic map employed specific locus amplified fragment sequencing (SLAF-seq) technology and based on an F1 pseudo-testcross population has been constructed. There were 11,544 SNPs assigned to 12 linkage groups (LGs), and the total length of the map was 2,062.85 cM with a mean distance of 0.37 cM between markers. According to the phenotypic variation analysis for three consecutive years, a total of seventeen QTLs for four traits were detected. Among 17 QTLs, there were six for plant height (Dh.16.1, Dh16.2, Dh17.1, Dh18.1–3), five for basal diameter (Dbd.17.1–5), four for needle length (Dnl17.1–3, Dnl18.1) and two for needle diameter (Dnd17.1 and Dnd18.1) respectively. These QTLs individually explained phenotypic variance from 11.0–16.3%, and the logarithm of odds (LOD) value ranged from 2.52 to 3.87.ConclusionsIn our study, a fine genetic map of Simao pine applied the technology of SLAF-seq has been constructed for the first time. Based on the map, a total of 17 QTLs for four growth-related traits were identified. It provides helpful information for genomic studies and marker-assisted selection (MAS) in Simao pine.

Investigation of genetic relationships within three Miscanthus species using SNP markers identified with SLAF-seq

BackgroundMiscanthus, which is a leading dedicated-energy grass in Europe and in parts of Asia, is expected to play a key role in the development of the future bioeconomy. However, due to its complex genetic background, it is difficult to investigate phylogenetic relationships in this genus. Here, we investigated 50 Miscanthus germplasms: 1 female parent (M. lutarioriparius), 30 candidate male parents (M. lutarioriparius, M. sinensis, and M. sacchariflorus), and 19 offspring. We used high-throughput Specific-Locus Amplified Fragment sequencing (SLAF-seq) to identify informative single nucleotide polymorphisms (SNPs) in all germplasms.ResultsWe identified 257,889 SLAF tags, of which 87,162 were polymorphic. Each tag was 264–364 bp long. The obtained 724,773 population SNPs were used to investigate genetic relationships within three species of Miscanthus. We constructed a phylogenetic tree of the 50 germplasms using the obtained SNPs and grouped them into two clades: one clade comprised of M. sinensis alone and the other one included the offspring, M. lutarioriparius, and M. sacchariflorus. Genetic cluster analysis had revealed that M. lutarioriparius germplasm C3 was the most likely male parent of the offspring.ConclusionsAs a high-throughput sequencing method, SLAF-seq can be used to identify informative SNPs in Miscanthus germplasms and to rapidly characterize genetic relationships within this genus. Our results will support the development of breeding programs with the focus on utilizing Miscanthus cultivars with elite biomass- or fiber-production potential for the developing bioeconomy.

Construction of a high-density genetic map based on specific-locus amplified fragment sequencing and identification of loci controlling anthocyanin pigmentation in Yunnan red radish.

Radish (Raphanus sativus L.) belongs to the family Brassicaceae. The Yunnan red radish variety contains relatively large amounts of anthocyanins, making them important raw materials for producing edible red pigment. However, the genetic mechanism underlying this pigmentation has not been fully characterized. Here, the radish inbred line YAAS-WR1 (white root skin and white root flesh) was crossed with the inbred line YAAS-RR1 (red root skin and red root flesh) to produce F1, F2, BC1P1, and BC1P2 populations. Genetic analyses revealed that the pigmented/non-pigmented and purple/red traits were controlled by two genetic loci. The F2 population and the specific-locus amplified fragment sequencing (SLAF-seq) technique were used to construct a high-density genetic map (1230.16 cM), which contained 4032 markers distributed in nine linkage groups, with a mean distance between markers of 0.31 cM. Additionally, two quantitative trait loci (QAC1 and QAC2) considerably affecting radish pigmentation were detected. A bioinformatics analysis of the QAC1 region identified 58 predicted protein-coding genes. Of these, RsF3′H, which is related to anthocyanin biosynthesis, was revealed as a likely candidate gene responsible for the purple/red trait. The results were further verified by analyzing gene structure and expression. Regarding QAC2, RsMYB1.3 was determined to be a likely candidate gene important for the pigmented/non-pigmented trait, with a 4-bp insertion in the first exon that introduced a premature termination codon in the YAAS-WR1 sequence. Assays demonstrated that RsMYB1.3 interacted with RsTT8 and activated RsTT8 and RsUFGT expression. These findings may help clarify the complex regulatory mechanism underlying radish anthocyanin synthesis. Furthermore, this study’s results may be relevant for the molecular breeding of radish to improve the anthocyanin content and appearance of the taproots.

Quantitative Trait Loci Mapping and Development of KASP Marker Smut Screening Assay Using High-Density Genetic Map and Bulked Segregant RNA Sequencing in Sugarcane (Saccharum spp.).

Sugarcane is one of the most important industrial crops globally. It is the second largest source of bioethanol, and a major crop for biomass-derived electricity and sugar worldwide. Smut, caused by Sporisorium scitamineum, is a major sugarcane disease in many countries, and is managed by smut-resistant varieties. In China, smut remains the single largest constraint for sugarcane production, and consequently it impacts the value of sugarcane as an energy feedstock. Quantitative trait loci (QTLs) associated with smut resistance and linked diagnostic markers are valuable tools for smut resistance breeding. Here, we developed an F1 population (192 progeny) by crossing two sugarcane varieties with contrasting smut resistance and used for genome-wide single nucleotide polymorphism (SNP) discovery and mapping, using a high-throughput genotyping method called “specific locus amplified fragment sequencing (SLAF-seq) and bulked-segregant RNA sequencing (BSR-seq). SLAF-seq generated 148,500 polymorphic SNP markers. Using SNP and previously identified SSR markers, an integrated genetic map with an average 1.96 cM marker interval was produced. With this genetic map and smut resistance scores of the F1 individuals from four crop years, 21 major QTLs were mapped, with a phenotypic variance explanation (PVE) > 8.0%. Among them, 10 QTLs were stable (repeatable) with PVEs ranging from 8.0 to 81.7%. Further, four QTLs were detected based on BSR-seq analysis. aligning major QTLs with the genome of a sugarcane progenitor Saccharum spontaneum, six markers were found co-localized. Markers located in QTLs and functional annotation of BSR-seq-derived unigenes helped identify four disease resistance candidate genes located in major QTLs. 77 SNPs from major QTLs were then converted to Kompetitive Allele-Specific PCR (KASP) markers, of which five were highly significantly linked to smut resistance. The co-localized QTLs, candidate resistance genes, and KASP markers identified in this study provide practically useful tools for marker-assisted sugarcane smut resistance breeding.

Genetic analysis of the early bud flush trait of tea plants (Camellia sinensis) in the cultivar 'Emei Wenchun' and its open-pollinated offspring.

The timing of bud flush (TBF) in the spring is one of the most important agronomic traits of tea plants (Camellia sinensis). In this study, we designed an open-pollination breeding program using ‘Emei Wenchun’ (EW, a clonal tea cultivar with extra-early TBF) as a female parent. A half-sib population (n = 388) was selected for genotyping using specific-locus amplified fragment sequencing. The results enabled the identification of paternity for 294 (75.8%) of the offspring, including 11 (2.8%) from EW selfing and 217 (55.9%) assigned to a common father, ‘Chuanmu 217’ (CM). The putative EW × CM full-sib population was used to construct a linkage map. The map has 4244 markers distributed in 15 linkage groups, with an average marker distance of 0.34 cM. A high degree of collinearity between the linkage map and physical map was observed. Sprouting index, a trait closely related to TBF, was recorded for the offspring population in 2020 and 2021. The trait had moderate variation, with coefficients of variation of 18.5 and 17.6% in 2020 and 2021, respectively. Quantitative trait locus (QTL) mapping that was performed using the linkage map identified two major QTLs and three minor QTLs related to the sprouting index. These QTLs are distributed on Chr3, Chr4, Chr5, Chr9, and Chr14 of the reference genome. A total of 1960 predicted genes were found within the confidence intervals of QTLs, and 22 key candidate genes that underlie these QTLs were preliminarily screened. These results are important for breeding and understanding the genetic base of the TBF trait of tea plants.

Genome-wide association study reveals growth-related SNPs and candidate genes in mandarin fish (Siniperca chuatsi)

Mandarin fish (Siniperca chuatsi) is a popular inland farmed fish species with high an economic value. Molecular selective breeding programs are urgently required to improve the growth traits of mandarin fish for farming industry. In this study, using 160 mandarin fish individuals, specific locus-amplified fragment sequencing and SNP genotyping, we performed a genome-wide association study (GWAS) to identify genomic regions and candidate genes that are potentially associated with various growth traits of mandarin fish, such as body weight (BW), body thickness (BT), body height (BH) total length (TL), slaughter weight (SW), and body length (BL). Through genotyping, 5205 high-quality SNPs were found located on 24 chromosomes. A total of 11 significant and 37 suggestive SNPs related to growth traits were obtained, among which five SNPs significantly related to BW were located in the linkage group (LG) 8, 17, 21, and 23, two SNPs significantly related to TL were located in LG 7 and 17, two SNPs significantly related to BH were located on LG 17 and 23, two SNPs significantly related to BT were located on LG 14 and 19, respectively. Furthermore, LG17_937,734 and LG23_23,856,613 were growth-related traits overlap. Within 300 kb upstream and downstream of these significant SNPs, 65 candidate genes were identified within the associated QTLs, many of which were related to bone differentiation, growth and development, cell division and neurogenesis. In particular, asns, tg, mkk6, htra1, rnf213, ttn, tgfbr2, and nck2, were shown to be closely associated with growth, fat, muscle and bone formation. To our knowledge, this is the first GWAS focusing on growth traits of mandarin fish. Our findings establish a foundation for molecular marker-assisted selecting, genomic selecting, and functional analyzing the growth traits of mandarin fish.

Candidate sex-associated gene identification in Trachinotus ovatus (Carangidae) using an integrated SLAF-seq and bulked segregant analysis approach.

It is difficult to distinguish the sexes of Trachinotus ovatus based on appearance, and little data about sex-determining genes are available for this species. Here, we generated 200 F2 individuals using the parents R404 and R403. DNA samples were collected from 50 individuals of each sex and aggregated into sex-specific DNA pools. Specific-locus amplified fragment sequencing was integrated with bulked segregant analysis to detect candidate sex-associated genes. Approximately 3,153,153 high-quality single-nucleotide polymorphism (SNP) markers and 135,363 high-quality insertion-deletion (Indel) markers were generated. Six candidate regions within chromosome 14, encompassing 132 candidate genes, were identified as closely related to sex. Based on annotations, six genes (EVM0019817, EVM0004192, EVM0001445, EVM0005260, EVM0014734, and EVM0009626) were predicted to be closely associated with sex. These results present an efficient genetic mapping approach that lays a foundation for molecular sex discrimination in T. ovatus.