Specific-locus Amplified Fragment Sequencing Research Articles

Genome-wide association study of body size traits in Wenshang Barred chickens based on the specific-locus amplified fragment sequencing technology.

Chicken body size (BS) is an economically important trait, which has been assessed in many studies for genetic selection. However, previous reports detected functional chromosome mutations or regions using gene chips. The present study used the specific-locus amplified fragment sequencing (SLAF-seq) technology to perform a genome-wide association study (GWAS) of purebred Wenshang Barred chickens. A total of 250 one-day-old male chickens were assessed in this study. Body size in individual birds was measured at 56days. SLAF-seq was used to genotype and GWAS analysis was carried out using the general linear model (GLM) of the TASSEL program. A total of 1,286,715 single-nucleotide polymorphisms (SNPs) were detected, of which 175,211 were tested as candidate SNPs for genome-wide association analysis using the TASSEL general linear model. Three SNPs markers reached genome-wide significance. Of these, chrZ:81729634, chrZ:81841715, and chrZ:81954149 at 81,729,634, 81,841,715, and 81,954,149bp of GGA Z were significantly associated with body diagonal length at 56days (BDL56); and tibia length at 56days (TL56). These SNPs were close to three genes, includingZCCHC7,PAX5,andMELK. These results open new horizons for studies on BS and should promote the use of Chinese chickens, especially Wenshang Barred chickens.

A High-Density Genetic Linkage Map of SLAFs and QTL Analysis of Grain Size and Weight in Barley (Hordeum vulgare L.).

Grain size is an important agronomic trait determines yield in barley, and a high-density genetic map is helpful to accurately detect quantitative trait loci (QTLs) related to grain traits. Using specific-locus amplified fragment sequencing (SLAF-seq) technology, a high-density genetic map was constructed with a population of 134 recombinant inbred lines (RILs) deriving from a cross between Golden Promise (GP) and H602, which contained 12,635 SLAFs with 26,693 SNPs, and spanned 896.74 cM with an average interval of 0.07 cM on seven chromosomes. Based on the map, a total of 16 QTLs for grain length (GL), grain width and thousand-grain weight were detected on 1H, 2H, 4H, 5H, and 6H. Among them, a major QTL locus qGL1, accounting for the max phenotypic variance of 16.7% was located on 1H, which is a new unreported QTL affecting GL. In addition, the other two QTLs, qGL5 and qTGW5, accounting for the max phenotypic variances of 20.7 and 21.1%, respectively, were identified in the same region, and sequencing results showed they are identical to HvDep1 gene. These results indicate that it is a feasible approach to construct a high-quality genetic map for QTL mapping by using SLAF markers, and the detected major QTLs qGL1, qGL5, and qTGW5 are useful for marker-assisted selection (MAS) of grain size in barley breeding.

Quantitative Trait Locus (QTLs) Mapping for Quality Traits of Wheat Based on High Density Genetic Map Combined With Bulked Segregant Analysis RNA-seq (BSR-Seq) Indicates That the Basic 7S Globulin Gene Is Related to Falling Number.

Numerous quantitative trait loci (QTLs) have been identified for wheat quality; however, most are confined to low-density genetic maps. In this study, based on specific-locus amplified fragment sequencing (SLAF-seq), a high-density genetic map was constructed with 193 recombinant inbred lines derived from Chuanmai 42 and Chuanmai 39. In total, 30 QTLs with phenotypic variance explained (PVE) up to 47.99% were identified for falling number (FN), grain protein content (GPC), grain hardness (GH), and starch pasting properties across three environments. Five NAM genes closely adjacent to QGPC.cib-4A probably have effects on GPC. QGH.cib-5D was the only one detected for GH with high PVE of 33.31–47.99% across the three environments and was assumed to be related to the nearest pina-D1 and pinb-D1genes. Three QTLs were identified for FN in at least two environments, of which QFN.cib-3D had relatively higher PVE of 16.58–25.74%. The positive effect of QFN.cib-3D for high FN was verified in a double-haploid population derived from Chuanmai 42 × Kechengmai 4. The combination of these QTLs has a considerable effect on increasing FN. The transcript levels of Basic 7S globulin and Basic 7S globulin 2 in QFN.cib-3D were significantly different between low FN and high FN bulks, as observed through bulk segregant RNA-seq (BSR). These QTLs and candidate genes based on the high-density genetic map would be beneficial for further understanding of the genetic mechanism of quality traits and molecular breeding of wheat.

Construction of the first high-density genetic map for growth related QTL analysis in Ancherythroculter nigrocauda

Ancherythroculter nigrocauda is a fish endemic to the upper areas of the Changjiang (Yangtze) River in China. Quantitative trait locus (QTL) mapping is a powerful tool to identify potential genes affecting traits of economic importance in domestic animals. In this study, a high-density genetic map was constructed with 5 901 single nucleotide polymorphism (SNP) makers by sequencing 92 individual fish from a F1 family using the specific-locus amplified fragment sequencing approach. Initially, 48 QTLs for total length, body length, body height, and body weight were identified according to the high density of the genetic map with 24 LGs, a total length of 3 839.4 cM, and marker spacing of about 0.82 cM. These QTLs explained 27.1%–49.9% of phenotypic variance. The results of this study suggest that major QTLs are responsible for the growth of A. nigrocauda, and these are potentially useful in comparative genomics research, genome assembly, and marker-assisted breeding programs for this species.

Identification and Validation of New Stable QTLs for Grain Weight and Size by Multiple Mapping Models in Common Wheat.

Improvement of grain weight and size is an important objective for high-yield wheat breeding. In this study, 174 recombinant inbred lines (RILs) derived from the cross between Jing 411 and Hongmangchun 21 were used to construct a high-density genetic map by specific locus amplified fragment sequencing (SLAF-seq). Three mapping methods, including inclusive composite interval mapping (ICIM), genome-wide composite interval mapping (GCIM), and a mixed linear model performed with forward–backward stepwise (NWIM), were used to identify QTLs for thousand grain weight (TGW), grain width (GW), and grain length (GL). In total, we identified 30, 15, and 18 putative QTLs for TGW, GW, and GL that explain 1.1–33.9%, 3.1%–34.2%, and 1.7%–22.8% of the phenotypic variances, respectively. Among these, 19 (63.3%) QTLs for TGW, 10 (66.7%) for GW, and 7 (38.9%) for GL were consistent with those identified by genome-wide association analysis in 192 wheat varieties. Five new stable QTLs, including 3 for TGW (Qtgw.ahau-1B.1, Qtgw.ahau-4B.1, and Qtgw.ahau-4B.2) and 2 for GL (Qgl.ahau-2A.1 and Qgl.ahau-7A.2), were detected by the three aforementioned mapping methods across environments. Subsequently, five cleaved amplified polymorphic sequence (CAPS) markers corresponding to these QTLs were developed and validated in 180 Chinese mini-core wheat accessions. In addition, 19 potential candidate genes for Qtgw.ahau-4B.2 in a 0.31-Mb physical interval were further annotated, of which TraesCS4B02G376400 and TraesCS4B02G376800 encode a plasma membrane H+-ATPase and a serine/threonine-protein kinase, respectively. These new QTLs and CAPS markers will be useful for further marker-assisted selection and map-based cloning of target genes.

Physiological characterization and gene mapping of a novel cuticular wax-related mutant in barley (Hordeum vulgare L.)

Cuticular wax is a type of lipid covering the surface of plants, which is directly related to crop stress resistance. Thus, it is important to study wax-related genes and their regulatory mechanism in wax biosynthesis pathway for improving stress resistance. In this study, a wax-deficient barley mutant barley cuticular wax1(bcw1)was identified, and genetic analysis indicated that the trait was controlled by a single recessive nuclear gene. Phenotype observations showed that the tubule-shaped waxy crystals covering the sheath and stem epidermis of mutants disappeared, but there were no significant differences were detected in the leaf epidermis between mutant and wild type. Water loss results confirmed that the cuticular waxes and cutins improved water-holding capacity of plant. By combining the bulk segregant analysis (BSA) and specific locus amplified fragment sequencing (SLAF-seq) strategy, the wax-related gene BCW1 was located on chromosome 2H with a total length of 15.10 Mb. No cuticular wax-related genes have been reported in the 9 regions, indicating that BCW1 is a novel gene that plays roles in cuticular wax biosynthesis and wax crystals formation.

Decryption of favourable haplotypes and potential candidate genes for five fibre quality properties using a relatively novel genome-wide association study procedure in upland cotton

To understand the genetic basis of crop economic characteristics, identifying whole-genome quantitative trait loci (QTLs) in breeding resources via genome-wide association studies (GWASs) is important, but conventional GWAS procedures have mainly concentrated on identifying regions of major QTLs. In this study, to reveal whole-genome QTLs controlling cotton fibre quality traits, a total of 1,236,418 single-nucleotide polymorphism (SNP) markers were explored in a natural population of 315 upland cotton accessions using the specific-locus amplified fragment sequencing (SLAF-seq) method, and 13,391 high-quality SNPs were organized into 9244 SNP linkage disequilibrium blocks (SNPLDBs) to develop molecular markers with multiple haplotypes/alleles. A relatively novel procedure for restricted two-stage multi-locus multi-allele GWAS (RTM-GWAS) involving two methods was applied, including multiple environments and four single environments. In total, 75, 77, 66, 66, and 60 significant SNPLDBs associated with the upper half mean length (UHML), fibre strength (FS), fibre micronaire (FM), fibre uniformity index (FUI), and fibre elongation (FE), respectively, were identified via the multiple-environment RTM-GWAS method. Based on these results, the haplotype/allele effects of the significant SNPLDB loci were estimated, and QTL-allele matrices were established to provide the abbreviated genetic composition of the population. Most importantly, nine stable SNPLDB loci were simultaneously detected in multiple environments and two or more single test environments with large –lg(P) values and percentages of explained phenotypic variation. Furthermore, favourable haplotypes/alleles of the nine stable SNPLDB loci were identified, and 79 potential candidate genes related to UHML and FS traits were predicted from RNA-seq data. Favourable haplotypes/alleles, potential candidate genes and QTL-allele matrices can offer important insights into the genetic basis of fibre quality traits in upland cotton and may facilitate the breeding of cotton varieties with excellent fibre quality properties.

Molecular diversity and genetic structure of wild rice accessions (Oryza rufipogon Griff.) in Guangdong Province, China, as revealed by SNP markers

Common wild rice (Oryza rufipogon Griff.) is a valuable germplasm resource for rice genetic improvement. However, it is endangered due to habitat loss. Assessment of its genetic diversity is essential for germplasm characterization, utilization, and conservation. In the present study, we developed a 79,422-single-nucleotide polymorphism genome-wide array by specific-locus amplified fragment sequencing (SLAF-seq) and used it to genotype 110 common wild rice accessions from different agroclimatic zones in Guangdong Province, China. The number of SNPs on each chromosome was consistent with the physical length of the chromosome, and the average marker density was approximately 4.83 kb/SNP. The genome-wide linkage disequilibrium decayed to its half maximum within 0.52 kb for O. rufipogon. The common wild rice accessions were clustered into four subgroups, with some overlap in principal component analysis and a neighbor-joining tree that was also confirmed by model-based admixture. Analysis of molecular variance revealed significant genetic differentiation among individuals, accounting for 90.64% of the total molecular variance; however, the differences between the subgroups (Enping, Lufeng, Suixi and Fogang) were not significant. The significant genetic variations in resources collected in different years were observed in Enping and Suixi subgroups, but there was no significant genetic variation in Fogang subgroup. The pairwise fixation index (FST) suggested that the populations from Enping and Lufeng were most differentiated while those from Enping and Fogang were least differentiated. Our results illustrated clear genetic relationships among 110 common wild rice accessions, showing a potential molecular marker-based strategy for preserving the genetic diversity and accelerating the systematic utilization of these important wild rice resources.

Loci and candidate gene identification for soybean resistance to Phytophthora root rot race 1 in combination with association and linkage mapping

As the second most destructive disease in soybean (Glycine max (L.) Merr), Phytophthora root or stem-rot (PRR) of soybean, which was caused by Phytophthora sojae (P. sojae), could lead to a 10–40% or complete yield loss annually. Researches were needed to perform to identify the P. sojae-resistant germplasm and to better understand the genetic basis of P. sojae resistance in soybean. A total of 225 diverse soybean cultivars and 109 recombinant inbred lines (RILs) derived from crossing ‘DongnongL-28’ (resistant to P. sojae race 1) and ‘Hefeng 25’ (susceptible to P. sojae race 1) were used to evaluate P. sojae race 1 resistance. These 225 soybean cultivars were sequenced using the Specific Locus Amplified Fragment Sequencing (SLAF-seq) approach, and 28,722 single-nucleotide polymorphisms (SNPs) were obtained from the mapping of resistant loci through genome-wide association. Eight quantitative trait nucleotides (QTNs) were associated with resistance to P. sojae race 1. The QTN on Chr.03 was colocalised according to linkage mapping of the RILs. A total of 18 candidate genes were predicted in the flanking region of the colocalised locus on Chr.03. There were stress response-related motifs, such as cis-acting regulatory elements involved in salicylic acid or MeJA responsiveness, in the 1-kb upstream region of sixteen genes. Quantitative RT-PCR showed that the Glyma.03G033700 was induced by P. sojae race 1. Association analysis for Rps loci showed that Glyma.03G033700 and Glyma.03G033800 were the candidates near peak SNP. The identified loci along with the candidate genes could be valuable for studying the molecular mechanisms underlying soybean resistance to P. sojae race 1 and breeding resistant varieties.

Construction of a high-density SNP-based genetic map and identification of fruit-related QTLs and candidate genes in peach [Prunus persica (L.) Batsch

BackgroundHigh-density genetic mapping is a valuable tool for mapping loci that control specific traits for perennial fruit trees. Peach is an economically important fruit tree and a model Rosaceae species for genomic and genetic research. In peach, even though many molecular markers, genetic maps and QTL mappings have been reported, further research on the improvement of marker numbers, map densities, QTL accuracy and candidate gene identification is still warranted.ResultsA high-density single nucleotide polymorphism (SNP)-based peach linkage map was constructed using specific locus amplified fragment sequencing (SLAF-seq). This genetic map consisted of 7998 SLAF markers, spanning 1098.79 cM with an average distance of 0.17 cM between adjacent markers. A total of 40 QTLs and 885 annotated candidate genes were detected for 10 fruit-related traits, including fruit weight (FW), fruit diameter (FD), percentage of red skin colour (PSC), eating quality (EQ), fruit flavour (FV), red in flesh (RF), red around pit (RP), adherence to pit (AP), fruit development period (FDP) and fruit fibre content (FFC). Eighteen QTLs for soluble solid content (SSC) were identified along LGs 1, 4, 5, and 6 in 2015 and 2016, and 540 genes were annotated in QTL intervals. Thirty-two QTLs for fruit acidity content (FA) were detected on LG1, and 2, 4, 5, 6, and 1232 candidate genes were identified. The expression profiles of 2 candidate genes for SSC and 4 for FA were analysed in parents and their offspring.ConclusionsWe constructed a high-density genetic map in peach based on SLAF-seq, which may contribute to the identification of important agronomic trait loci. Ninety QTLs for 12 fruit-related traits were identified, most of which overlapped with previous reports, and some new QTLs were obtained. A large number of candidate genes for fruit-related traits were screened and identified. These results may improve our understanding of the genetic control of fruit quality traits and provide useful information in marker-assisted selection for fruit quality in peach breeding programmes.

Identification of Loci and Candidate Genes Analyses for Tocopherol Concentration of Soybean Seed.

Tocopherol (Toc) occurs in soybean seeds and is extracted together with the soybean oil. Toc is utilized as an antioxidant in food and an additive in animal feed. A total of 180 representative accessions and 144 recombinant inbred lines (RILs) from the cross of ‘Hefeng 25’ and ‘OAC Bayfield’ were selected to evaluate individuals and total Toc concentrations in soybean seeds. The 180 soybean samples were sequenced by the approach of Specific Locus Amplified Fragment Sequencing (SLAF-seq). A total of 22,611 single nucleotide polymorphisms (SNPs) were developed. Nineteen quantitative trait nucleotides (QTNs) were identified associated with individual or total-Toc based on genome-wide association analysis (GWAS). Among them, three QTNs located near known QTLs, and 16 were novel. Eighteen QTLs and nine eQTLs were also detected by linkage mapping. The QTN rs9337368 on Chr.02 was colocalized according to the linkage mapping of the RILs and genome-wide association analysis and regarded as a stable locus for mining the candidate genes in association with Toc. A total of 42 candidate genes near the 200 kbp flanking region of this identified locus were found. Upon a gene-based association, 11 SNPs from five genes out of the 42 candidates were detected. Expression level analysis of five candidate genes revealed that two genes were significantly related to Toc content. The identified loci, along with the candidate genes, might be valuable for increasing the Toc concentration in soybean seeds and improving the nutritional value of soybean oil.

Cytogenetic Analysis and Molecular Marker Development for a New Wheat-Thinopyrum ponticum 1Js (1D) Disomic Substitution Line With Resistance to Stripe Rust and Powdery Mildew.

Thinopyrum ponticum (2n = 10x = 70), a member of the tertiary gene pool of wheat (Triticum aestivum L.), harbors many biotic and abiotic stress resistance genes. CH10A5, a novel disomic substitution line from a cross of T. aestivum cv. 7182 and Th. ponticum, was characterized by cytogenetic identification, in situ hybridization, molecular marker analysis, and morphological investigation of agronomic traits and disease resistance. Cytological observations showed that CH10A5 contained 42 chromosomes and formed 21 bivalents at meiotic metaphase I. Genome in situ hybridization (GISH) analysis indicated that two of its chromosomes came from the Js genome of Th. ponticum, and wheat 15K array mapping and fluorescence in situ hybridization (FISH) revealed that chromosome 1D was absent from CH10A5. Polymorphic analysis of molecular markers indicated that the pair of alien chromosomes belonged to homoeologous group one, designated as 1Js. Thus, CH10A5 was a wheat–Th. ponticum 1Js (1D) disomic substitution line. Field disease resistance trials demonstrated that the introduced Th. ponticum chromosome 1Js was probably responsible for resistance to both stripe rust and powdery mildew at the adult stage. Based on specific-locus amplified fragment sequencing (SLAF-seq), 507 STS molecular markers were developed to distinguish chromosome 1Js genetic material from that of wheat. Of these, 49 STS markers could be used to specifically identify the genetic material of Th. ponticum. CH10A5 will increase the resistance gene diversity of wheat breeding materials, and the markers developed here will permit further tracing of heterosomal chromosome fragments in the future.

Genome sequencing and population genomics modeling provide insights into the local adaptation of weeping forsythia

Understanding the genetic basis underlying the local adaptation of nonmodel species is a fundamental goal in evolutionary biology. In this study, we explored the genetic mechanisms of the local adaptation of Forsythia suspensa using genome sequence and population genomics data obtained from specific-locus amplified fragment sequencing. We assembled a high-quality reference genome of weeping forsythia (Scaffold N50 = 7.3 Mb) using ultralong Nanopore reads. Then, genome-wide comparative analysis was performed for 15 natural populations of weeping forsythia across its current distribution range. Our results revealed that candidate genes associated with local adaptation are functionally correlated with solar radiation, temperature and water variables across heterogeneous environmental scenarios. In particular, solar radiation during the period of fruit development and seed drying after ripening, cold, and drought significantly contributed to the adaptive differentiation of F. suspensa. Natural selection exerted by environmental factors contributed substantially to the population genetic structure of F. suspensa. Our results supported the hypothesis that adaptive differentiation should be highly pronounced in the genes involved in signal crosstalk between different environmental variables. Our population genomics study of F. suspensa provides insights into the fundamental genetic mechanisms of the local adaptation of plant species to climatic gradients.

A Genomewide Scan for Genetic Structure and Demographic History of Two Closely Related Species, Rhododendron dauricum and R. mucronulatum (Rhododendron, Ericaceae).

Understanding the processes of divergence and speciation is an important task for evolutionary research, and climate oscillations play a pivotal role. We estimated the genetic structure and demographic history of two closely related species of Rhododendron, R. dauricum, and R. mucronulatum, distributed in northeastern China using 664,406 single nucleotide polymorphic loci of specific-locus amplified fragment sequencing (SLAF-seq) and 4 chloroplast DNA (cpDNA) fragments, sampling 376 individuals from 39 populations of these two species across their geographic distributions. The geographical distribution of cpDNA haplotypes revealed that R. dauricum and R. mucronulatum have different spatial genetic structures and haplotype diversity. Analysis of molecular variance (AMOVA) results showed that these two species have significant genetic differentiation and that the phylogeny demonstrates that these two species clustered a monophyletic group based on SLAF data, respectively, but not in cpDNA data. The evidence of significant gene flow was also detected from R. mucronulatum to R. dauricum. A deep divergence between the two species was observed and occurred during the early Oligocene. The niche models showed that the two species have different demographic histories. Thus, our results imply that geography and climate changes played important roles in the evolutionary process of R. dauricum and R. mucronulatum, and although there was an interspecific gene flow, the divergence was maintained by natural selection.

High-density genetic map construction and identification of loci controlling flower-type traits in Chrysanthemum (Chrysanthemum \xd7 morifolium Ramat.)

Flower type is an important and extremely complicated trait of chrysanthemum. The corolla tube merged degree (CTMD) and the relative number of ray florets (RNRF) are the two key factors affecting chrysanthemum flower type. However, few reports have clarified the inheritance of these two complex traits, which limits directed breeding for flower-type improvement. In this study, 305 F1 hybrids were obtained from two parents with obvious differences in CTMD and RNRF performance. Using specific-locus amplified fragment sequencing (SLAF-seq) technology, we constructed a high-density genetic linkage map with an average map distance of 0.76 cM. Three major QTLs controlling CTMD and four major QTLs underlying RNRF were repeatedly detected in the 2 years. Moreover, the synteny between the genetic map and other Compositae species was investigated, and weak collinearity was observed. In QTL regions with a high degree of genomic collinearity, eight annotated genes were probed in the Helianthus annuus L. and Lactuca sativa L. var. ramosa Hort. genomes. Furthermore, 20 and 11 unigenes were identified via BLAST searches between the SNP markers of the QTL regions and the C. vestitum and C. lavandulifolium transcriptomes, respectively. These results lay a foundation for molecular marker-assisted breeding and candidate gene exploration in chrysanthemum without a reference assembly.

Genome wide association mapping for tocopherol concentration in soybean seeds across multiple environments

Tocopherol (Toc) in soybean seeds has value for foods, medicines, cosmetics, and animal husbandry. Soybean breeders and soybean-derived food industries have thus focused on breeding new soybean cultivars with higher seed Toc content. In this study, an association panel consisting of 180 diverse soybean accessions were used to detect quantitative trait nucleotides (QTNs) and scan possible candidate genes based on genome-wide association analysis (GWAS) and high-throughput single-nucleotide polymorphisms (SNP). Approximately 22,611 SNPs were identified using the specific locus amplified fragment sequencing (SLAF-seq) approach and based on minor allele frequencies (MAFs) > 5 %, which spanned 84.10 % of the soybean whole genome. For the three individuals including α-tocopherol (α-Toc), γ- tocopherol (γ-Toc), δ- tocopherol (δ-Toc), and total tocopherol (total Toc) levels, a total of 18 quantitative trait nucleotides (QTNs) distributed on the 18 genomic regions of 10 chromosomes were verified via GWAS. Seven QTNs (rs55811336, rs7975863, rs37094897, rs7097743, rs17125409, rs330000, and rs4730199) could be identified in more than three environments, and the other eleven could be identified in least two environments. A total of 6, 8, 4, and 10 QTNs were novel loci for α-Toc, γ-Toc, δ-Toc, and total Toc, respectively. Nine QTNs (rs37505279, rs37686178, rs36359429, rs6898494, rs7097743, rs17125409, rs10200201, rs21080726, and rs5680781) were associated with more than one Toc component. A total of 216 candidate genes near the 200-kb flanking region of these identified QTNs were identified as potential candidate genes associated with soybean seed Toc levels. Of these genes, Glyma.17G061900 (located near QTN rs4730199 on Chr.17), a homogentisate phytyltransferase 1 that catalytic homogentisate to 2-methyl-6-phyl-1,4-benzoquinol in Toc biosynthesis, was detected through association analysis, haplotype analysis, and expression pattern analysis. The identified QTNs with beneficial alleles and the candidate genes would be valuable for improving Toc levels in soybean seeds and improving the nutritional value of soybean oil.

QTL mapping of drought tolerance traits in soybean with SLAF sequencing

Drought stress is an important factor affecting soybean yield. Improving drought tolerance of soybean varieties can increase yield and yield stability when the stress occurs. Identifying QTL related to drought tolerance using molecular marker-assisted selection is able to facilitate the development of drought-tolerant soybean varieties. In this study, we used a high-yielding and drought-sensitive cultivar ‘Zhonghuang 35’ and a drought-tolerant cultivar ‘Jindou 21’ to establish F6:9 recombinant inbred lines. We constructed a high-density genetic map using specific locus amplified fragment sequencing (SLAF-Seq) technology. The genetic map contained 8078 SLAF markers distributing across 20 soybean chromosomes with a total genetic distance of 3780.98 cM and an average genetic distance of 0.59 cM between adjacent markers. Two treatments (irrigation and drought) were used in the field tests, the Additive-Inclusive Composite Interval Mapping (ICIM-ADD) was used to call QTL, and plant height and seed weight per plant were used as the indicators of drought tolerance. We identified a total of 23 QTL related to drought tolerance. Among them, seven QTL (qPH2, qPH6, qPH7, qPH17, qPH19-1, qPH19-2, and qPH19-3) on chromosomes 2, 6, 7, 17, and 19 were related to plant height, and five QTL (qSWPP2, qSWPP6, qSWPP13, qSWPP17, and qSWPP19) on chromosomes 2, 6, 13, 17, and 19 were related to seed weight and could be considered as the major QTL. In addition, three common QTL (qPH6/qSWPP6, qPH17/qSWPP17, and qPH19-3/qSWPP19) for both plant height and seed weight per plant were located in the same genomic regions on the same chromosomes. Three (qPH2, qPH17, and qPH19-2) and four novel QTL (qSWPP2, qSWPP13, qSWPP17, and qSWPP19) were identified for plant height and seed weight per plant, respectively. Two pairs of QTL (qPH2/qSWPP2 and qPH17/qSWPP17) were also common for both plant height and seed weight per plant. These QTL and closely linked SLAF markers could be used to accelerate breeding for drought tolerant cultivars via MAS.

Molecular Cytogenetic Identification of Wheat-Aegilops Biuncialis 5Mb Disomic Addition Line with Tenacious and Black Glumes.

Production of wheat-alien disomic addition lines is of great value to the exploitation and utilization of elite genes originated from related species to wheat. In this study, a novel wheat-Aegilops biuncialis 5Mb disomic addition line WA317 was characterized by in situ hybridization (ISH) and specific-locus amplified fragment sequencing (SLAF-seq) markers. Compared to its parent Chinese Spring (CS), the glumes of WA317 had black color and were difficult to remove after harvesting, suggesting chromosome 5Mb carried gene(s) related to glume development and Triticeae domestication process. A total of 242 Ae. biuncialis SLAF-based markers (298 amplified patterns) were developed and further divided into four categories by Ae. biuncialis Y17, Ae. umbellulata Y139 and Ae. comosa Y258, including 172 markers amplifying the same bands of U and M genome, six and 102 markers amplifying U-specific and M-specific bands, respectively and eighteen markers amplifying specific bands in Y17. Among them, 45 markers had the specific amplifications in WA317 and were 5Mb specific markers. Taken together, line WA317 with tenacious and black glumes should serve as the foundation for understanding of the Triticeae domestication process and further exploitation of primitive alleles for wheat improvement. Ae. biuncialis SLAF-based markers can be used for studying syntenic relationships between U and M genomes as well as rapid tracking of U and M chromosomal segments in wheat background.

The bHLH transcription factor PPLS1 regulates the color of pulvinus and leaf sheath in foxtail millet (Setaria italica).

The bHLH transcription factor, PPLS1, interacts with SiMYB85 to control the color of pulvinus and leaf sheath by regulating anthocyanin biosynthesis in foxtail millet (Setaria italica). Foxtail millet (Setaria italica), a self-pollinated crop with numerous small florets, is difficult for cross-pollination. The color of pulvinus and leaf sheath with purple being dominant to green is an indicative character and often used for screening authentic hybrids in foxtail millet crossing. Deciphering molecular mechanism controlling this trait would greatly facilitate genetic improvement of cultivars in foxtail millet. Here, using the F2 bulk specific-locus amplified fragment sequencing approach, we mapped the putative causal gene for the purple color of pulvinus and leaf sheath (PPLS) trait to a 100Kb region on chromosome 7. Expression analyses of the 15 genes in this region revealed that Seita.7G195400 (renamed here as PPLS1) was differentially expressed between purple and green cultivars. PPLS1 encodes a bHLH transcription factor and is localized in the nucleus with a transactivation activity. Furthermore, we observed that expression of a MYB transcription factor gene, SiMYB85 (Seita.4G086300) involved in anthocyanin biosynthesis, shows a totally positive association with that of PPLS1. Heterologous co-expression of both PPLS1 and SiMYB85 in tobacco leaves led to elevated anthocyanin accumulation and expression of some anthocyanin-related genes. Furthermore, PPLS1 physically interacts with SiMYB85. Taken together, our results suggest that PPLS1 interacts with SiMYB85 to control the color of pulvinus and leaf sheath by regulating anthocyanin biosynthesis in foxtail millet.

Relationships among Chinese Boehmeria species and the evolution of various clade

It is important to provide insights into the relationships among members of Boehmeria in China and the course of evolutionary of various taxa, thus deepening understanding of Boehmeria.In this study, 34 plants (33 specimens of Boehmeria and one of Oreocnide) were sequenced using specific-locus amplified fragment sequencing (SLAF-seq) technology, and 55.76 M reads with a mean sequencing depth of 17.24x and an average Q30 of 94.03 % were obtained. Based on 1,283,459 polymorphic SLAF tags, 964,957 SNPs were discovered using the two methods and a set of 22,967 SNPs with integrity > 0.5 and minor allele frequencies (MAF) > 0.05 was selected. Neighbor-joining (NJ) tree analyses were performed. Six clades of Chinese Boehmeria were observed in the NJ tree. Those results showed that Chinese Boehmeria be divided into six clades: sect. Boehmeria, sect. Tilocnide, sect. Siamensis, sect. Duretia, sect. Phyllostachys, and sect. Silvestri. Additionally, evolution in Chinese Boehmeria occurred in the following manner: sect. Silvestri and sect. Phyllostachys originated from sect. Duretia; sect. Duretia originated from sect. Siamensis; sect. Siamensis and sect. Tilocnide originated from sect. Boehmeria. Furthermore, evolution in sect. Tilocnide occurred by B. nivea var. Nipononivea and B. nivea var. Viridula originating from ramie, whereas ramie originating from B. nivea var. tenacissima.