Specific Length Amplified Fragment Sequencing Research Articles

The constructed high-density genetic map helps to explore the genetic regulation of erucic acid, oleic acid, and linolenic acid contents in Brassica juncea

Rapeseed mustard (Brassica juncea L.) is the third most important oilseed crop in the world but the genetic mechanism underlying its massive phenotypic variation remains largely unexplored. In this study, specific length amplified fragment sequencing (SLAF-Seq) was used to resequence a population comprising 197 F8 recombinant inbred lines (RILs), derived from a cross between vegetable-type Qichi881 and oilseed-type YufengZC in B. juncea. In total, 438,895 high-quality SLAFs were discovered, of which 47,644 were polymorphic, and 3,887 of the polymorphic markers met the requirements for genetic map construction. The final map included 3,887 markers on 18 linkage groups and was 1,830.23 cM in length, with an average distance of 0.47 cM between adjacent markers. Using the newly constructed high-density genetic map, a total of 53 QTLs for erucic acid (EA), oleic acid (OA), and linolenic acid (LNA) were detected and integrated into 8 consensus QTLs with two for each of these traits. For each of these three traits, two candidate genes were cloned and sequence analyzed, indicating colocalization with their respective consensus QTLs. The co-dominant allele-specific markers for Bju.FAD3.A03 and Bju.FAD3.B07 were developed and showed co-localization with their consensus QTL and co-segregation with LNA content, further supporting the results of QTL mapping and bioinformatic analysis. The expression level for the cloned homologous genes was also identified, which was tightly correlated with the EA, OA and LNA contents of different lines. The results would facilitate the improvement of fatty acid traits and molecular breeding of B. juncea. More use of the high-density genetic map created in this study is also discussed.

Revealing the Genetic Diversity and Population Structure of Garlic Resource Cultivars and Screening of Core Cultivars Based on Specific Length Amplified Fragment Sequencing (SLAF-Seq).

Garlic is an important vegetable and condiment that has good medical and health care effects. At present, the origin of Chinese garlic and its association with other types of quality are limited to the molecular marker level, and there are few reports at the genome level. Therefore, this study is based on the specific length amplified fragment sequencing (SLAF-seq) of 102 copies of garlic germplasm resources, the group structure, and further screening of the core germplasm. SLAF-seq of 102 garlic cultivars yielded 1949.85 Mb of clean data and 526,432,275 SNPs. Through principal component analysis, evolutionary tree, population structure, and genetic relationship analysis, all garlic cultivars were divided into 3 groups. Among them, Group 1 contains 45 Chinese cultivars and 1 Egyptian cultivar, which are distributed mainly in the coastal and central areas of China. Group 2 contains 36 Chinese cultivars and 1 U.S. cultivar, which are distributed mainly in Northwest China. Group 3 contains 19 Chinese cultivars, which are distributed mainly in Xinjiang, China. The genetic diversity results indicate that the fixation index (Fst) values of Group 1 and Group 2 are lower than those of Group 1 and Group 3 and that the diversity of nucleotides (π) of Group 3 is greater than those of Group 2 and Group 1. Finally, the 30 parts of the cultivars were used as the core germplasms, and there was no difference between the two cultivars in terms of core quality. In summary, this study provides tags for the determination of garlic molecular markers and genotypes and provides a theoretical basis for subsequent resource protection and utilization, genetic positioning of important agronomic traits, and molecular marking agglomeration breeding.

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.

Fine mapping and analysis of a candidate gene controlling Phytophthora blight resistance in cucumber.

Cucumber blight is a destructive disease. The best way to control this disease is resistance breeding. This study focuses on disease resistance gene mapping and molecular marker development. We used the resistant cucumber, JSH, and susceptible cucumber, B80, as parents to construct F2 populations. Bulked segregant analysis (BSA) combined with specific length amplified fragment sequencing (SLAF-seq) were used, from which we developed cleaved amplified polymorphic sequence (CAPs) markers to map the resistance gene. Resistance in F2 individuals showed a segregation ratio of resistance:susceptibility close to 3:1. The gene in JSH resistant cucumber was mapped to an interval of 9.25 kb, and sequencing results for the three genes in the mapped region revealed three mutations at base sites 225, 302, and 591 in the coding region of Csa5G139130 between JSH and B80, but no mutations in coding regions of Csa5G139140 and Csa5G139150. The mutations caused changes in amino acids 75 and 101 of the protein encoded by Csa5G139130, suggesting that Csa5G139130 is the most likely resistance candidate gene. We developed a molecular marker, CAPs-4, as a closely linked marker for the cucumber blight resistance gene. This is the first report on mapping of a cucumber blight resistance gene and will provideg a useful marker for molecular breeding of cucumber resistance to Phytophthora blight.

Multi-Omics Analyses Unravel Genetic Relationship of Chinese Coffee Germplasm Resources

The genetic relationships between Coffea arabica resources were analyzed via specific length amplified fragment sequencing (SLAF-seq) and transcriptome sequencing to provide the theoretical basis for breeding new varieties. Twenty C. arabica accessions were used to analyze genetic diversity on the basis of SNPs identified in SLAFs and the transcriptome data. For the SLAF-seq analysis of 20 C. arabica accessions, two Coffea canephora accessions, one Coffea liberica accession, and one Coffea racemosa accession, the number of reads ranged from 2,665,424 to 7,210,310, with a GC content of 38.49%–40.91% and a Q30 value of 94.99%–96.36%. A total of 3,347,069 SLAF tags were obtained, with an average sequencing depth of 13.90×. Moreover, the 1,048,575 SNPs identified in the polymorphic SLAFs were filtered, then the remaining 198,955 SNPs were used to construct a phylogenetic tree, perform a principal component analysis, and characterize the population structure. For the transcriptome analysis, 128.50 Gb clean reads were generated for the 20 C. arabica accessions, with a GC content of 44.36%–51.09% and a Q30 value of 94.55%–95.40%. Furthermore, 25,872 genes’ expression levels were used for the correlation analysis. The phylogenetic relationships as well as the results of the principal component analysis, population structure analysis, and correlation analysis clearly distinguished C. arabica Typica-type accessions from the C. arabica Bourbon-type accessions. Notably, several C. arabica local selections with unknown genetic backgrounds were classified according to all four clustering results.

High-Density Genetic Map and QTL Analysis in Cunninghamia lanceolate: Insights into Growth and Wood-Color Traits

Cunninghamia lanceolata is one of the most important tree species in China due to its significance both in economy and ecology. The aims of the present study were to construct a high-density genetic map and identify a quantitative trait locus (QTL) for C. lanceolata. In this study, an F1 population comprising 81 individuals was developed. Using specific length amplified fragment sequencing (SLAF-seq) technology, a total of 254,899 loci were found to be polymorphic. After linkage analysis, 2574 markers were used to construct genetic linkage maps. Specifically, 1632 markers were allocated to 11 linkage groups (LGs) for the female map, 1038 for the male map, and 2574 for the integrated map. The integrated map consisted of 4596 single-nucleotide polymorphisms (SNPs) loci, resulting in an average of 1.79 SNP loci per SLAF marker. The marker coverage was 1665.76 cM for the female map, 1436.39 cM for the male map, and 1748.40 cM for the integrated map. The average interval between two adjacent mapped markers was 1.03 cM, 1.40 cM, and 0.68 cM for the female map, male map, and integrated map, respectively. Using the integrated map, we performed interval mapping (logarithm of odds, LOD > 2.0) to detect traits of interest. We identified a total of 2, 1, 2, 5, 1, 2, 1, and 3 QTLs for diameter at breast height, heartwood diameter, heartwood proportion, heartwood a*, heartwood b*, heartwood L*, sapwood a*, and sapwood L*, respectively. The number of markers associated with each QTL ranged from 1 to 14, and each marker explained phenotypic variances ranging from 12.70% to 23.60%. Furthermore, a common QTL was identified for diameter at breast height and heartwood color a*, while another common QTL was observed for heartwood color L* and heartwood color a*. These findings suggest possible pleiotropic effects of the same genes on these traits. In conclusion, we successfully constructed high-density genetic maps for C. lanceolata using the SLAF-seq method with an F1 population. Notably, these linkage maps represent the most comprehensive and densest ones available to date for C. lanceolata and will facilitate future chromosome assignments for C. lanceolata whole-genome sequencing. These identified QTLs will serve as a valuable resource for conducting fine-scale QTL mapping and implementing marker-assisted selection in C. lanceolata, particularly for growth and wood-color traits.

A major QTL identification and candidate gene analysis of watermelon fruit cracking using QTL-seq and RNA-seq.

Fruit cracking decreases the total production and the commercial value of watermelon. The molecular mechanisms of fruit cracking are unknown. In this study, 164 recombinant inbred lines (RILs) of watermelon, derived from the crossing of the WQ1 (cracking-sensitive) and WQ2 (cracking-tolerant) lines, were sequenced using specific length amplified fragment sequencing (SLAF-seq). A high-density genetic linkage map was constructed with 3,335 markers spanning 1,322.74 cM, at an average 0.40 cM across whole-genome flanking markers. The cracking tolerance capacity (CTC), depth of fruit cracking (DFC), rind thickness (RT), and rind hardness (RH) were measured for quantitative trait locus (QTL) analysis. Of the four traits analyzed, one major QTL with high phenotypic variation (41.04%-61.37%) was detected at 76.613-76.919 cM on chromosome 2, which contained 104 annotated genes. Differential gene expression analysis with RNA sequencing (RNA-seq) data between the two parents identified 4,508 differentially expressed genes (DEGs). Comparison of the genes between the QTL region and the DEGs obtained eight coexisting genes. Quantitative real-time PCR (qRT-PCR) analysis revealed that these genes were significant differentially expressed between the two parents. These results provide new insights into the identification of QTLs or genes and marker-assisted breeding in watermelon.

Specific-Locus Amplified Fragment Sequencing (SLAF-Seq).

Specific length amplified fragment sequencing (SLAF-seq) technology is a simplified genome sequencing technology based on next-generation sequencing. SLAF-seq technology has several distinguishing characteristics: 1. Deep sequencing to ensure accuracy of genotyping; 2. Effectively reduce sequencing costs; 3. Pre-designed simplified representation scheme to optimize marker efficiency; 4. Doubled barcode system for large populations. The advantages and technical process of SLAF-seq are described briefly with summarized results for the application of SLAF-seq in development of molecular markers, construction of high-density genetic map and gene mapping in ornamental plants. Finally, the difficulties and prospects of this method are discussed in application.

Cytogenetic characterization and molecular marker development for a wheat-T. boeoticum 4Ab (4B) disomic substitution line with stripe rust resistance.

Triticum boeoticum (2n = 2x = 14, AbAb) is an important relative of wheat. This species tolerates many different types of environmental stresses, including drought, salt, and pathogenic infection, and is lower in dietary fiber and higher in antioxidants, protein (15-18%), lipids, and trace elements than common wheat. However, the gene transfer rate from this species to common wheat is low, and few species-specific molecular markers are available. In this study, the wheat-T. boeoticum substitution line Z1889, derived from a cross between the common wheat cultivar Crocus and Triticum boeoticum line G52, was identified using multicolor fluorescence in situ hybridization (mc-FISH), multicolor genomic in situ hybridization (mc-GISH) and a 55K single nucleotide polymorphism (SNP) array. Z1889 was revealed to be a 4Ab (4B) substitution line with a high degree of resistance to stripe rust pathogen strains prevalent in China. In addition, 22 4Ab chromosome-specific molecular markers and 11 T. boeoticum genome-specific molecular markers were developed from 1145 4Ab chromosome-specific fragments by comparing the sequences generated by specific-length amplified fragment sequencing (SLAF-seq), with an efficiency of up to 55.0%. Furthermore, the specificity of these markers was verified in four species containing the Ab genome. These markers can not only be used for the detection of the 4Ab chromosome but also provide a basis for molecular marker-assisted selection-based breeding in wheat.

Identification of a genomic region linked with sex determination of Undaria pinnatifida (Alariaceae) through genomic resequencing and genetic linkage analyses of a segregating gametophyte family.

Different from the traditional knowledge about kelp, three sexual phenotypes (female, male, and monoecious) exist in the haploid gametophytes of Undaria pinnatifida. However, the sex-determining mechanisms remain unknown. Genetic linkage mapping is an efficient tool to identify sex-linked regions. In the present study, we resequenced a segregating gametophyte family based on the male genome of U. pinnatifida. A high-density genetic linkage map was constructed using 9887 SNPs, with an average distance of 0.41 cM between adjacent SNPs. On the basis of this genetic map and using the composite interval mapping method, we identified 62 SNPs significantly linked with the sexual phenotype. They were located at a position of 67.67 cM on the linkage group 23, corresponding to a physical range of 14.67 Mbp on the HiC_Scaffold_23 of the genome. Reanalysis of the previous specific length amplified fragment sequencing data according to the reference genome led to the identification of a sex-linked genomic region that encompassed the above-mentioned 14.67 Mbp region. Hence, this overlapped genomic range was likely the sex-determining region. Within this region, 129 genes were retrieved and 39 of them were annotated with explicit function, including the potential male sex-determining gene-encoding high mobility group (HMG) domain protein. Relative expression analysis of the HMG gene showed that its expression was higher in male gametophytes during the vegetative phase and monoecious gametophytes during both the vegetative and gametogenesis phases, but significantly lower in male gametophytes during the gametogenesis phase. These results provide a foundation for deciphering the sex-determining mechanism of U. pinnatifida.

Fine mapping of the reduced height gene Rht22 in tetraploid wheat landrace Jianyangailanmai (Triticum turgidum L.)

Rht22 was fine mapped in the interval of 0.53-1.48Mb on 7AS, which reduces cell number of internode to cause semi-dwarfism in Jianyangailanmai. As a valuable germplasm resource for wheat genetic improvement, tetraploid wheat has several reduced height (Rht) and enhanced harvest index genes. Rht22, discovered in Jianyangailanmai (JAM, Triticum turgidum L., 2n = 4x = 28, AABB), significantly increases the spikelet number per spike, but its accurate chromosomal position is still unknown. In this study, a high-density genetic map was constructed using specific-length amplified fragment sequencing in an F7 RIL_DJ population, which was derived from a cross between dwarf Polish wheat (T. polonicum L., 2n = 4x = 28, AABB) and JAM. Two plant height loci, Qph.sicau-4B and Qph.sicau-7A, were mapped on chromosomes 4BS and 7AS, respectively. Qph.sicau-7A was mapped to the 0.33-4.46Mb interval on 7AS and likely represents the candidate region of Rht22. Fine mapping confirmed and narrowed Rht22 on chromosome arm 7AS between Xbag295.s53 and Xb295.191 in three different populations. The physical region ranged from 0.53 to 1.48Mb and included 18 candidate genes. Transcriptome analysis of two pairs of near-isogenic lines revealed that 135 differentially expressed genes (DEGs) were associated with semi-dwarfism. Of these, the expression of 83 annotated DEGs involved in hormones synthesis and signal transduction, cell wall composition, DNA replication, microtubule and phragmoplast arrays was significantly down-regulated in the semi-dwarf line. Therefore, Rht22 causes semi-dwarfism in JAM by disrupting these cellular processes, which impairs cell proliferation and reduces internode cell number.

GWAS and bulked segregant analysis reveal the Loci controlling growth habit-related traits in cultivated Peanut (Arachis hypogaea L.)

BackgroundPeanut (Arachis hypogaea L.) is a grain legume crop that originated from South America and is now grown around the world. Peanut growth habit affects the variety’s adaptability, planting patterns, mechanized harvesting, disease resistance, and yield. The objective of this study was to map the quantitative trait locus (QTL) associated with peanut growth habit-related traits by combining the genome-wide association analysis (GWAS) and bulked segregant analysis sequencing (BSA-seq) methods.ResultsGWAS was performed with 17,223 single nucleotide polymorphisms (SNPs) in 103 accessions of the U.S. mini core collection genotyped using an Affymetrix version 2.0 SNP array. With a total of 12,342 high-quality polymorphic SNPs, the 90 suggestive and significant SNPs associated with lateral branch angle (LBA), main stem height (MSH), lateral branch height (LBL), extent radius (ER), and the index of plant type (IOPT) were identified. These SNPs were distributed among 15 chromosomes. A total of 597 associated candidate genes may have important roles in biological processes, hormone signaling, growth, and development. BSA-seq coupled with specific length amplified fragment sequencing (SLAF-seq) method was used to find the association with LBA, an important trait of the peanut growth habit. A 4.08 Mb genomic region on B05 was associated with LBA. Based on the linkage disequilibrium (LD) decay distance, we narrowed down and confirmed the region within the 160 kb region (144,193,467–144,513,467) on B05. Four candidate genes in this region were involved in plant growth. The expression levels of Araip.E64SW detected by qRT-PCR showed significant difference between ‘Jihua 5’ and ‘M130’.ConclusionsIn this study, the SNP (AX-147,251,085 and AX-144,353,467) associated with LBA by GWAS was overlapped with the results in BSA-seq through combined analysis of GWAS and BSA-seq. Based on LD decay distance, the genome range related to LBA on B05 was shortened to 144,193,467–144,513,467. Three candidate genes related to F-box family proteins (Araip.E64SW, Araip.YG1LK, and Araip.JJ6RA) and one candidate gene related to PPP family proteins (Araip.YU281) may be involved in plant growth and development in this genome region. The expression analysis revealed that Araip.E64SW was involved in peanut growth habits. These candidate genes will provide molecular targets in marker-assisted selection for peanut growth habits.

Combined use of specific length amplified fragment sequencing (SLAF-seq) and bulked segregant analysis (BSA) for rapid identification of genes influencing fiber content of hemp (Cannabis sativa L.)

Hemp (Cannabis sativa L.), an ancient crop, is a significant source of high-quality fiber that primarily caters to the textile industry worldwide. Fiber content is a crucial quantitative trait for evaluating fiber yield in hemp. Understanding the genetic mechanisms involved in hemp breeding is essential for improving yield. In this study, we developed 660 F1 plants from a cross between Jindao-15 (high fiber content fiber-use variety) and Fire No.1 (low fiber content fiber-use variety), and thirty plants each with high and low fiber content were selected from 305 monoecious plants of this population according to 5%-10% of population size for quantitative traits. The DNA from these plants was extracted to establish two bulk DNA pools and then subjected to the restriction digestion by the enzymes RsaI and HaeIII to obtain 314–364 bp digestion fragments and subjected to sequencing using specific length amplified fragment sequencing (SLAF-seq). Finally, we successfully developed 368,404 SLAF tags, which led to the detection of 25,133 high-quality SNPs. Combing with the resequencing results of parents, the SNPs of mixed pools were then subjected to the SNP-Index correlation algorithm, which revealed four candidate regions related to fiber content traits on Chromosome 1, with a length of 8.68 Mb and containing 389 annotated genes. The annotation information and the comparison results identified 15 genes that were highly likely to modulate the fiber content of hemp. Further, qPCR validation identified six genes (LOC115705530, LOC115705875, LOC115704794, LOC115705371, LOC115705688 and LOC115707511) that were highly positively correlated with influencing the hemp fiber content. These genes were involved in the transcription regulation, auxin and water transportion, one carbon and sugar metabolism. And non-synnoumous mutation SNPs which may play vital role in influencing the fiber content were detected in LOC115705875, LOC115704794, LOC115705688 and LOC115707511. Thus, our study highlights the importance of the combined use of SLAF-Seq and Bulked Segregant analysis (BSA) to locate genes related to hemp fiber content rapidly. Hence, our study provides novel mechanistic inputs for the fast identification of genes related to important agronomic traits of hemp and other crops catering to the textile industry.

High-Density Single Nucleotide Polymorphisms Genetic Map Construction and Quantitative Trait Locus Mapping of Color-Related Traits of Purple Sweet Potato [Ipomoea batatas (L.) Lam.

Flesh color (FC), skin color (SC), and anthocyanin content (AC) are three important traits being used for commodity evaluation in purple-fleshed sweet potato. However, to date, only a few reports are available on the inheritance of these traits. In this study, we used a biparental mapping population of 274 F1 progeny generated from a cross between a dark purple-fleshed (Xuzishu8) and white-fleshed (Meiguohong) sweet potato variety for genetic analyses. Correlation analysis showed a significant positive correlation among AC, SC, and FC. Medium-to-high heritability was observed for these traits. We detected single nucleotide polymorphisms (SNPs) by specific length amplified fragment sequencing (SLAF-seq) with the average sequencing depth of 51.72 and 25.76 for parents and progeny, respectively. Then we constructed an integrated genetic map consisting of 15 linkage groups (LGS) of sweet potato spanning on 2,233.66 cm with an average map distance of 0.71 cm between adjacent markers. Based on the linkage map, ten major quantitative trait loci (QTLs) associated to FC, SC, and AC were identified on LG12 between 0 and 64.97 cm distance, such as one QTL for SC and FC, respectively, which explained 36.3 and 45.9% of phenotypic variation; eight QTLs for AC, which explained 10.5–28.5% of the variation. These major QTLs were highly consistent and co-localized on LG12. Positive correlation, high heritability, and co-localization of QTLs on the same LG group confirm the significance of this study to establish a marker-assisted breeding program for sweet potato improvement.

Rapid Identification of a Stripe Rust Resistance Gene YrXK in Chinese Wheat Line Xike01015 Using Specific Locus Amplified Fragment (SLAF) Sequencing.

Wheat stripe rust, an airborne fungal disease caused by Puccinia striiformis Westend. f. sp. tritici, is one of the most devastating diseases of wheat. Chinese wheat cultivar Xike01015 displays high levels of all-stage resistance (ASR) to the current predominant P. striiformis f. sp. tritici race CYR33. In this study, a single dominant gene, designated YrXk, was identified in Xike01015 conferring resistance to CYR33 with genetic analysis of F2 and BC1 populations from a cross of Mingxian169 (susceptible) and Xike01015. The specific length amplified fragment sequencing (SLAF-seq) strategy was used to construct a linkage map in the F2 population. Quantitative trait loci (QTL) analysis mapped YrXk to a 12.4-Mb segment on chromosome1 BS, explaining >86.96% of the phenotypic variance. Gene annotation in the QTL region identified three differential expressed candidate genes, TraesCS1B02G168600.1, TraesCS1B02G170200.1, and TraesCS1B02G172400.1. The qRT-PCR results showed that TraesCS1B02G172400.1 and TraesCS1B02G168600.1 are upregulated and that TraesCS1B02G170200.1 is slightly downregulated after inoculation with CYR33 in the seedling stage, which indicates that these genes may function in wheat resistance to stripe rust. The results of this study can be used in wheat breeding for improving resistance to stripe rust.

Identification of quantitative trait loci underlying five major agronomic traits of soybean in three biparental populations by specific length amplified fragment sequencing (SLAF-seq).

Flowering time, plant height, branch number, node numbers of main stem and pods per plant are important agronomic traits related to photoperiodic sensitivity, plant type and yield of soybean, which are controlled by multiple genes or quantitative trait loci (QTL). The main purpose of this study is to identify new QTL for five major agronomic traits, especially for flowering time. Three biparental populations were developed by crossing cultivars from northern and central China. Specific loci amplified fragment sequencing (SLAF-seq) was used to construct linkage map and QTL mapping was carried out. A total of 10 QTL for flowering time were identified in three populations, some of which were related to E1 and E2 genes or the other reported QTL listed in Soybase. In the Y159 population (Xudou No.9 × Kenfeng No.16), QTL for flowering time on chromosome 4, qFT4_1 and qFT4_2 were new. Compared with the QTL reported in Soybase, 1 QTL for plant height (PH), 3 QTL for branch number (BR), 5 QTL for node numbers of main stem, and 3 QTL for pods per plant were new QTL. Major E genes were frequently detected in different populations indicating that major the E loci had a great effect on flowering time and adaptation of soybean. Therefore, in order to further clone minor genes or QTL, it may be of great significance to carefully select the genotypes of known loci. These results may lay a foundation for fine mapping and clone of QTL/genes related to plant-type, provided a basis for high yield breeding of soybean.

Genome wide association study of plant height and tiller number in hulless barley

Hulless barley (Hordeum vulgare L. var. nudum), also called naked barley, is a unique variety of cultivated barley. The genome-wide specific length amplified fragment sequencing (SLAF-seq) method is a rapid deep sequencing technology that is used for the selection and identification of genetic loci or markers. In this study, we collected 300 hulless barley accessions and used the SLAF-seq method to identify candidate genes involved in plant height (PH) and tiller number (TN). We obtained a total of 1407 M paired-end reads, and 228,227 SLAF tags were developed. After filtering using an integrity threshold of >0.8 and a minor allele frequency of >0.05, 14,504,892 single-nucleotide polymorphisms (SNP) loci were screened out. The remaining SNPs were used for the construction of a neighbour-joining phylogenetic tree, and the three subcluster members showed no obvious differentiation among regional varieties. We used a genome wide association study approach to identify 1006 and 113 SNPs associated with TN and PH, respectively. Based on best linear unbiased predictors (BLUP), 41 and 29 SNPs associated with TN and PH, respectively. Thus, several of genes, including Hd3a and CKX5, may be useful candidates for the future genetic breeding of hulless barley. Taken together, our results provide insight into the molecular mechanisms controlling barley architecture, which is important for breeding and yield.

QTL analysis of flowering‐related traits by specific length amplified fragment sequencing in melon

AbstractMelon (Cucumis melo L., 2n = 24) belongs to the family Cucurbitaceae and is an important vegetable crop. This study aimed to investigate the quantitative trait loci (QTL) of flowering‐related traits to understand the genetic inheritance and candidate gene of flowering‐related traits. The specific length amplified fragment sequencing (SLAF‐seq) technology was used to construct a genetic map of 115 individual plants in the melon F2 population. A total of 82.6 Gb of data were generated using SLAF‐seq. A genetic map was constructed with 10,596 specific length amplified fragment markers in 12 linkage groups, which spanned 1,383.88 cM with an average distance of 0.13 cM between markers. Ten QTL of flowering‐related traits were detected, which explained 2.16–19.34% of the phenotypic variance. The logarithm of the odds (LOD) values ranged from 3.5 to 24.9. Ninety‐four F2:3 families were used for mapping mft and fft in candidate regions by simple sequence repeat (SSR) and cleaved amplified polymorphic sequences (CAPS) markers. The mapping results showed that mft2.1 and fft2.1 were located in the same region, between CmSSR07071 and CmSSR07102. By gene annotation, 36 candidate genes were detected in this region, 26 of which were annotated. Quantitative reverse transcription–polymerase chain reaction (PCR) showed that candidate genes, including WRKY transcription factor and AP2/ERF and B3 domain‐containing transcription factor, were differentially expressed in female and male flowers in the flower budding stages. These genes may provide the theoretical foundation for gene cloning and genetic transformation in melons.

Revealing the coexistence of differentiation and communication in an endemic hare, Lepus yarkandensis (Mammalia, Leporidae) using specific-length amplified fragment sequencing

BackgroundThe Yarkand hare (Lepus yarkandensis Günther, 1875) is endemic to oasis and desert areas around the Tarim Basin in the Xinjiang Uyghur Autonomous Region of northwest China; however, genome-wide information for this species remains limited. Moreover, the genetic variation, genetic structure, and phylogenetic relationships of Yarkand hare from the plateau mountain regions have not been reported. Thus, we used specific-length amplified fragment sequencing (SLAF-seq) technology to evaluate the genetic diversity of 76 Yarkand hares from seven geographic populations in the northern and southwestern parts of the Tarim Basin to investigate single-nucleotide polymorphism (SNP) marker-based population differentiation and evolutionary processes. Selective sweep analysis was conducted to identify genetic differences between populations.ResultsUsing SLAF-seq, a total of 1,835,504 SNPs were initially obtained, of which 308,942 high-confidence SNPs were selected for further analysis. Yarkand hares exhibited a relatively high degree of genetic diversity at the SNP level. Based on pairwise FST estimates, the north and southwest groups showed a moderate level of genetic differentiation. Phylogenetic tree and population structure analyses demonstrated evident systematic phylogeographical structure patterns consistent with the geographical distribution of the hares. Hierarchical analysis of molecular variation further indicated that genetic variation was mainly observed within populations. Low to moderate genetic differentiation also occurred among populations despite a common genomic background, likely due to geographical barriers, genetic drift, and differential selection pressure of distinct environments. Nevertheless, the observed lineage-mixing pattern, as indicated by the evolutionary tree, principal component analysis, population structure, and TreeMix analyses, suggests a certain degree of gene flow between the north and southwest groups. This may be related to the migration of hares to high-altitude water sources southwest of the basin during glacial climatic oscillations, as well as river re-diffusion and oasis restoration in the basin following the glacial period. We also identified candidate genes, and their associated gene ontology terms and pathways, related to the adaptation of Yarkand hares to different environmental habitats.ConclusionsThe identified genome-wide SNPs, genetic diversity, and population structure of Yarkand hares expand our understanding of the genetic background of this endemic species and provide valuable insights into its environmental adaptation, allowing for further exploration of the underlying mechanisms.

Identify of Fast-Growing Related Genes Especially in Height Growth by Combining QTL Analysis and Transcriptome in Salix matsudana (Koidz).

The study on the fast-growing traits of trees, mainly valued by tree height (TH) and diameter at breast height (DBH), is of great significance to promote the development of the forest industry. Quantitative trait locus (QTL) mapping based on high-density genetic maps is an efficient approach to identify genetic regions for fast-growing traits. In our study, a high-density genetic map for the F1 population was constructed. The genetic map had a total size of 5,484.07 centimorgan (cM), containing 5,956 single nucleotide polymorphisms (SNPs) based on Specific Length Amplified Fragment sequencing. Six fast-growing related stable QTL were identified on six chromosomes, and five stable QTL were identified by a principal component analysis (PCA). By combining the RNA-seq analysis for the two parents and two progenies with the qRT-PCR analysis, four candidate genes, annotated as DnaJ, 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO1), Caffeic acid 3-O-methyltransferase 1 (COMT1), and Dirigent protein 6 (DIR6), that may regulate height growth were identified. Several lignin biosynthesis-related genes that may take part in height growth were detected. In addition, 21 hotspots in this population were found. The results of this study will provide an important foundation for further studies on the molecular and genetic regulation of TH and DBH.