Functional Endophytes Regulating Plant Secondary Metabolism: Current Status, Prospects and Applications.

Endophytes, which are widely found in host plants and have no harmful effects, are a vital biological resource. Plant endophytes promote plant growth and enhance plants' resistance to diseases, pests, and environmental stresses. In addition, they enhance the synthesis of important secondary metabolites in plants and improve the potential applicability of plants in agriculture, medicine, food, and horticulture. In this review, we summarize the recent progress in understanding the interaction between endophytes and plants and summarize the construction of synthetic microbial communities (SynComs) and metaomics analysis of the interaction between endophytes and plants. The application and development prospects of endophytes in agriculture, medicine, and other industries are also discussed to provide a reference for further study of the interaction between endophytes and plants and further development and utilization of endophytes.

Development and validation of high-throughput and low-cost STARP assays for genes underpinning economically important traits in wheat.

KEY MESSAGE: We developed and validated 56 gene-specific semi-thermal asymmetric reverse PCR (STARP) markers for 46 genes of important wheat quality, biotic and abiotic stress resistance, grain yield, and adaptation-related traits for marker-assisted selection in wheat breeding. Development of high-throughput, low-cost, gene-specific molecular markers is important for marker-assisted selection in wheat breeding. In this study, we developed 56 gene-specific semi-thermal asymmetric reverse PCR (STARP) markers for wheat quality, tolerance to biotic and abiotic stresses, grain yield, and adaptation-related traits. The STARP assays were validated by (1) comparison of the assays with corresponding diagnostic STS/CAPS markers on 40 diverse wheat cultivars and (2) characterization of allelic effects based on the phenotypic and genotypic data of three segregating populations and 305 diverse wheat accessions from China and 13 other countries. The STARP assays showed the advantages of high-throughput, accuracy, flexibility, simple assay design, low operational costs, and platform compatibility. The state-of-the-art assays of this study provide a robust and reliable molecular marker toolkit for wheat breeding programs.

Genetic architecture of grain yield in bread wheat based on genome-wide association studies.

BACKGROUND: Identification of loci for grain yield (GY) and related traits, and dissection of the genetic architecture are important for yield improvement through marker-assisted selection (MAS). Two genome-wide association study (GWAS) methods were used on a diverse panel of 166 elite wheat varieties from the Yellow and Huai River Valleys Wheat Zone (YHRVWD) of China to detect stable loci and analyze relationships among GY and related traits. RESULTS: A total of 326,570 single nucleotide polymorphism (SNP) markers from the wheat 90 K and 660 K SNP arrays were chosen for GWAS of GY and related traits, generating a physical distance of 14,064.8 Mb. One hundred and twenty common loci were detected using SNP-GWAS and Haplotype-GWAS, among which two were potentially functional genes underpinning kernel weight and plant height (PH), eight were at similar locations to the quantitative trait loci (QTL) identified in recombinant inbred line (RIL) populations in a previous study, and 78 were potentially new. Twelve pleiotropic loci were detected on eight chromosomes; among these the interval 714.4-725.8 Mb on chromosome 3A was significantly associated with GY, kernel number per spike (KNS), kernel width (KW), spike dry weight (SDW), PH, uppermost internode length (UIL), and flag leaf length (FLL). GY shared five loci with thousand kernel weight (TKW) and PH, indicating significantly affected by two traits. Compared with the total number of loci for each trait in the diverse panel, the average number of alleles for increasing phenotypic values of GY, TKW, kernel length (KL), KW, and flag leaf width (FLW) were higher, whereas the numbers for PH, UIL and FLL were lower. There were significant additive effects for each trait when favorable alleles were combined. UIL and FLL can be directly used for selecting high-yielding varieties, whereas FLW can be used to select spike number per unit area (SN) and KNS. CONCLUSIONS: The loci and significant SNP markers identified in the present study can be used for pyramiding favorable alleles in developing high-yielding varieties. Our study proved that both GWAS methods and high-density genetic markers are reliable means of identifying loci for GY and related traits, and provided new insight to the genetic architecture of GY.

Genetic dissection of a major QTL for kernel weight spanning the Rht-B1 locus in bread wheat.

KEY MESSAGE: Genetic dissection uncovered a major QTL QTKW.caas-4BS corresponding with a 483 kb deletion that included genes ZnF, EamA and Rht-B1. This deletion was associated with increased grain weight and semi-dwarf phenotype. Previous studies identified quantitative trait loci (QTL) for thousand kernel weight (TKW) in the region spanning the Rht-B1 locus in wheat (Triticum aestivum L.). We recently mapped a major QTL QTKW.caas-4BS for TKW spanning the Rht-B1 locus in a recombinant inbred line (RIL) population derived from Doumai/Shi 4185 using the wheat 90K array. The allele from Doumai at QTKW.caas-4BS significantly increased TKW and kernel number per spike, and conferred semi-dwarf trait, which was beneficial to improve grain yield without a penalty to lodging. To further dissect QTKW.caas-4BS, we firstly re-investigated the genotypes and phenotypes of the RILs and confirmed the QTL using cleaved amplified polymorphic sequence (CAPS) markers developed from flanking SNP markers IWA102 and IWB54814. The target sequences of the CAPS markers were used as queries to BLAST the wheat reference genome RefSeq v1.0 and hit an approximate 10.4 Mb genomic region. Based on genomic mining and SNP loci from the wheat 660K SNP array in the above genomic region, we developed eight new markers and narrowed QTKW.caas-4BS to a genetic interval of 1.5 cM. A 483 kb deletion in Doumai corresponded with QTKW.caas-4BS genetically, including three genes ZnF, EamA and Rht-B1. The other 15 genes with either differential expressions and/or sequence variations between parents were also potential candidate genes for QTKW.caas-4BS. The findings not only provide a toolkit for marker-assisted selection of QTKW.caas-4BS but also defined candidate genes for further functional analysis.

Genome-wide variation patterns between landraces and cultivars uncover divergent selection during modern wheat breeding.

KEY MESSAGE: Genetic diversity, population structure, LD decay, and selective sweeps in 687 wheat accessions were analyzed, providing relevant guidelines to facilitate the use of the germplasm in wheat breeding. Common wheat (Triticum aestivum L.) is one of the most widely grown crops in the world. Landraces were subjected to strong human-mediated selection in developing high-yielding, good quality, and widely adapted cultivars. To investigate the genome-wide patterns of allelic variation, population structure and patterns of selective sweeps during modern wheat breeding, we tested 687 wheat accessions, including landraces (148) and cultivars (539) mainly from China and Pakistan in a wheat 90 K single nucleotide polymorphism array. Population structure analysis revealed that cultivars and landraces from China and Pakistan comprised three relatively independent genetic clusters. Cultivars displayed lower nucleotide diversity and a wider average LD decay across whole genome, indicating allelic erosion and a diversity bottleneck due to the modern breeding. Analysis of genetic differentiation between landraces and cultivars from China and Pakistan identified allelic variants subjected to selection during modern breeding. In total, 477 unique genome regions showed signatures of selection, where 109 were identified in both China and Pakistan germplasm. The majority of genomic regions were located in the B genome (225), followed by the A genome (175), and only 77 regions were located in the D genome. EigenGWAS was further used to identify key selection loci in modern wheat cultivars from China and Pakistan by comparing with global winter wheat and spring wheat diversity panels, respectively. A few known functional genes or loci found within these genome regions corresponded to known phenotypes for disease resistance, vernalization, quality, adaptability and yield-related traits. This study uncovered molecular footprints of modern wheat breeding and explained the genetic basis of polygenic adaptation in wheat. The results will be useful for understanding targets of modern wheat breeding, and in devising future breeding strategies to target beneficial alleles currently not pursued.

Genome-wide linkage mapping of yield-related traits in three Chinese bread wheat populations using high-density SNP markers.

KEY MESSAGE: We identified 21 new and stable QTL, and 11 QTL clusters for yield-related traits in three bread wheat populations using the wheat 90 K SNP assay. Identification of quantitative trait loci (QTL) for yield-related traits and closely linked molecular markers is important in order to identify gene/QTL for marker-assisted selection (MAS) in wheat breeding. The objectives of the present study were to identify QTL for yield-related traits and dissect the relationships among different traits in three wheat recombinant inbred line (RIL) populations derived from crosses Doumai × Shi 4185 (D × S), Gaocheng 8901 × Zhoumai 16 (G × Z) and Linmai 2 × Zhong 892 (L × Z). Using the available high-density linkage maps previously constructed with the wheat 90 K iSelect single nucleotide polymorphism (SNP) array, 65, 46 and 53 QTL for 12 traits were identified in the three RIL populations, respectively. Among them, 34, 23 and 27 were likely to be new QTL. Eighteen common QTL were detected across two or three populations. Eleven QTL clusters harboring multiple QTL were detected in different populations, and the interval 15.5-32.3 cM around the Rht-B1 locus on chromosome 4BS harboring 20 QTL is an important region determining grain yield (GY). Thousand-kernel weight (TKW) is significantly affected by kernel width and plant height (PH), whereas flag leaf width can be used to select lines with large kernel number per spike. Eleven candidate genes were identified, including eight cloned genes for kernel, heading date (HD) and PH-related traits as well as predicted genes for TKW, spike length and HD. The closest SNP markers of stable QTL or QTL clusters can be used for MAS in wheat breeding using kompetitive allele-specific PCR or semi-thermal asymmetric reverse PCR assays for improvement of GY.

Genome-wide association mapping of black point reaction in common wheat (Triticum aestivum L.).

BACKGROUND: Black point is a serious threat to wheat production and can be managed by host resistance. Marker-assisted selection (MAS) has the potential to accelerate genetic improvement of black point resistance in wheat breeding. We performed a genome-wide association study (GWAS) using the high-density wheat 90 K and 660 K single nucleotide polymorphism (SNP) assays to better understand the genetic basis of black point resistance and identify associated molecular markers. RESULTS: Black point reactions were evaluated in 166 elite wheat cultivars in five environments. Twenty-five unique loci were identified on chromosomes 2A, 2B, 3A, 3B (2), 3D, 4B (2), 5A (3), 5B (3), 6A, 6B, 6D, 7A (5), 7B and 7D (2), respectively, explaining phenotypic variation ranging from 7.9 to 18.0%. The highest number of loci was detected in the A genome (11), followed by the B (10) and D (4) genomes. Among these, 13 were identified in two or more environments. Seven loci coincided with known genes or quantitative trait locus (QTL), whereas the other 18 were potentially novel loci. Linear regression showed a clear dependence of black point scores on the number of favorable alleles, suggesting that QTL pyramiding will be an effective approach to increase resistance. In silico analysis of sequences of resistance-associated SNPs identified 6 genes possibly involved in oxidase, signal transduction and stress resistance as candidate genes involved in black point reaction. CONCLUSION: SNP markers significantly associated with black point resistance and accessions with a larger number of resistance alleles can be used to further enhance black point resistance in breeding. This study provides new insights into the genetic architecture of black point reaction.

Genome-wide linkage mapping of QTL for black point reaction in bread wheat (Triticum aestivum L.).

KEY MESSAGE: Nine QTL for black point resistance in wheat were identified using a RIL population derived from a Linmai 2/Zhong 892 cross and 90K SNP assay. Black point, discoloration of the embryo end of the grain, downgrades wheat grain quality leading to significant economic losses to the wheat industry. The availability of molecular markers will accelerate improvement of black point resistance in wheat breeding. The aims of this study were to identify quantitative trait loci (QTL) for black point resistance and tightly linked molecular markers, and to search for candidate genes using a high-density genetic linkage map of wheat. A recombinant inbred line (RIL) population derived from the cross Linmai 2/Zhong 892 was evaluated for black point reaction during the 2011-2012, 2012-2013 and 2013-2014 cropping seasons, providing data for seven environments. A high-density linkage map was constructed by genotyping the RILs with the wheat 90K single nucleotide polymorphism (SNP) chip. Composite interval mapping detected nine QTL on chromosomes 2AL, 2BL, 3AL, 3BL, 5AS, 6A, 7AL (2) and 7BS, designated as QBp.caas-2AL, QBp.caas-2BL, QBp.caas-3AL, QBp.caas-3BL, QBp.caas-5AS, QBp.caas-6A, QBp.caas-7AL.1, QBp.caas-7AL.2 and QBp.caas-7BS, respectively. All resistance alleles, except for QBp.caas-7AL.1 from Linmai 2, were contributed by Zhong 892. QBp.caas-3BL, QBp.caas-5AS, QBp.caas-7AL.1, QBp.caas-7AL.2 and QBp.caas-7BS probably represent new loci for black point resistance. Sequences of tightly linked SNPs were used to survey wheat and related cereal genomes identifying three candidate genes for black point resistance. The tightly linked SNP markers can be used in marker-assisted breeding in combination with the kompetitive allele specific PCR technique to improve black point resistance.

Genome-wide linkage mapping of flour color-related traits and polyphenol oxidase activity in common wheat.

KEY MESSAGE: Fifty-six QTL for flour color-related traits and polyphenol oxidase activity were identified using a genome-wide linkage mapping of data from a RIL population derived from a Gaocheng 8901/Zhoumai 16 cross. ABSTRACT: Flour color-related traits, including L*, a*, b*, yellow pigment content (YPC), and polyphenol oxidase (PPO) activity are important parameters influencing the quality of wheat end-use products. Mapping quantitative trait loci (QTL) for these traits and characterization of candidate genes are important for improving wheat quality. The aims of this study were to identify QTL for flour color-related traits and PPO activity and to characterize candidate genes using a high-density genetic linkage map in a common wheat recombinant inbred line (RIL) population derived from a cross between Gaocheng 8901 and Zhoumai 16. A linkage map was constructed by genotyping the RILs with the wheat 90 K iSelect array. Fifty-six QTL were mapped on 35 chromosome regions on homoeologous groups 1, 2, 5 and 7 chromosomes, and chromosomes 3B, 4A, 4B and 6B. Four QTL were for PPO activity, and the others were for flour color-related traits. Compared with previous studies, five QTL for a*, two for b*, one for L*, one for YPC and one for PPO activity were new. The new QTL on chromosome 2DL was involved in both a* and YPC, and another on chromosome 7DS affected both a* and L*. The scan for SNP sequences tightly linked to QTL for flour color-related traits against the wheat and/or related cereals genomes identified six candidate genes significantly related to these traits, and five of them were associated with the terpenoid backbone biosynthesis pathway. The high-density genetic linkage map of Gaocheng 8901/Zhoumai 16 represents a useful tool to identify QTL for important quality traits and candidate genes.

Development and validation of KASP assays for genes underpinning key economic traits in bread wheat.

KEY MESSAGE: We developed and validated a robust marker toolkit for high-throughput and cost-effective screening of a large number of functional genes in wheat. Functional markers (FMs) are the most valuable markers for crop breeding programs, and high-throughput genotyping for FMs could provide an excellent opportunity to effectively practice marker-assisted selection while breeding cultivars. Here we developed and validated kompetitive allele-specific PCR (KASP) assays for genes that underpin economically important traits in bread wheat including adaptability, grain yield, quality, and biotic and abiotic stress resistances. In total, 70 KASP assays either developed in this study or obtained from public databases were validated for reliability in application. The validation of KASP assays were conducted by (a) comparing the assays with available gel-based PCR markers on 23 diverse wheat accessions, (b) validation of the derived allelic information using phenotypes of a panel comprised of 300 diverse cultivars from China and 13 other countries, and (c) additional testing, where possible, of the assays in four segregating populations. All KASP assays being reported were significantly associated with the relevant phenotypes in the cultivars panel and bi-parental populations, thus revealing potential application in wheat breeding programs. The results revealed 45 times superiority of the KASP assays in speed than gel-based PCR markers. KASP has recently emerged as single-plex high-throughput genotyping technology; this is the first report on high-throughput screening of a large number of functional genes in a major crop. Such assays could greatly accelerate the characterization of crossing parents and advanced lines for marker-assisted selection and can complement the inflexible, high-density SNP arrays. Our results offer a robust and reliable molecular marker toolkit that can contribute towards maximizing genetic gains in wheat breeding programs.

Cloning of TaSST genes associated with water soluble carbohydrate content in bread wheat stems and development of a functional marker.

KEY MESSAGE: We cloned TaSST genes, developed a gene-specific marker for TaSST-D1, and identified three QTL in the Doumai/Shi 4185 RIL population. TaSST-D1 is within one of the three QTL. Sucrose:sucrose-1-fructosyltransferase (1-SST), a critical enzyme in the fructan biosynthetic pathway, is significantly and positively associated with water soluble carbohydrate (WSC) content in bread wheat stems. In the present study, wheat 1-SST genes (TaSST) were isolated and located on chromosomes 4A, 7A and 7D. Sequence analysis of TaSST-D1 revealed 15 single nucleotide polymorphisms (SNP) in the third exon between cultivars with higher and lower WSC content. A cleaved amplified polymorphism sequence (CAPS) marker, WSC7D, based on the polymorphism at position 1216 (C-G) was developed to discriminate the two alleles. WSC7D was located on chromosome 7DS using a recombinant inbred line (RIL) population from a Doumai/Shi 4185 cross, and a set of Chinese Spring nullisomic-tetrasomic lines. TaSST-D1 co-segregated with the CAPS marker WSC7D and was linked to SNP marker BS00108793_51 on chromosome 7DS at a genetic distance of 6.1 cM. It explained 8.8, 10.9, and 11.3% of the phenotypic variances in trials at Beijing and Shijiazhuang as well as the averaged data from those environments, respectively. Two additional QTL (QWSC.caas-4BS and QWSC.caas-7AS) besides TaSST-D1 were mapped in the RIL population. One hundred and forty-nine Chinese wheat cultivars and advanced lines tested in four environments were used to validate a highly significant (P < 0.01) association between WSC7D and WSC content in wheat stems. WSC7D can be used as a gene-specific marker for improvement of stem WSC content in wheat breeding programs.

Mapping quantitative trait loci for peroxidase activity and developing gene-specific markers for TaPod-A1 on wheat chromosome 3AL.

KEY MESSAGE: Three novel QTL for peroxidase activity were mapped, and gene-specific markers for TaPod-A1 were developed and validated using RILs derived from the Doumai/Shi 4185 cross and 281 wheat cultivars. TaPod-A1 is within one of the three QTL. Peroxidase (POD) activity in grain is an important factor determining the color of flour and end-use products of wheat, such as noodles and steamed bread. Mapping QTL for POD activity, characterization of POD genes and development of gene-specific markers are important for molecular marker-assisted selection in wheat breeding. Quantitative trait loci (QTL) for POD activity in common wheat were mapped using a recombinant inbred line (RIL) population derived from a Doumai/Shi 4185 cross grown in four environments and genotyped using the wheat 90 K iSelect assay. Three novel QTL for POD activity, QPod.caas-3AL, QPod.caas-4BS and QPod.caas-5AS, were identified on chromosomes 3AL, 4BS and 5AS, explaining 5.3-21.2% of phenotypic variance across environments. The full-length genomic DNA (gDNA) sequence of a POD gene, designated TaPod-A1, on chromosome 3A was characterized by homolog cloning and PCR verification. Two complementary dominant sequence-tagged site (STS) markers, POD-3A1 and POD-3A2, were developed based on single nucleotide polymorphisms (SNPs) between two alleles at the TaPod-A1 locus, amplifying 291- and 766-bp fragments in cultivars with lower and higher POD activities, respectively. The two gene-specific markers were mapped on chromosome 3AL using a set of Chinese Spring (CS) nulli-tetrasomic lines, and ditelosomic lines 3AL and 3AS. QTL analysis indicated that QPod.caas-3AL co-segregated with the gene-specific markers POD-3A1 and POD-3A2. POD-3A1 and POD-3A2 were verified on 281 wheat cultivars and advanced lines, and showed significant (P < 0.05) associations with POD activities. POD-3A1 and POD-3A2 may be useful as markers for improving color attributes in wheat breeding programs.

Biosynthetic Mechanisms of Secondary Metabolites Promoted by the Interaction Between Endophytes and Plant Hosts.

Endophytes is a kind of microorganism resource with great potential medicinal value. The interactions between endophytes and host not only promote the growth and development of each other but also drive the biosynthesis of many new medicinal active substances. In this review, we summarized recent reports related to the interactions between endophytes and hosts, mainly regarding the research progress of endophytes affecting the growth and development of host plants, physiological stress and the synthesis of new compounds. Then, we also discussed the positive effects of multiomics analysis on the interactions between endophytes and their hosts, as well as the application and development prospects of metabolites synthesized by symbiotic interactions. This review may provide a reference for the further development and utilization of endophytes and the study of their interactions with their hosts.

Dendrobine biosynthesis in Dendrobium nobile in four different habitats is affected by the variations in the endophytic fungal community.

Dendrobium nobile, an epiphytic plant, is a traditional medicinal herb with abundant endophytes. It is unclear whether the variation in the diversity and abundance of endophytes could stimulate the biosynthesis of medicinal compounds in the plant. In this study, we collected fresh stems of D. nobile from four habitats for investigating the fungal community structure, dendrobine content, and environment factors and their correlations. The results indicated no significant difference in endophytic fungal diversity among the habitats; however, different dominant or special endophytic genera were observed in the hosts from different habitats. The altitude was observed to be positively related to the dendrobine content, as the stems collected from the altitude of 692 m exhibited the highest level of dendrobine. Furthermore, the relative abundance of Toxicocladosporium was found to be positively correlated with the altitude and dendrobine content. The epiphytic matrix exhibited a significant negative correlation with the relative abundance of the endophytic fungus Gibberella but did not exhibit any significant correlation with the dendrobine content. The results indicated that the abundance of endophytes in D. nobile was affected by the altitude and epiphytic matrix and that high Toxicocladosporium abundance and high altitude were conducive to dendrobine production.

Genome-Wide Identification of Genes Related to Biosynthesis of Phenolic Acid Derivatives in Bletilla striata at Different Suspension Culture Stages.

To screen the genes regulating the biosynthesis of phenolic acid derivatives from the genome of Bletilla striata, we designed a suspension culture system to sample the cells for the following experiments. The contents of four phenolic acid derivatives were determined by high-performance liquid chromatography, and several full-length transcriptome sequencings of RNA samples at 10 time points were performed for bioinformatics analysis. The correlation analysis was used to identify and verify the key DEGs involved in the biosynthesis of the four phenolic acid derivatives. The results showed that the contents of p-hydroxybenzylalcohol (HBA), Dactylorhin A, Militarine, and Coelonin peaked at 33 days postinoculation (Dpi), 18 Dpi, 39 Dpi, and 39 Dpi of the culture system, respectively. Based on transcriptome data, 80 DEGs involved in the biosynthesis of phenolic acid derivatives were obtained. The KEGG pathway enrichment analysis classified them mostly into five metabolic pathways: phenylpropane biosynthesis, starch and sucrose metabolic, cyanoamino acid metabolism, gluconeogenesis and glycolysis, and phenylalanine metabolism. qPCR analysis revealed that the relative gene expression levels were consistent with the overall trend of transcriptome sequencing results. Among them, 14, 18, 23, and 41 unigenes were found to be involved in the synthesis of HBA, Dactylorhin A, Coelonin, and Militarine, respectively. These unigenes laid a solid foundation for elucidating the biosynthesis mechanism of phenolic acid derivatives in suspension cells of B. striata.

Construction of Consensus Genetic Map With Applications in Gene Mapping of Wheat (Triticum aestivum L.) Using 90K SNP Array.

Wheat is one of the most important cereal crops worldwide. A consensus map combines genetic information from multiple populations, providing an effective alternative to improve the genome coverage and marker density. In this study, we constructed a consensus map from three populations of recombinant inbred lines (RILs) of wheat using a 90K single nucleotide polymorphism (SNP) array. Phenotypic data on plant height (PH), spike length (SL), and thousand-kernel weight (TKW) was collected in six, four, and four environments in the three populations, and then used for quantitative trait locus (QTL) mapping. The mapping results obtained using the constructed consensus map were compared with previous results obtained using individual maps and previous studies on other populations. A simulation experiment was also conducted to assess the performance of QTL mapping with the consensus map. The constructed consensus map from the three populations spanned 4558.55 cM in length, with 25,667 SNPs, having high collinearity with physical map and individual maps. Based on the consensus map, 21, 27, and 19 stable QTLs were identified for PH, SL, and TKW, much more than those detected with individual maps. Four PH QTLs and six SL QTLs were likely to be novel. A putative gene called TraesCS4D02G076400 encoding gibberellin-regulated protein was identified to be the candidate gene for one major PH QTL located on 4DS, which may enrich genetic resources in wheat semi-dwarfing breeding. The simulation results indicated that the length of the confidence interval and standard errors of the QTLs detected using the consensus map were much smaller than those detected using individual maps. The consensus map constructed in this study provides the underlying genetic information for systematic mapping, comparison, and clustering of QTL, and gene discovery in wheat genetic study. The QTLs detected in this study had stable effects across environments and can be used to improve the wide adaptation of wheat cultivars through marker-assisted breeding.

Full Transcriptome Analysis of Callus Suspension Culture System of Bletilla striata.

BACKGROUND: Bletilla striata has been widely used in the pharmacology industry. To effectively produce the secondary metabolites through suspension cultured cells of B. striata, it is important to exploring the full-length transcriptome data and the genes related to cell growth and chemical producing of all culture stages. We applied a combination of Real-Time Sequencing of Single Molecule (SMRT) and second-generation sequencing (SGS) to generate the complete and full-length transcriptome of B. striata suspension cultured cells. METHODS: The B. striata transcriptome was formed in de novo way by using PacBio isoform sequencing (Iso-Seq) on a pooled RNA sample derived from 23 samples of 10 culture stages, to explore the potential for capturing full-length transcript isoforms. All unigenes were obtained after splicing, assembling, and clustering, and corrected by the SGS results. The obtained unigenes were compared with the databases, and the functions were annotated and classified. RESULTS AND CONCLUSIONS: A total of 100,276 high-quality full-length transcripts were obtained, with an average length of 2530 bp and an N50 of 3302 bp. About 52% of total sequences were annotated against the Gene Ontology, 53,316 unigenes were hit by KOG annotations and divided into 26 functional categories, 80,020 unigenes were mapped by KEGG annotations and clustered into 363 pathways. Furthermore, 15,133 long-chain non-coding RNAs (lncRNAs) were detected. And 68,996 coding sequences were identified based on SSR analysis, among which 31 pairs of primers selected at random were amplified and obtained stable bands. In conclusion, our results provide new full-length transcriptome data and genetic resources for identifying growth and metabolism-related genes, which provide a solid foundation for further research on its growth regulation mechanisms and genetic engineering breeding mechanisms of B. striata.

Identification and bioinformatic analysis of Aux/IAA family based on transcriptome data of Bletilla striata.

Auxin/Indole-3-Acetic Acid (Aux/IAA) genes are involved in auxin signaling pathway and play an important role in plant growth and development. However, many studies focus on Aux/IAA gene families and much less known in Bletilla striata. In this study, a total of 27 Aux/IAA genes (BsIAA1-27) were cloned from the transcriptome of Bletilla striata. Based on a phylogenetic analysis of the Aux/IAA protein sequences from B. striata, Arabidopsis thaliana and Dendrobium officinale, the Aux/IAA genes of B. striata (BsIAAs) were categorized into 2 subfamilies and 9 groups. While BsIAAs were more closer to those of D. officinale compared to A. thaliana. EST-SSR marker mining test showed that 4 markers could be stably amplified with obvious polymorphisms among 4 landraces. Our results suggested that BsIAAs were involved in the process of tuber development and provided insights into functional roles of Aux/IAA genes in B. striata and other plants.

A Genome-Wide Association Study Reveals a Rich Genetic Architecture of Flour Color-Related Traits in Bread Wheat.

Flour color-related traits, including brightness (L*), redness (a*), yellowness (b*) and yellow pigment content (YPC), are very important for end-use quality of wheat. Uncovering the genetic architecture of these traits is necessary for improving wheat quality by marker-assisted selection (MAS). In the present study, a genome-wide association study (GWAS) was performed on a collection of 166 bread wheat cultivars to better understand the genetic architecture of flour color-related traits using the wheat 90 and 660 K SNP arrays, and 10 allele-specific markers for known genes influencing these traits. Fifteen, 28, 25, and 32 marker-trait associations (MTAs) for L*, a*, b*, and YPC, respectively, were detected, explaining 6.5-20.9% phenotypic variation. Seventy-eight loci were consistent across all four environments. Compared with previous studies, Psy-A1, Psy-B1, Pinb-D1, and the 1B•1R translocation controlling flour color-related traits were confirmed, and four loci were novel. Two and 11 loci explained much more phenotypic variation of a* and YPC than phytoene synthase 1 gene (Psy1), respectively. Sixteen candidate genes were predicted based on biochemical information and bioinformatics analyses, mainly related to carotenoid biosynthesis and degradation, terpenoid backbone biosynthesis and glycolysis/gluconeogenesis. The results largely enrich our knowledge of the genetic basis of flour color-related traits in bread wheat and provide valuable markers for wheat quality improvement. The study also indicated that GWAS was a powerful strategy for dissecting flour color-related traits and identifying candidate genes based on diverse genotypes and high-throughput SNP arrays.

A High-Density Consensus Map of Common Wheat Integrating Four Mapping Populations Scanned by the 90K SNP Array.

A high-density consensus map is a powerful tool for gene mapping, cloning and molecular marker-assisted selection in wheat breeding. The objective of this study was to construct a high-density, single nucleotide polymorphism (SNP)-based consensus map of common wheat (Triticum aestivum L.) by integrating genetic maps from four recombinant inbred line populations. The populations were each genotyped using the wheat 90K Infinium iSelect SNP assay. A total of 29,692 SNP markers were mapped on 21 linkage groups corresponding to 21 hexaploid wheat chromosomes, covering 2,906.86 cM, with an overall marker density of 10.21 markers/cM. Compared with the previous maps based on the wheat 90K SNP chip detected 22,736 (76.6%) of the SNPs with consistent chromosomal locations, whereas 1,974 (6.7%) showed different chromosomal locations, and 4,982 (16.8%) were newly mapped. Alignment of the present consensus map and the wheat expressed sequence tags (ESTs) Chromosome Bin Map enabled assignment of 1,221 SNP markers to specific chromosome bins and 819 ESTs were integrated into the consensus map. The marker orders of the consensus map were validated based on physical positions on the wheat genome with Spearman rank correlation coefficients ranging from 0.69 (4D) to 0.97 (1A, 4B, 5B, and 6A), and were also confirmed by comparison with genetic position on the previously 40K SNP consensus map with Spearman rank correlation coefficients ranging from 0.84 (6D) to 0.99 (6A). Chromosomal rearrangements reported previously were confirmed in the present consensus map and new putative rearrangements were identified. In addition, an integrated consensus map was developed through the combination of five published maps with ours, containing 52,607 molecular markers. The consensus map described here provided a high-density SNP marker map and a reliable order of SNPs, representing a step forward in mapping and validation of chromosomal locations of SNPs on the wheat 90K array. Moreover, it can be used as a reference for quantitative trait loci (QTL) mapping to facilitate exploitation of genes and QTL in wheat breeding.