Bulked Segregant RNA Sequencing Research Articles

QTL mapping and BSR-seq revealed loci and candidate genes associated with the sporadic multifoliolate phenotype in soybean (Glycine max)

Key messageThe QTLs and candidate genes governing the multifoliolate phenotype were identified by combining linkage mapping with BSR-seq, revealing a possible interplay between genetics and the environment in soybean leaf development.Soybean, as a legume, is typified by trifoliolate leaves. Although multifoliolate leaves (compound leaves with more than three leaflets each) have been reported in soybean, including sporadic appearances in the first compound leaves in a recombinant inbred line (RIL) population from a cross between cultivated soybean C08 and wild soybean W05 from this study, the genetic basis of this phenomenon is still unclear. Here, we integrated quantitative trait locus (QTL) mapping with bulked segregant RNA sequencing (BSR-seq) to identify the genetic loci associated with the multifoliolate phenotype in soybean. Using linkage mapping, ten QTLs related to the multifoliolate trait were identified. Among these, a significant and major QTL, qMF-2-1 on chromosome 2 and consistently detected across biological replicates, explained more than 10% of the phenotypic variation. Together with BSR-seq analyses, which analyzed the RILs with the highest multifoliolate frequencies and those with the lowest frequencies as two distinct bulks, two candidate genes were identified: Glyma.06G204300 encoding the transcription factor TCP5, and Glyma.06G204400 encoding LONGIFOLIA 2 (LNG2). Transcriptome analyses revealed that stress-responsive genes were significantly differentially expressed between high-multifoliolate occurrence lines and low occurrence ones, indicating environmental factors probably influence the appearance of multifoliolate leaves in soybean through stress-responsive genes. Hence, this study offers new insights into the genetic mechanism behind the multifoliolate phenotype in soybean.

Identification of Rapeseed (Brassica napus L.) Plant Height-Associated QTL Using BSA-seq and RNA-seq.

Plant height (PH) is a critical agronomic trait in Brassica napus, significantly impacting yield. Consequently, identifying genes associated with plant height is a pivotal objective in oilseed rape breeding. This study employed a combination of bulk segregant analysis sequencing (BSA-seq) and RNA sequencing (RNA-seq) for analysis. A novel quantitative trait locus (QTL), qPH_C02, was identified between 63,989,634 and 64,945,122 bp on chromosome C02, from which eight candidate genes were screened. The Gene Ontology (GO) analysis revealed enrichment in peroxisomes, while the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated enrichment in the oxidative phosphorylation (OP) pathway. It is hypothesized that the observed differences in plant height and silique length may be attributed to the regulation of peroxidase activity in the OP pathway, which in turn alters plant energy metabolism and controls nutrient uptake. Subsequently, we will further test this hypothesis. The results of this study will contribute to our understanding of the genetic basis for differences in plant height and provide a foundation for the selection and breeding of Brassica napus varieties with desired plant shapes.

Genetic mapping and molecular marker development for white flesh color in tomato.

Fruit color significantly influences the quality of horticultural crops, which affects phytochemical diversity and consumer preferences. Despite its importance, the genetic basis of the white-colored fruit in tomatoes remains poorly understood. In this study, we demonstrate that white-fleshed tomato varieties accumulate fewer carotenoids than yellow-fleshed varieties. We developed various segregating populations by hybridizing red, yellow, and white fruit tomato cultivars. Genetic analysis revealed that the white fruit color trait is controlled by a single gene that dominates both red and yellow fruits. Bulk segregant RNA sequencing provided a preliminary map of a 3.17 Mb region on chromosome 3 associated with the white color trait. Based on kompetitive allele-specific PCR (KASP) markers, we narrowed the candidate gene region to 819 kb. Within this region, we identified a 4906-bp sequence absence variation near Phytoene Synthase 1 (SlPSY1) specific to white-colored tomatoes. Genotyping of the progeny and natural populations using a single nucleotide polymorphism adjacent to this absence of variation confirmed its key role in white fruit formation. Collectively, our findings provide insights into white fruit trait formation in tomatoes, enabling tomato breeders to precisely introduce white fruit traits for commercial exploitation.

Characterization of a Powdery Mildew Resistance Gene in Wheat Breeding Line Jingzi 102 Using Bulk Segregant RNA Sequencing.

Wheat (Triticum aestivum L.) is one of the most important crops worldwide. Powdery mildew caused by Blumeria graminis f. sp. tritici is a destructive disease threatening wheat yield and quality. The utilization of resistant genes and cultivars is considered the most economical, environmentally friendly, and effective method to control powdery mildew. Wheat breeding line Jingzi 102 was highly resistant to powdery mildew at both seedling and adult plant stages. Genetic analysis of F1, F2, and F2:3 populations of "Jingzi 102 × Shixin 828" showed that the resistance of Jingzi 102 against powdery mildew isolate E09 at the seedling stage was controlled by a single dominant gene, temporarily designated PmJZ. Using bulked segregant RNA sequencing combined with molecular markers analysis, PmJZ was located on the long arm of chromosome 2B and flanked by markers BJK695-1 and CIT02g-20 with the genetic distances of 1.2 and 0.5 centimorgan, respectively, corresponding to the bread wheat genome of Chinese Spring (International Wheat Genome Sequencing Consortium RefSeq v2.1) 703.8 to 707.6 Mb. PmJZ is most likely different from the documented Pm genes on chromosome 2BL based on their physical positions, molecular markers analysis, and resistance spectrum. Based on the gene annotation information, five genes related to disease resistance could be considered as the candidate genes of PmJZ. To accelerate the application of PmJZ, the flanking markers BJK695-1 and CIT02g-20 can serve for marker-assisted selection of PmJZ in wheat disease-resistance breeding.

Identification and functional marker development of SbPLSH1 conferring purple leaf sheath in sorghum.

We identified a SbPLSH1gene conferring purple leaf sheath in sorghum (sorghumbicolor(L.) Moench)and developed a functional markerfor it. The purple leaf sheath of sorghum, a trait mostly related to anthocyanin deposition, is a visually distinguishable morphological marker widely used to evaluate the purity of crop hybrids. We aimed to dissect the genetic mechanism for leaf sheath color to mine the genes regulating this trait. In this study, two F2 populations were constructed by crossing a purple leaf sheath inbred line (Gaoliangzhe) with two green leaf sheath inbred lines (BTx623 and Silimei). Based on the results of bulked-segregant analysis sequencing, bulk-segregant RNA sequencing, and map-based cloning, SbPLSH1 (Sobic.006G175700), which encodes a bHLH transcription factor on chromosome 6, was identified as the candidate gene for purple leaf sheath in sorghum. Genetic analysis demonstrated that overexpression of SbPLSH1 in Arabidopsis resulted in anthocyanin deposition and purple petiole, while two single-nucleotide polymorphism (SNP) variants on the exon 6 resulted in loss of function. Further haplotype analysis revealed that there were two missense mutations and one cis-acting element mutation in SbPLSH1, which are closely associated with leaf sheath color in sorghum. Based on the variations, a functional marker (LSC4-2) for marker-assisted selection was developed, which has a broad-spectrum capability of distinguishing leaf sheath color in natural variants. In summary, this study lays a foundation for analyzing the genetic mechanism for sorghum leaf sheath color.

PE (Prickly Eggplant) encoding a cytokinin-activating enzyme responsible for the formation of prickles in eggplant.

Eggplant is one of the most important vegetables worldwide, with some varieties displaying prickles. These prickles, present on the leaves, stems, and fruit calyxes, posing challenges during cultivation, harvesting, and transportation, making them an undesirable agronomic trait. However, the genetic mechanisms underlying prickle morphogenesis in eggplant remain poorly understood, impeding genetic improvements. In this study, genetic analyses revealed that prickle morphogenesis is governed by a single dominant nuclear gene, termed PE (Prickly Eggplant). Subsequent bulk segregant RNA-sequencing (BSR-seq) and linkage analysis preliminarily mapped PE to chromosome 6. This locus was then fine mapped to a 9233 bp interval in a segregating population of 1109 plants, harboring only one candidate gene, SmLOG1, which encodes a LONELY GUY (LOG)-family cytokinin biosynthetic enzyme. Expression analyses via transcriptome and qRT-PCR demonstrate that SmLOG1 is predominantly expressed in immature prickles. CRISPR-Cas9 knockout experiments targeting SmLOG1 in prickly parental line 'PI 381159' abolished prickles across all tissues, confirming its critical role in prickle morphogenesis. Sequence analysis of SmLOG1 pinpointed variations solely within the non-coding region. We developed a cleaved amplified polymorphic sequences (CAPS) marker from a distinct SNP located at -735-bp within the SmLOG1 promoter, finding significant association with prickle variation in 190 eggplant germplasms. These findings enhance our understanding of the molecular mechanisms governing prickle development in eggplant and facilitate the use of marker-assisted selection (MAS) for breeding prickleless cultivars.

RNA-Seq Bulked Segregant Analysis of an Exotic B. napus ssp. napobrassica (Rutabaga) F2 Population Reveals Novel QTLs for Breeding Clubroot-Resistant Canola.

In this study, a rutabaga (Brassica napus ssp. napobrassica) donor parent FGRA106, which exhibited broad-spectrum resistance to 17 isolates representing 16 pathotypes of Plasmodiophora brassicae, was used in genetic crosses with the susceptible spring-type canola (B. napus ssp. napus) accession FG769. The F2 plants derived from a clubroot-resistant F1 plant were screened against three P. brassicae isolates representing pathotypes 3A, 3D, and 3H. Chi-square (χ2) goodness-of-fit tests indicated that the F2 plants inherited two major clubroot resistance genes from the CR donor FGRA106. The total RNA from plants resistant (R) and susceptible (S) to each pathotype were pooled and subjected to bulked segregant RNA-sequencing (BSR-Seq). The analysis of gene expression profiles identified 431, 67, and 98 differentially expressed genes (DEGs) between the R and S bulks. The variant calling method indicated a total of 12 (7 major + 5 minor) QTLs across seven chromosomes. The seven major QTLs included: BnaA5P3A.CRX1.1, BnaC1P3H.CRX1.2, and BnaC7P3A.CRX1.1 on chromosomes A05, C01, and C07, respectively; and BnaA8P3D.CRX1.1, BnaA8P3D.RCr91.2/BnaA8P3H.RCr91.2, BnaA8P3H.Crr11.3/BnaA8P3D.Crr11.3, and BnaA8P3D.qBrCR381.4 on chromosome A08. A total of 16 of the DEGs were located in the major QTL regions, 13 of which were on chromosome C07. The molecular data suggested that clubroot resistance in FGRA106 may be controlled by major and minor genes on both the A and C genomes, which are deployed in different combinations to confer resistance to the different isolates. This study provides valuable germplasm for the breeding of clubroot-resistant B. napus cultivars in Western Canada.

Map-based cloning and characterization reveal that an R2R3 MYB gene confers red glume in wheat

Pigment accumulation is an important trait related to wheat domestication, but there remains a limited understanding of its molecular mechanism. The genetic control of the red glume trait by a dominant allele, Rg-B1, on 1BS was reported in the last century, but the underlying gene and its molecular basis remained elusive. Here, we identified TraesTSP1B01G005700 (G57) encoding an R2R3-MYB transcription factor (TF) as the candidate Rg-B1 gene controlling red glume color by a combination of genome-wide association study (GWAS), bulked segregant RNA-sequencing (BSR-Seq), map-based cloning, and RNA-seq. TheRg-B1locus had zero to five duplicate copies only one of whichhadhightranscriptional activity. Genetic evidence suggested that promoter sequence variation in G57 in the glumes leads to high expression of G57, resulting in the red glume phenotype. G57 could bind to the promoters of anthocyanin synthesis genes TaCHS, TaF3ʹH, and TaUFGT, activating their expression and contributing to anthocyanin accumulation in wheat glume. G57 also played a pivotal role in up-regulating expression of genes TaDREB1C and TaFLO2 associated with increased grain weight, thereby causing increased grain weight. Our research offers a better understanding of the molecular basis of red glume in bread wheat.

Identification of Yellow Seed Color Genes Using Bulked Segregant RNA Sequencing in Brassica juncea L.

Yellow seed breeding is an effective method to improve oil yield and quality in rapeseed (Brassica napus L.). However, naturally occurring yellow-seeded genotypes have not been identified in B. napus. Mustard (Brassica juncea L.) has some natural, yellow-seeded germplasms, yet the molecular mechanism underlying this trait remains unclear. In this study, a BC9 population derived from the cross of yellow seed mustard "Wuqi" and brown seed mustard "Wugong" was used to analyze the candidate genes controlling the yellow seed color of B. juncea. Subsequently, yellow-seeded (BY) and brown-seeded (BB) bulks were constructed in the BC9 population and subjected to bulked segregant RNA sequencing (BSR-Seq). A total of 511 differentially expressed genes (DEGs) were identified between the brown and yellow seed bulks. Enrichment analysis revealed that these DEGs were involved in the phenylpropanoid biosynthetic process and flavonoid biosynthetic process, including key genes such as 4CL, C4H, LDOX/TT18, PAL1, PAL2, PAL4, TT10, TT12, TT4, TT8, BAN, DFR/TT3, F3H/TT6, TT19, and CHI/TT5. In addition, 111,540 credible single-nucleotide polymorphisms (SNPs) and 86,319 INDELs were obtained and used for quantitative trait locus (QTL) identification. Subsequently, two significant QTLs on chromosome A09, namely, qSCA09-3 and qSCA09-7, were identified by G' analysis, and five DEGs (BjuA09PAL2, BjuA09TT5, BjuA09TT6, BjuA09TT4, BjuA09TT3) involved in the flavonoid pathway were identified as hub genes based on the protein-to-protein network. Among these five genes, only BjuA09PAL2 and BjuA09F3H had SNPs between BY and BB bulks. Interestingly, the majority of SNPs in BjuA09PAL2 were consistent with the SNPs identified between the high-quality assembled B. juncea reference genome "T84-66" (brown-seed) and "AU213" (yellow-seed). Therefore, BjuA09PAL2, which encodes phenylalanine lyase, was considered as the candidate gene associated with yellow seed color of B. juncea. The identification of a novel gene associated with the yellow seed coloration of B. juncea through this study may play a significant role in enhancing yellow seed breeding in rapeseed.

Integrated BSA-seq and RNA-seq analysis to identify candidate genes associated with nitrogen utilization efficiency (NUtE) in rapeseed (Brassica napus L.)

Rapeseed (Brassica napus L.) is one of the important oil crops, with a high demand for nitrogen (N). It is essential to explore the potential of rapeseed to improve nitrogen utilization efficiency (NUtE). Rapeseed is an allotetraploid crop with a relatively large and complex genome, and there are few studies on the mapping of genes related to NUtE regulation. In this study, we used the combination of bulk segregant analysis sequencing (BSA-Seq) and RNA sequencing (RNA-Seq) to analyze the N-efficient genotype ‘Zheyou 18’ and N-inefficient genotype ‘Sollux’, to identify the genetic regulatory mechanisms. Several candidate genes were screened, such as the high-affinity nitrate transporter gene NRT2.1 (BnaC08g43370D) and the abscisic acid (ABA) signal transduction-related genes (BnaC02g14540D, BnaA03g20760D, and BnaA05g01330D). BnaA05g01330D was annotated as ABA-INDUCIBLE bHLH-TYPE TRANSCRIPTION FACTOR (AIB/bHLH17), which was highly expressed in the root. The results showed that the primary root length of the ataib mutant was significantly longer than that of the wild type under low N conditions. Overexpression of BnaA5.AIB could reduce the NUtE under low N levels in Arabidopsis (Arabidopsis thaliana). Candidate genes identified in this study may be involved in the regulation of NUtE in rapeseed, and new functions of AIB in orchestrating N uptake and utilization have been revealed. It is indicated that BnaA5.AIB may be the key factor that links ABA to N signaling and a negative regulator of NUtE. It will provide a theoretical basis and application prospect for resource conservation, environmental protection, and sustainable agricultural development.

Genetic analysis of stripe rust resistance in the common wheat line Kfa/2*Kachu under a Chinese rust environment.

We mapped a new race-specific seedling stripe rust resistance gene on wheat chromosome 5BL and a new APR locus QYr.hazu-2BS from CIMMYT wheat line Kfa/2*Kachu. Breeding resistant wheat (Triticum aestivum) varieties is the most economical and efficient way to manage wheat stripe rust, but requires the prior identification of new resistance genes and development of associated molecular markers for marker-assisted selection. To map stripe rust resistance loci in wheat, we used a recombinant inbred line population generated by crossing the stripe rust-resistant parent 'Kfa/2*Kachu' and the susceptible parent 'Apav#1'. We employed genotyping-by-sequencing and bulked segregant RNA sequencing to map a new race-specific seedling stripe rust resistance gene, which we designated YrK, to wheat chromosome arm 5BL. TraesCS5B02G330700 encodes a receptor-like kinase and is a high-confidence candidate gene for YrK based on virus-induced gene silencing results and the significant induction of its expression 24h after inoculation with wheat stripe rust. To assist breeding, we developed functional molecular markers based on the polymorphic single nucleotide polymorphisms in the coding sequence region of YrK. We also mapped four adult plant resistance (APR) loci to wheat chromosome arms 1BL, 2AS, 2BS and 4AL. Among these APR loci, we determined that QYr.hazu-1BL and QYr.hazu-2AS are allelic to the known pleiotropic resistance gene Lr46/Yr29/Pm39 and the race-specific gene Yr17, respectively. However, QYr.hazu-2BS is likely a new APR locus, for which we converted closely linked SNP polymorphisms into breeder-friendly Kompetitive allele-specific PCR (KASP) markers. In the present study, we provided new stripe rust resistance locus/gene and molecular markers for wheat breeder to develop rust-resistant wheat variety.

Isolation and characterization of the gene HvFAR1 encoding acyl-CoA reductase from the cer-za.227 mutant of barley (Hordeum vulgare) and analysis of the cuticular barrier functions.

The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley (Hordeum vulgare). The barley eceriferum mutants cer-za.227 and cer-ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. Cuticular waxes and permeabilities were measured in cer-za.227 and cer-ye.267. The mutant loci were isolated by bulked segregant RNA sequencing. New cer-za alleles were generated by genome editing. The CER-ZA protein was characterized after expression in yeast and Arabidopsis cer4-3. Cer-za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl-CoA reductase (FAR1). The cer-ye.267 mutation is located to HORVU4Hr1G063420 encoding β-ketoacyl-CoA synthase (KAS1) and is allelic to cer-zh.54. The amounts of intracuticular waxes were strongly decreased in cer-ye.267. The cuticular water loss and permeability of cer-za.227 were similar to wild-type (WT), but were increased in cer-ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. The differential decrease in intracuticular waxes between cer-za.227 and cer-ye.267, and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.

Identification of candidate gene for the defective kernel phenotype using bulked segregant RNA and exome capture sequencing methods in wheat.

Wheat is a significant source of protein and starch worldwide. The defective kernel (Dek) mutant AK-3537, displaying a large hollow area in the endosperm and shrunken grain, was obtained through ethyl methane sulfonate (EMS) treatment of the wheat cultivar Aikang 58 (AK58). The mode of inheritance of the AK-3537 grain Dek phenotype was determined to be recessive with a specific statistical significance level. We used bulked segregant RNA-seq (BSR-seq), BSA-based exome capture sequencing (BSE-seq), and the ΔSNP-index algorithm to identify candidate regions for the grain Dek phenotype. Two major candidate regions, DCR1 (Dek candidate region 1) and DCR2, were identified on chromosome 7A between 279.98 and 287.93 Mb and 565.34 and 568.59 Mb, respectively. Based on transcriptome analysis and previous reports, we designed KASP genotyping assays based on SNP variations in the candidate regions and speculated that the candidate gene is TraesCS7A03G0625900 (HMGS-7A), which encodes a 3-hydroxy-3-methylglutaryl-CoA synthase. One SNP variation located at position 1,049 in the coding sequence (G>A) causes an amino acid change from Gly to Asp. The research suggests that functional changes in HMGS-7A may affect the expression of key enzyme genes involved in wheat starch syntheses, such as GBSSII and SSIIIa.

A cell wall invertase modulates resistance to fusarium crown rot and sharp eyespot in common wheat.

Fusarium crown rot (FCR) and sharp eyespot (SE) are serious soil-borne diseases in wheat and its relatives that have been reported to cause wheat yield losses in many areas. In this study, the expression of a cell wall invertase gene, TaCWI-B1, was identified to be associated with FCR resistance through a combination of bulk segregant RNA sequencing and genome resequencing in a recombinant inbred line population. Two bi-parental populations were developed to further verify TaCWI-B1 association with FCR resistance. Overexpression lines and ethyl-methanesulfonate (EMS) mutants revealed TaCWI-B1 positively regulating FCR resistance. Determination of cell wall thickness and components showed that the TaCWI-B1-overexpression lines exhibited considerably increased thickness and pectin and cellulose contents. Furthermore, we found that TaCWI-B1 directly interacted with an alpha-galactosidase (TaGAL). EMS mutants showed that TaGAL negatively modulated FCR resistance. The expression of TaGAL is negatively correlated with TaCWI-B1 levels, thus may reduce mannan degradation in the cell wall, consequently leading to thickening of the cell wall. Additionally, TaCWI-B1-overexpression lines and TaGAL mutants showed higher resistance to SE; however, TaCWI-B1 mutants were more susceptible to SE than controls. This study provides insights into a FCR and SE resistance gene to combat soil-borne diseases in common wheat. Short Summary: A cell wall invertase gene TaCWI was cloned and associated with resistance against Fusarium crown rot and sharp eyespot in common wheat, and an underlying molecular mechanism was proposed, in which TaCWI conferring thickened cell wall counteracts the effects of TaGAL, an alpha-galactosidase gene associated with susceptibility to the two diseases. This article is protected by copyright. All rights reserved.

BrCWM Mutation Disrupted Leaf Flattening in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Leaf flattening plays a vital role in the establishment of plant architecture, which is closely related to plant photosynthesis and, thus, influences the product yield and quality of Chinese cabbage. In this study, we used the doubled haploid line 'FT' of Chinese cabbage as the wild type for ethyl methanesulfonate (EMS) mutagenesis and obtained a mutant cwm with stably inherited compact and wrinkled leaves. Genetic analysis revealed that the mutated trait was controlled by a single recessive nuclear gene, Brcwm. Brcwm was preliminarily mapped to chromosome A07 based on bulked segregant RNA sequencing (BSR-seq) and fine-mapped to a 205.66 kb region containing 39 genes between Indel12 and Indel21 using SSR and Indel analysis. According to the whole-genome re-sequencing results, we found that there was only one nonsynonymous single nucleotide polymorphism (SNP) (C to T) within the target interval on exon 4 of BraA07g021970.3C, which resulted in a proline to serine amino acid substitution. The mutated trait co-segregated with the SNP. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed that BraA07g021970.3C expression was dramatically higher in 'FT' leaves than that in cwm leaves. BraA07g021970.3C is homologous to AT3G55000 encoding a protein related to cortical microtubule organization. A similar phenotype of dwarfism and wrinkled leaves was observed in the recessive homozygous mutant cwm-f1 of AT3G55000, and its T3 transgenic lines were restored to the Arabidopsis wild-type phenotype through ectopic overexpression of BraA07g021970.3C. These results verified that BraA07g021970.3C was the target gene essential for leaf flattening in Chinese cabbage.

TaTPP-7A positively feedback regulates grain filling and wheat grain yield through T6P-SnRK1 signalling pathway and sugar-ABA interaction.

Grain size and filling are two key determinants of grain thousand-kernel weight (TKW) and crop yield, therefore they have undergone strong selection since cereal was domesticated. Genetic dissection of the two traits will improve yield potential in crops. A quantitative trait locus significantly associated with wheat grain TKW was detected on chromosome 7AS flanked by a simple sequence repeat marker of Wmc17 in Chinese wheat 262 mini-core collection by genome-wide association study. Combined with the bulked segregant RNA-sequencing (BSR-seq) analysis of an F2 genetic segregation population with extremely different TKW traits, a candidate trehalose-6-phosphate phosphatase gene located at 135.0 Mb (CS V1.0), designated as TaTPP-7A, was identified. This gene was specifically expressed in developing grains and strongly influenced grain filling and size. Overexpression (OE) of TaTPP-7A in wheat enhanced grain TKW and wheat yield greatly. Detailed analysis revealed that OE of TaTPP-7A significantly increased the expression levels of starch synthesis- and senescence-related genes involved in abscisic acid (ABA) and ethylene pathways. Moreover, most of the sucrose metabolism and starch regulation-related genes were potentially regulated by SnRK1. In addition, TaTPP-7A is a crucial domestication- and breeding-targeted gene and it feedback regulates sucrose lysis, flux, and utilization in the grain endosperm mainly through the T6P-SnRK1 pathway and sugar-ABA interaction. Thus, we confirmed the T6P signalling pathway as the central regulatory system for sucrose allocation and source-sink interactions in wheat grains and propose that the trehalose pathway components have great potential to increase yields in cereal crops.

Brcd1 Is Associated with Plant Height through the Gibberellin Pathway in Brassica rapa L.

In crops such as Brassica rapa, the agronomic trait plant height affects the leaf light absorption rate, benefits substance accumulation, and increases production by increasing the biological yield of the population. The mechanism of plant height was adequately studied in Brassica napus and Arabidopsis, while Brassica rapa had been rarely reported. Herein, we studied a B. rapa dwarf mutant Brcd1YS. Compared with its wild-type, Yellow sarson, Brcd1YS showed shorter hypocotyls and slow growth, with inhibited root elongation and reduced numbers of lateral roots. Chlorophyll content determination and pollen viability detection indicated that the mutant’s chlorophyll content was higher than that of the wild-type; however, its pollen was inactive. Cytological identification showed that the number of cells in Brcd1YS leaves was significantly reduced and were arranged irregularly compared with those of the wild-type. Bulked Segregant RNA sequencing combined with conventional linkage mapped the dwarf mutation Brcd1YS to Chromosome A06, at position 21,621,766 to 24,683,923 bp. Application of exogenous gibberellic acid (GA) partially rescued the dwarf phenotype of Brcd1YS. GA-related genes BraA06g034110.3C and BraA06g033010.3C were identified as the most promising candidate genes. These results make a valuable contribution to our understanding of the mechanism of plant height determination in B. rapa, paving the way for further breeding of dwarf B. rapa.

Mapping Stripe Rust Resistance QTL in 'N2496', a Synthetic Hexaploid Wheat Derivative.

Stripe rust is a destructive disease that affects plant growth and substantially reduces wheat yields globally. An economically and environmentally friendly way to control this disease is to use resistant cultivars. 'N2496' is a synthetic hexaploid wheat derivative that exhibits high resistance and could serve as a source of resistance for breeding programs. We developed three recombinant inbred lines (RILs) populations by crossing 'N2496' with common wheat cultivars 'CN16', 'CM107', and 'MM37'. Stripe rust responses were evaluated in all three populations using a mixture of current predominant Chinese Puccinia striiformis f. sp. tritici races. A stripe rust resistance quantitative trait locus (QTL) in the 'N2496'/'CN16' RIL population was mapped on chromosome arm 6BL at 519.35 to 526.55 Mb using bulked segregant RNA sequencing. The population was genotyped using simple sequence repeats and kompetitive allele-specific polymerase (KASP) markers. The QTL QYr.sicau-6B was localized to a 1.19-cM interval flanked by markers KASP-TXK-10 and KASP-TXK-6. The genetic effect of QYr.sicau-6B was validated in the 'N2496' × 'CM107' and 'N2496' × 'MM37' RILs populations and explained up to 63.16% of the phenotypic variation. RNA sequencing and quantitative real-time polymerase chain reaction identified two differentially expressed candidate genes in the physical interval of QYr.sicau-6B.

Recognition of Pep-13/25 MAMPs of Phytophthora localizes to an RLK locus in Solanum microdontum.

Pattern-triggered immunity (PTI) in plants is mediated by cell surface-localized pattern recognition receptors (PRRs) upon perception of microbe-associated molecular pattern (MAMPs). MAMPs are conserved molecules across microbe species, or even kingdoms, and PRRs can confer broad-spectrum disease resistance. Pep-13/25 are well-characterized MAMPs in Phytophthora species, which are renowned devastating oomycete pathogens of potato and other plants, and for which genetic resistance is highly wanted. Pep-13/25 are derived from a 42 kDa transglutaminase GP42, but their cognate PRR has remained unknown. Here, we genetically mapped a novel surface immune receptor that recognizes Pep-25. By using effectoromics screening, we characterized the recognition spectrum of Pep-13/25 in diverse Solanaceae species. Response to Pep-13/25 was predominantly found in potato and related wild tuber-bearing Solanum species. Bulk-segregant RNA sequencing (BSR-Seq) and genetic mapping the response to Pep-25 led to a 0.081 cM region on the top of chromosome 3 in the wild potato species Solanum microdontum subsp. gigantophyllum. Some BAC clones in this region were isolated and sequenced, and we found the Pep-25 receptor locates in a complex receptor-like kinase (RLK) locus. This study is an important step toward the identification of the Pep-13/25 receptor, which can potentially lead to broad application in potato and various other hosts of Phytophthora species.

Copy number variation of B1 controls awn length in wheat

Wheat awns contribute to photosynthesis and grain production. In this study, an F2 population and F2:3 families from a cross between the awned line 7D12 and the Chinese awnless variety Shiyou 20 (SY20) were used to identify loci associated with awn length. Bulked-segregant RNA sequencing and linkage mapping identified a single dominant locus in a 0.3 cM interval on chromosome 5AL. Five genes were in the interval, including the recently cloned awn inhibitor B1. Although a single copy of the B1 gene was detected in 7D12, SY20 carried five copies of the gene. Increased copy number of B1 in SY20 enhanced gene expression. Based on sequence variation among the promoter regions of five B1 gene copies in SY20, two dominant markers were developed and found to cosegregate with B1 in a population of 931 wheat accessions. All 77 awnless accessions harbored sequence variations in the B1 promoter regions similar to those of SY20 and thus carried multiple copies of the gene, whereas 15 randomly selected awned wheats carried only one copy. These results suggest that an increase in copy number of the B1 gene is associated with inhibition of awn length.