Bulked Segregant RNA-Seq Research Articles

Unveiling resistance expression profile to powdery mildew in wheat via Bulked Segregant RNA-Seq.

Developing wheat cultivars with durable resistance to powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is crucial for sustainable agriculture. The wheat genotype MYC exhibited high resistance to the Bgt isolate E09 at the seedling stage. Genetic analysis identified a recessive gene, temporarily named PmMYC, responsible for this resistance. Understanding the molecular mechanisms underlying this resistance is essential for advancing breeding programs. Bulked Segregant RNA-Seq revealed numerous alternative splicing events generated following Bgt infection, suggesting powdery mildew may disrupt alternative splicing and affect immune responses. Gene Ontology (GO) analysis indicated significant enrichment of differentially expressed genes in "response to stimuli" and "immune system processes", implying their roles in disease defense. BSR-Seq analysis identified two high-confidence candidate regions for PmMYC on chromosome 2B, spanning 40,451,950 - 102,426,703bp and 421,707,046-449,840,516bp. Within these intervals, 740 genes were identified, with nonsynonymous mutations in 46 genes in the parents and bulks. Real-time PCR showed distinct expression profiles in four genes in resistant MYC compared to susceptible Yannong 21. KEGG and COG analyses of differentially expressed genes in candidate intervals revealed enrichment in immune processes related to plant-pathogen interactions, confirming that PmMYC initiated a broad immune response to prevent Bgt invasion. The study identified key genetic intervals and genes involved in the resistance of wheat genotype MYC to Bgt. The identified genes, particularly those with altered expression profiles, could serve as valuable targets for breeding programs aimed at developing wheat cultivars with durable resistance to powdery mildew. These findings enhanced our understanding of plant-pathogen interactions and provided a foundation for future genetic and functional studies.

Tyrp1 is the mendelian determinant of the Axolotl (Ambystoma mexicanum) copper mutant

Several dozen Mendelian mutants have been discovered in axolotl (Ambystoma mexicanum) populations, including several that affect pigmentation. Four recessive mutants have been described in the scientific literature and genes for three of these have been identified. Here we describe and genetically dissect copper, a mutant with an albino-like phenotype known only from the pet trade. We performed a cross segregating copper and wildtype color phenotypes and used bulked segregant RNA-Seq to identify a region on chromosome 6 that was enriched for single-nucleotide polymorphisms (SNPs) between the color phenotypes. This region included Tyrosinase-like Protein 1 (Tyrp1), a melanin synthesis protein that when mutated, is associated with lighter than black melanin coloration in animal models and oculocutaneous albinism in humans. Inspection of RNA-Seq reads identified a single nucleotide deletion that is predicted to change the coding frame, introduce a premature stop codon in exon 6 and yield a truncated Tyrp1 protein in copper individuals. Using CRISPR-Cas9 editing, we show that wildtype Tyrp1 crispants exhibit copper pigmentation, thus confirming Tyrp1 as the copper locus. Our results suggest that commercial and hobbyist axolotl populations may harbor useful mutants for biological research.

QSCR4.05 and qSCR4.08, two QTLs on chromosome 4 are associated with resistance to southern corn rust in maize

Southern corn rust (SCR), caused by the fungal pathogen Puccinia polysora, is one of the most serious threats to maize production. Identification of stable QTLs is a prerequisite in maize resistance breeding programs. Bulked Segregant RNA-Seq (BSR-Seq) and QTL mapping are two effective approaches to detect genomic regions responsible for traits of interest. In the present study, BSR-Seq analysis and QTL mapping were used to detect the genomic regions/QTLs associated with the resistance to SCR. By using BSR-Seq analysis, totally 2 and 3 genomic regions were detected in F2 and BC1 populations, respectively. By using QTL mapping analysis, 3 resistance QTLs (qSCR3.02/03, qSCR4.05 and qSCR4.08) were identified in F2:3 population. Among them, qSCR4.05 and qSCR4.08 overlapped with the regions detected by using BSR-Seq method. Moreover, the two QTLs were further validated by using near isogenic lines. The identification of the two stable QTLs (qSCR4.05 and qSCR4.08) lay the foundation for further application in resistance breeding programs.

Use of Maize (Zea maysL.) Mutator Transposon-Induced Mutants of theBonnMuResource for Forward and Reverse Genetics Studies

TheBonnMuresource represents a tagged collection of maize (Zea maysL.)Mutator(Mu) transposon-induced mutants, designed for functional genomics studies. Here, we describe the use of theBonnMucollection for identifying and characterizing mutations. Specifically, we describe workflows for use in both reverse and forward genetics strategies in maize. For reverse genetics, users first acquire aBonnMuF2stock of interest based on data accessible at the Maize Genetics and Genomics Database (MaizeGDB). We provide details here for their subsequent propagation and for the confirmation ofMuinsertions by genotyping via PCR, with the ultimate goal of establishing genotype–phenotype relationships of interest. For forward genetics studies, we describe a workflow that involves a combined approach of Mutant-Seq (Mu-Seq) and bulked segregant RNA-seq (BSR-Seq), to identify the causal gene underlying a mutant phenotype of interest.

Bulked Segregant RNA-Seq Analysis of Pollinated Pistils Reveals Genes Influencing Spikelet Fertility in Rice

Prezygotic isolation is important for successful fertilization in rice, significantly affecting yield. This study focused on F5:6 generation plants derived from inter-subspecific crosses (Nipponbare × KDML105) with low (LS) and high seed-setting rates (HS), in which normal pollen fertility was observed. However, LS plants showed a reduced number of pollen grains adhering to the stigma and fewer pollen tubes reaching the ovules at 4‒5 h post-pollination, compared with HS plants. Bulked segregant RNA-Seq analysis of pollinated pistils from the HS and LS groups revealed 249 and 473 differentially expressed genes (DEGs), respectively. Kyoto Encyclopedia of Genes and Genomes analysis of the HS and LS- specific DEGs indicated enrichment in metabolic pathways, pentose and glucuronate interconversions, and flavonoid biosynthesis. Several of these DEGs exhibited co-expression with pollen development genes and formed extensive clusters of co-expression networks. Compared with LS pistils, enzyme genes controlling pectin degradation, such as OsPME35 and OsPLL9, showed similar expression patterns, with higher levels in HS pistils pre-pollination. Os02g0467600, similar to cinnamate 4-hydroxylase gene (CYP73), involved in flavonoid biosynthesis, displayed higher expression in HS pistils post-pollination. Our findings suggest that OsPME35, OsPLL9, and Os02g0467600 contribute to prezygotic isolation by potentially modifying the stigma cell wall (OsPME35 and OsPLL9) and controlling later processes such as pollen-stigma adhesion (Os02g0467600) genes. Furthermore, several DEGs specific to HS and LS were co-localized with QTLs and functional genes associated with spikelet fertility. These findings provide valuable insights for further research on rice spikelet fertility, ultimately contributing to the development of high-yielding rice varieties.

Bulked segregant RNA-seq reveals complex resistance expression profile to powdery mildew in wild emmer wheat W762.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive fungal diseases threatening global wheat production. Exploring powdery mildew resistance (Pm) gene(s) and dissecting the molecular mechanism of the host resistance are critical to effectively and reasonably control this disease. Durum wheat (Triticum turgidum L. var. durumDesf.) is an important gene donor for wheat improvement against powdery mildew. In this study, a resistant durum wheat accession W762 was used to investigate its potential resistance component(s) and profile its expression pattern in responding to Bgt invasion using bulked segregant RNA-Seq (BSR-Seq) and further qRT-PCR verification. Genetic analysis showed that the powdery mildew resistance in W762 did not meet monogenic inheritance and complex genetic model might exist within the population of W762 × Langdon (susceptible durum wheat). After BSR-Seq, 6,196 consistently different single nucleotide polymorphisms (SNPs) were called between resistant and susceptible parents and bulks, and among them, 763 SNPs were assigned to the chromosome arm 7B. Subsequently, 3,653 differentially expressed genes (DEGs) between resistant and susceptible parents and bulks were annotated and analyzed by Gene Ontology (GO), Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. The potential regulated genes were selected and analyzed their temporal expression patterns following Bgt inoculation. As a result, nine disease-related genes showed distinctive expression profile after Bgt invasion and might serve as potential targets to regulate the resistance against powdery mildew in W762. Our study could lay a foundation for analysis of the molecular mechanism and also provide potential targets for the improvement of durable resistance against powdery mildew.

Bulked Segregant RNA-Seq Reveals Different Gene Expression Patterns and Mutant Genes Associated with the Zigzag Pattern of Tea Plants (Camellia sinensis).

The unique zigzag-patterned tea plant is a rare germplasm resource. However, the molecular mechanism behind the formation of zigzag stems remains unclear. To address this, a BC1 genetic population of tea plants with zigzag stems was studied using histological observation and bulked segregant RNA-seq. The analysis revealed 1494 differentially expressed genes (DEGs) between the upright and zigzag stem groups. These DEGs may regulate the transduction and biosynthesis of plant hormones, and the effects on the phenylpropane biosynthesis pathways may cause the accumulation of lignin. Tissue sections further supported this finding, showing differences in cell wall thickness between upright and curved stems, potentially due to lignin accumulation. Additionally, 262 single-nucleotide polymorphisms (SNPs) across 38 genes were identified as key SNPs, and 5 genes related to zigzag stems were identified through homologous gene function annotation. Mutations in these genes may impact auxin distribution and content, resulting in the asymmetric development of vascular bundles in curved stems. In summary, we identified the key genes associated with the tortuous phenotype by using BSR-seq on a BC1 population to minimize genetic background noise.

Genetic Mapping of a Novel Gene PmAege7M from Aegilops geniculata Conferring Resistance to Wheat Powdery Mildew.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most damaging foliage diseases of wheat across the world. Aegilops geniculata Roth is a valuable gene resource for enhancing wheat resistance to powdery mildew. This study identified Ae. geniculata accession PI 487224 as immune and PI 487228 as susceptible to powdery mildew. Genetic analysis of the F1, F2, and F2:3 progeny derived from PI 487224 × PI 487228 showed that powdery mildew resistance in PI 487224 was controlled by two independent dominant genes located on two different nonhomologous chromosomes. By combing bulked segregant RNA-Seq, genetic linkage analysis of a single resistance gene segregation population, and marker analysis of a set of 14 wheat-Ae. geniculata chromosome addition lines, one of the resistance genes, temperately designated PmAege7M, was mapped to a 4.9-cM interval flanked by markers STS7-55926 and SNP7-45792/STS7-65911 on the long arm of chromosome 7 Mg of PI 487224, spanning 604.73 to 622.82 Mb on the 7D long arm based on the Ae. tauschii reference genome (Aet_v4.0). The map and closely linked markers of PmAege7M from Ae. geniculata in this study will facilitate the transfer of PmAege7M into common wheat and fine mapping of the gene.

TaACTIN7-D regulates plant height and grain shape in bread wheat

Exploitation of new gene resources and genetic networks contributing to the control of crop yield-related traits, such as plant height, grain size, and shape, may enable us to breed modern high-yielding wheat varieties through molecular methods. In this study, via ethylmethanesulfonate mutagenesis, we identify a wheat mutant plant, mu-597, that shows semi-dwarf plant architecture and round grain shape. Through bulked segregant RNA-seq and map-based cloning, the causal gene for the semi-dwarf phenotype of mu-597 is located. We find that a single-base mutation in the coding region of TaACTIN7-D (TaACT7-D), leading to a Gly-to-Ser (G65S) amino acid mutation at the 65th residue of the deduced TaACT7-D protein, can explain the semi-dwarfism and round grain shape of mu-597. Further evidence shows that the G65S mutation in TaACT7-D hinders the polymerization of actin from monomeric (G-actin) to filamentous (F-actin) status while attenuates wheat responses to multiple phytohormones, including brassinosteroids, auxin, and gibberellin. Together, these findings not only define a new semi-dwarfing gene resource that can be potentially used to design plant height and grain shape of bread wheat but also establish a direct link between actin structure modulation and phytohormone signal transduction.

Identification of the Solid Stem Suppressor Gene Su-TdDof in Synthetic Hexaploid Wheat Syn-SAU-117.

Lodging is one of the most important factors affecting the high and stable yield of wheat worldwide. Solid-stemmed wheat has higher stem strength and lodging resistance than hollow-stemmed wheat does. There are many solid-stemmed varieties, landraces, and old varieties of durum wheat. However, the transfer of solid stem genes from durum wheat is suppressed by a suppressor gene located on chromosome 3D in common wheat, and only hollow-stemmed lines have been created. However, synthetic hexaploid wheat can serve as a bridge for transferring solid stem genes from tetraploid wheat to common wheat. In this study, the F1, F2, and F2:3 generations of a cross between solid-stemmed Syn-SAU-119 and semisolid-stemmed Syn-SAU-117 were developed. A single dominant gene, which was tentatively designated Su-TdDof and suppresses stem solidity, was identified in synthetic hexaploid wheat Syn-SAU-117 by using genetic analysis. By using bulked segregant RNA-seq (BSR-seq) analysis, Su-TdDof was mapped to chromosome 7DS and flanked by markers KASP-669 and KASP-1055 within a 4.53 cM genetic interval corresponding to 3.86 Mb and 2.29 Mb physical regions in the Chinese Spring (IWGSC RefSeq v1.1) and Ae. tauschii (AL8/78 v4.0) genomes, respectively, in which three genes related to solid stem development were annotated. Su-TdDof differed from a previously reported solid stem suppressor gene based on its origin and position. Su-TdDof would provide a valuable example for research on the suppression phenomenon. The flanking markers developed in this study might be useful for screening Ae. tauschii accessions with no suppressor gene (Su-TdDof) to develop more synthetic hexaploid wheat lines for the breeding of lodging resistance in wheat and further cloning the suppressor gene Su-TdDof.

Identification and fine mapping of PmNJ3946 for powdery mildew resistance in einkorn wheat

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive wheat disease. Although it can be easily overcome by deployment of resistance genes, the resistance is often quickly compromised by pathogen virulence. Thus, exploration and characterization of new resistance genes is always ongoing. Line NJ3946 derived from a cross of einkorn wheat accessions TA2032 and M389 showed resistance to powdery mildew. Inheritance analysis of an F2 population derived from a cross of NJ3946 and M389 suggested that the resistance was conferred by a dominant allele. With polymorphic markers identified through bulked segregant analysis (BSA), this gene was mapped to a novel locus on chromosome 3A, and was designated as PmNJ3946. Bulked segregant RNA-seq analysis (BSR-seq) was conducted to obtain more closely linked markers, which allowed delimitation of the PMNJ3946 locus to a 0.9 cM interval covering a physical distance of less than 1 Mb. PMNJ3946 was flanked by Xwgrc5153 and SNP-derived marker CHS21_3A008915069, and co-segregated with SNP-derived markers CHS21_3A008939814 and CHS21_3A008943175. The PmNJ3946 discovery expands the diversity of powdery mildew resistance genes and is useful for wheat breeding.

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.

The putative vacuolar processing enzyme gene TaVPE3cB is a candidate gene for wheat stem pith-thickness

Key messageThe vacuolar processing enzyme gene TaVPE3cB is identified as a candidate gene for a QTL of wheat pith-thickness on chromosome 3B by BSR-seq and differential expression analyses.The high pith-thickness (PT) of the wheat stem could greatly enhance stem mechanical strength, especially the basal internodes which support the heavier upper part, such as upper stems, leaves and spikes. A QTL for PT in wheat was previously discovered on 3BL in a double haploid population of ‘Westonia’ × ‘Kauz’. Here, a bulked segregant RNA-seq analysis was applied to identify candidate genes and develop associated SNP markers for PT. In this study, we aimed at screening differentially expressed genes (DEGs) and SNPs in the 3BL QTL interval. Sixteen DEGs were obtained based on BSR-seq and differential expression analyses. Twenty-four high-probability SNPs in eight genes were identified by comparing the allelic polymorphism in mRNA sequences between the high PT and low PT samples. Among them, six genes were confirmed to be associated with PT by qRT-PCR and sequencing. A putative vacuolar processing enzyme gene TaVPE3cB was screened out as a potential PT candidate gene in Australian wheat ‘Westonia’. A robust SNP marker associated with TaVPE3cB was developed, which can assist in the introgression of TaVPE3cB.b in wheat breeding programs. In addition, we also discussed the function of other DEGs which may be related to pith development and programmed cell death (PCD). A five-level hierarchical regulation mechanism of stem pith PCD in wheat was proposed.

Leukocyte Tyrosine Kinase (Ltk) Is the Mendelian Determinant of the Axolotl Melanoid Color Variant.

The great diversity of color patterns observed among amphibians is largely explained by the differentiation of relatively few pigment cell types during development. Mexican axolotls present a variety of color phenotypes that span the continuum from leucistic to highly melanistic. The melanoid axolotl is a Mendelian variant characterized by large numbers of melanophores, proportionally fewer xanthophores, and no iridophores. Early studies of melanoid were influential in developing the single-origin hypothesis of pigment cell development, wherein it has been proposed that all three pigment cell types derive from a common progenitor cell, with pigment metabolites playing potential roles in directing the development of organelles that define different pigment cell types. Specifically, these studies identified xanthine dehydrogenase (XDH) activity as a mechanism for the permissive differentiation of melanophores at the expense of xanthophores and iridophores. We used bulked segregant RNA-Seq to screen the axolotl genome for melanoid candidate genes and identify the associated locus. Dissimilar frequencies of single-nucleotide polymorphisms were identified between pooled RNA samples of wild-type and melanoid siblings for a region on chromosome 14q. This region contains gephyrin (Gphn), an enzyme that catalyzes the synthesis of the molybdenum cofactor that is required for XDH activity, and leukocyte tyrosine kinase (Ltk), a cell surface signaling receptor that is required for iridophore differentiation in zebrafish. Wild-type Ltk crispants present similar pigment phenotypes to melanoid, strongly implicating Ltk as the melanoid locus. In concert with recent findings in zebrafish, our results support the idea of direct fate specification of pigment cells and, more generally, the single-origin hypothesis of pigment cell development.

Identification of the candidate gene controlling tiller angle in common wheat through genome-wide association study and linkage analysis

Wheat tiller angle (TA) is an important agronomic trait that contributes to grain production by affecting plant architecture. It also plays a crucial role in high-yield wheat breeding. An association panel and a recombinant inbred line (RIL) population were used to map quantitative trait loci (QTL) for TA. Results showed that 470 significant SNPs with 10.4%–28.8% phenotypic variance explained (PVE) were detected in four replicates by a genome-wide association study (GWAS). Haplotype analysis showed that the TA_Hap_4B1 locus on chromosome 4B was a major QTL to regulate wheat TA. Ten QTL were totally detected by linkage mapping with the RIL population, and QTA.hau-4B.1 identified in six environments with the PVE of 7.88%–18.82% was a major and stable QTL. A combined analysis demonstrated that both TA_Hap_4B1 and QTA.hau-4B.1 were co-located on the same region. Moreover, QTA.hau-4B.1 was confirmed by bulked segregant RNA-Seq (BSR-Seq) analysis. Phenotypic analysis showed that QTA.hau-4B.1 was also closely related to yield traits. Furthermore, TraesCS4B02G049700 was considered as a candidate gene through analysis of gene sequence and expression. This study can be potentially used in cloning key genes modulating wheat tillering and provides valuable genetic resources for improvement of wheat plant architecture.

Identification of a Wheat Powdery Mildew Dominant Resistance Gene in the Pm5 Locus for High-Throughput Marker-Assisted Selection.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), poses a severe threat to wheat yield and quality worldwide. Rapid identification and the accurate transference of effective resistance genes are important to the development of resistant cultivars and the sustainable control of this disease. In the present study, the wheat line AL11 exhibited high levels of resistance to powdery mildew at both the seedling and adult plant stages. Genetic analysis of the AL11 × 'Shixin 733' mapping population revealed that its resistance was controlled by a single dominant gene, tentatively designated PmAL11. Using bulked segregant RNA-Seq and molecular marker analysis, PmAL11 was mapped to the Pm5 locus on chromosome 7B where it cosegregated with the functional marker Pm5e-KASP. Sequence alignment analysis revealed that the Pm5e-homologous sequence in AL11 was identical to the reported recessive gene Pm5e in wheat landrace 'Fuzhuang 30'. It appears that PmAL11 was most probably Pm5e, but it was mediated by a dominant inheritance pattern, so it should provide a valuable resistance resource for both genetic study and wheat breeding. To efficiently use and trace PmAL11 in breeding, a new kompetitive allele-specific PCR marker AL11-K2488 that cosegregated with this gene was developed and confirmed to be applicable in the different wheat backgrounds, thus promoting its use in the marker-assisted selection of PmAL11.

Screening of Candidate Genes Associated with Brown Stripe Resistance in Sugarcane via BSR-seq Analysis.

Sugarcane brown stripe (SBS), caused by the fungal pathogen Helminthosporium stenospilum, is one of the most serious threats to sugarcane production. However, its outbreaks and epidemics require suitable climatic conditions, resulting in the inefficient improvement of the SBS resistance by phenotype selection. The sugarcane F1 population of SBS-resistant YT93-159 × SBS-susceptible ROC22 was used for constructing the bulks. Bulked segregant RNA-seq (BSR-seq) was then performed on the parents YT93-159 (T01) and ROC22 (T02), and the opposite bulks of 30 SBS-susceptible individuals mixed bulk (T03) and 30 SBS-resistant individuals mixed bulk (T04) collected from 287 F1 individuals. A total of 170.00 Gb of clean data containing 297,921 SNPs and 70,426 genes were obtained. Differentially expressed genes (DEGs) analysis suggested that 7787 and 5911 DEGs were identified in the parents (T01 vs. T02) and two mixed bulks (T03 vs. T04), respectively. In addition, 25,363 high-quality and credible SNPs were obtained using the genome analysis toolkit GATK for SNP calling. Subsequently, six candidate regions with a total length of 8.72 Mb, which were located in the chromosomes 4B and 7C of sugarcane wild species Saccharum spontaneum, were identified, and 279 genes associated with SBS-resistance were annotated by ED algorithm and ΔSNP-index. Furthermore, the expression profiles of candidate genes were verified by quantitative real-time PCR (qRT-PCR) analysis, and the results showed that eight genes (LRR-RLK, DHAR1, WRKY7, RLK1, BLH4, AK3, CRK34, and NDA2) and seven genes (WRKY31, CIPK2, CKA1, CDPK6, PFK4, CBL2, and PR2) of the 20 tested genes were significantly up-regulated in YT93-159 and ROC22, respectively. Finally, a potential molecular mechanism of sugarcane response to H. stenospilum infection is illustrate that the activations of ROS signaling, MAPK cascade signaling, Ca2+ signaling, ABA signaling, and the ASA-GSH cycle jointly promote the SBS resistance in sugarcane. This study provides abundant gene resources for the SBS resistance breeding in sugarcane.

SlGH9-15 regulates tomato fruit cracking with hormonal and abiotic stress responsiveness cis-elements

Fruit cracking occurs easily during the late period of fruit development when plants encounter an unsuitable environment, dramatically affecting fruit production and marketing. This study conducted the bulked segregant RNA-Seq (BSR) to identify the key regulatory gene of fruit cracking in tomatoes. BSR-Seq analysis illustrated that two regions associated with irregularly cracking were located on chromosomes 9 and 11, containing 127 candidate genes. Further, through differentially expression analysis and qRT-PCR in cracking-susceptible and cracking-resistant genotypes, the candidate gene SlGH9-15 (Solyc09g010210) with significantly differential expression levels was screened. Bioinformatics analysis of the GH9 gene family revealed that 20 SlGH9 genes were divided into three groups. The phylogenetic analysis showed that SlGH9-15 was closely related to cell wall construction-associated genes AtGH9B1, AtGH9B6, OsGH9B1, and OsGH9B3. The cis-acting elements analysis revealed that SlGH9-15 was activated by various hormones (ethylene and ABA) and abiotic stresses. The expression pattern indicated that 13 SlGH9 genes, especially SlGH9-15, were highly expressed in the cracking-susceptible genotype. Its expression level gradually increased during fruit development and achieved maximum value at the red ripe stage. Additionally, the cracking-susceptible tomato showed higher cellulase activity and lower cellulose content than the cracking-resistant tomato, particularly at the red ripe stage. This study identified SlGH9-15 as a key gene associated with fruit cracking in tomatoes for the first time and gives new insights for understanding the molecular mechanism and complex regulatory network of fruit cracking.

Identification of Novel Genes Associated with Partial Resistance to Aphanomyces Root Rot in Field Pea by BSR-Seq Analysis.

Aphanomyces root rot, caused by Aphanomyces euteiches, causes severe yield loss in field pea (Pisum sativum). The identification of a pea germplasm resistant to this disease is an important breeding objective. Polygenetic resistance has been reported in the field pea cultivar ‘00-2067’. To facilitate marker-assisted selection (MAS), bulked segregant RNA-seq (BSR-seq) analysis was conducted using an F8 RIL population derived from the cross of ‘Carman’ × ‘00-2067’. Root rot development was assessed under controlled conditions in replicated experiments. Resistant (R) and susceptible (S) bulks were constructed based on the root rot severity in a greenhouse study. The BSR-seq analysis of the R bulks generated 44,595,510~51,658,688 reads, of which the aligned sequences were linked to 44,757 genes in a reference genome. In total, 2356 differentially expressed genes were identified, of which 44 were used for gene annotation, including defense-related pathways (jasmonate, ethylene and salicylate) and the GO biological process. A total of 344.1 K SNPs were identified between the R and S bulks, of which 395 variants were located in 31 candidate genes. The identification of novel genes associated with partial resistance to Aphanomyces root rot in field pea by BSR-seq may facilitate efforts to improve management of this important disease.

Fine mapping of a stripe rust resistance gene YrZM175 in bread wheat.

A stripe rust resistance gene YrZM175 in Chinese wheat cultivar Zhongmai 175 was mapped to a genomic interval of 636.4 kb on chromosome arm 2AL, and a candidate gene was predicted. Stripe rust, caused by Puccinia striiformis f. sp. tritici (PST), is a worldwide wheat disease that causes large losses in production. Fine mapping and cloning of resistance genes are important for accurate marker-assisted breeding. Here, we report the fine mapping and candidate gene analysis of stripe rust resistance gene YrZM175 in a Chinese wheat cultivar Zhongmai 175. Fifteen F1, 7,325 F2 plants and 117 F2:3 lines derived from cross Avocet S/Zhongmai 175 were inoculated with PST race CYR32 at the seedling stage in a greenhouse, and F2:3 lines were also evaluated for stripe rust reaction in the field using mixed PST races. Bulked segregant RNA-seq (BSR-seq) analyses revealed 13 SNPs in the region 762.50-768.52Mb on chromosome arm 2AL. By genome mining, we identified SNPs and InDels between the parents and contrasting bulks and mapped YrZM175 to a 0.72-cM, 636.4-kb interval spanned by YrZM175-InD1 and YrZM175-InD2 (763,452,916-764,089,317bp) including two putative disease resistance genes based on IWGSC RefSeq v1.0. Collinearity analysis indicated similar target genomic intervals in Chinese Spring, Aegilops tauschii (2D: 647.7-650.5Mb), Triticum urartu (2A: 750.7-752.3Mb), Triticum dicoccoides (2A: 771.0-774.5Mb), Triticum turgidum (2B: 784.7-788.2Mb), and Triticum aestivum cv. Aikang 58 (2A: 776.3-778.9Mb) and Jagger (2A: 789.3-791.7Mb). Through collinearity analysis, sequence alignments of resistant and susceptible parents and gene expression level analysis, we predicted TRITD2Bv1G264480 from Triticum turgidum to be a candidate gene for map-based cloning of YrZM175. A gene-specific marker for TRITD2Bv1G264480 co-segregated with the resistance gene. Molecular marker analysis and stripe rust response data revealed that YrZM175 was different from genes Yr1, Yr17, Yr32, and YrJ22 located on chromosome 2A. Fine mapping of YrZM175 lays a solid foundation for functional gene analysis and marker-assisted selection for improved stripe rust resistance in wheat.