Rising temperature is a major threat to the normal growth and development of maize, resulting in low yield production and quality. The mechanism of maize in response to heat stress remains uncertain. In this study, a maize mutant Zmhsl-1 (heat sensitive leaves) with wilting and curling leaves under high temperatures was identified from maize Zheng 58 (Z58) mutant lines generated by ethyl methanesulfonate (EMS) mutagenesis. The Zmhsl-1 plants were more sensitive to increased temperature than Z58 in the field during growth season. The Zmhsl-1 plants had lower plant height, lower yield, and lower content of photosynthetic pigments. A bulked segregant analysis coupled with whole-genome sequencing (BSA-seq) enabled the identification of the corresponding gene, named ZmHSL, which encodes an endo-β-1,4-xylanase from the GH10 family. The loss-of-function of ZmHSL resulted in reduced lignin content in Zmhsl-1 plants, leading to defects in water transport and more severe leaf wilting with the increase in temperature. RNA-seq analysis revealed that the differentially expressed genes identified between Z58 and Zmhsl-1 plants are mainly related to heat stress-responsive genes and unfolded protein response genes. All these data indicated that ZmHSL plays a key role in lignin synthesis, and its defective mutation causes changes in the cell wall structure and gene expression patterns, which impedes water transport and confers higher sensitivity to high-temperature stress.

As a Brassica crop, Brassica napus typically has single flowers that contain four petals. The double-flower phenotype of rapeseed has been a desirable trait in China because of its potential commercial value in ornamental tourism. However, few double-flowered germplasms have been documented in B. napus, and knowledge of the underlying genes is limited. Here, B. napus D376 was characterized as a double-flowered strain that presented an average of 10.92 ± 1.40 petals and other normal floral organs. F1, F2 and BC1 populations were constructed by crossing D376 with a single-flowered line reciprocally. Genetic analysis revealed that the double-flower trait was a recessive trait controlled by multiple genes. To identify the key genes controlling the double-flower trait, bulk segregant analysis sequencing (BSA-seq) and RNA-seq analyses were conducted on F2 individual bulks with opposite extreme phenotypes. Through BSA-seq, one candidate interval was mapped at the region of chromosome C05: 14.56-16.17 Mb. GO and KEGG enrichment analyses revealed that the DEGs were significantly enriched in carbohydrate metabolic processes, notably starch and sucrose metabolism. Interestingly, five and thirty-six DEGs associated with floral development were significantly up- and down-regulated, respectively, in the double-flowered plants. A combined analysis of BSA-seq and RNA-seq data revealed that five genes were candidates associated with the double flower trait, and BnaC05.ERS2 was the most promising gene. These findings provide novel insights into the breeding of double-flowered varieties and lay a theoretical foundation for unveiling the molecular mechanisms of floral development in B. napus.

UNASSIGNED: Fusarium head blight (FHB) has a large influence on both the yield and quality of wheat grain worldwide. Host resistance is the most effective method for controlling FHB, but unfortunately, very few genetic resources on FHB resistance are available; therefore, identifying novel resistance genes or quantitative trait loci (QTLs) is valuable.
UNASSIGNED: Here, a recombinant inbred line (RIL) population containing 451 lines derived from the cross L661/PI672538 was sown in four different environments (2019CZa, 2019CZb, 2021QL and 2021WJ).
UNASSIGNED: Five QTLs, consisting of two previously reported QTLs (FhbL693a and FhbL693b) and three new QTLs (FhbL693c, FhbL693d and FhbL693e), were identified. Further investigation revealed that FhbL693b, FhbL693c and FhbL693d could be detected in all four environments, and FhbL693a and FhbL693e were detected only in 2019CZb and 2021WJ, respectively. Among the QTLs, the greatest contribution (10.5%) to the phenotypic variation effect (PVE) was FhbL693d in 2021WJ, while the smallest (1.2%) was FhbL693e and FhbL693a in 2019CZb. The selection of 5Dindel-4 for FhbL693d, 4Aindel-7 for FhbL693c and 3Bindel-24 for FhbL693b decreased the number of damaged spikelets by 2.1, and a new line resistant to FHB named H140-2 was developed by marker-assisted selection (MAS).
UNASSIGNED: These results could help to further improve FHB resistance in the future.

Fruit size is a crucial agronomic trait in bottle gourd, impacting both yield and utility. Despite its significance, the regulatory mechanism governing fruit size in bottle gourd remains largely unknown. In this study, we used bottle gourd (small-fruited H28 and large-fruited H17) parent plants to measure the width and length of fruits at various developmental stages, revealing a single \'S\' growth curve for fruit expansion. Paraffin section observations indicated that both cell number and size significantly influence bottle gourd fruit size. Through bulked segregant analysis and combined genotype-phenotype analysis, the candidate interval regulating fruit size was pinpointed to 17,747,353 bp-18,185,825 bp on chromosome 9, encompassing 0.44 Mb and including 44 genes. Parental fruits in the rapid expansion stage were subjected to RNA-seq, highlighting that differentially expressed genes were mainly enriched in pathways related to cell wall biosynthesis, sugar metabolism, and hormone signaling. Transcriptome and resequencing analysis, combined with gene function annotation, identified six genes within the localized region as potential regulators of fruit size. This study not only maps the candidate interval of genes influencing fruit size in bottle gourd through forward genetics, but also offers new insights into the potential molecular mechanisms underlying this trait through transcriptome analysis.

BACKGROUND: The cold tolerance of rice is closely related to its production and geographic distribution. The identification of cold tolerance-related genes is of important significance for developing cold-tolerant rice. Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) is well-adapted to the cold climate of northernmost-latitude habitats ever found in the world, and is one of the most valuable rice germplasms for cold tolerance improvement.
RESULTS: Transcriptome analysis revealed genes differentially expressed between Xieqingzao B (XB; a cold sensitive variety) and 19H19 (derived from an interspecific cross between DXWR and XB) in the room temperature (RT), low temperature (LT), and recovery treatments. The results demonstrated that chloroplast genes might be involved in the regulation of cold tolerance in rice. A high-resolution SNP genetic map was constructed using 120 BC5F2 lines derived from a cross between 19H19 and XB based on the genotyping-by-sequencing (GBS) technique. Two quantitative trait loci (QTLs) for cold tolerance at the early seedling stage (CTS), qCTS12 and qCTS8, were detected. Moreover, a total of 112 candidate genes associated with cold tolerance were identified based on bulked segregant analysis sequencing (BSA-seq). These candidate genes were divided into eight functional categories, and the expression trend of candidate genes related to \'oxidation-reduction process\' and \'response to stress\' differed between XB and 19H19 in the RT, LT and recovery treatments. Among these candidate genes, the expression level of LOC_Os12g18729 in 19H19 (related to \'response to stress\') decreased in the LT treatment but restored and enhanced during the recovery treatment whereas the expression level of LOC_Os12g18729 in XB declined during recovery treatment. Additionally, XB contained a 42-bp deletion in the third exon of LOC_Os12g18729, and the genotype of BC5F2 individuals with a survival percentage (SP) lower than 15% was consistent with that of XB. Weighted gene coexpression network analysis (WGCNA) and modular regulatory network learning with per gene information (MERLIN) algorithm revealed a gene interaction/coexpression network regulating cold tolerance in rice. In the network, differentially expressed genes (DEGs) related to \'oxidation-reduction process\', \'response to stress\' and \'protein phosphorylation\' interacted with LOC_Os12g18729. Moreover, the knockout mutant of LOC_Os12g18729 decreased cold tolerance in early rice seedling stage signifcantly compared with that of wild type.
CONCLUSIONS: In general, study of the genetic basis of cold tolerance of rice is important for the development of cold-tolerant rice varieties. In the present study, QTL mapping, BSA-seq and RNA-seq were integrated to identify two CTS QTLs qCTS8 and qCTS12. Furthermore, qRT-PCR, genotype sequencing and knockout analysis indicated that LOC_Os12g18729 could be the candidate gene of qCTS12. These results are expected to further exploration of the genetic mechanism of CTS in rice and improve cold tolerance of cultivated rice by introducing the cold tolerant genes from DXWR through marker-assisted selection.

Fruit length (FL) is an important economical trait that affects fruit yield and appearance. Pumpkin (Cucurbita moschata Duch) contains a wealth genetic variation in fruit length. However, the natural variation underlying differences in pumpkin fruit length remains unclear. In this study, we constructed a F2 segregate population using KG1 producing long fruit and MBF producing short fruit as parents to identify the candidate gene for fruit length. By bulked segregant analysis (BSA-seq) and Kompetitive Allele-Specific PCR (KASP) approach of fine mapping, we obtained a 50.77 kb candidate region on chromosome 14 associated with the fruit length. Then, based on sequence variation, gene expression and promoter activity analyses, we identified a candidate gene (CmoFL1) encoding E3 ubiquitin ligase in this region may account for the variation of fruit length. One SNP variation in promoter of CmoFL1 changed the GT1CONSENSUS, and DUAL-LUC assay revealed that this variation significantly affected the promoter activity of CmoFL1. RNA-seq analysis indicated that CmoFL1 might associated with the cell division process and negatively regulate fruit length. Collectively, our work identifies an important allelic affecting fruit length, and provides a target gene manipulating fruit length in future pumpkin breeding.

Globe artichoke (Cynara cardunculus var. scolymus; 2n = 2x = 34) is a food crop consumed for its immature flower heads. Traditionally, globe artichoke varietal types are vegetatively propagated. However, seed propagation makes it possible to treat the crop as annual, increasing field uniformity and reducing farmers costs, as well as pathogens diffusion. Despite globe artichoke\'s significant agricultural value and the critical role of heterosis in the development of superior varieties, the production of hybrids remains challenging without a reliable system for large-scale industrial seed production. Male sterility (MS) presents a promising avenue for overcoming these challenges by simplifying the hybridization process and enabling cost-effective seed production. However, within the Cynara genus, genic male sterility has been linked to three recessive loci in globe artichoke, with no definitive genetic mechanism elucidated to date. A 250 offsprings F2 population, derived from a cross between a MS globe artichoke and a male fertile (MF) cultivated cardoon (C. cardunculus var. altilis) and fitting a monogenic segregation model (3:1), was analyzed through BSA-seq, aiming at the identification of genomic regions/genes affecting male sterility. Four QTL regions were identified on chromosomes 4, 12, and 14. By analyzing the sequence around the highest pick on chromosome 14, a cytochrome P450 (CYP703A2) was identified, carrying a deleterious substitution (R/Q) fixed in the male sterile parent. A single dCAPS marker was developed around this SNP, allowing the discrimination between MS and MF genotypes within the population, suitable for applications in plant breeding programs. A 3D model of the protein was generated by homology modeling, revealing that the mutated amino acid is part of a highly conserved motif crucial for protein folding.

The soil-borne pathogen Plasmodiophora brassicae is the causal agent of clubroot, a major disease in Chinese cabbage (Brassica rapa ssp. pekinensis). The host\'s resistance genes often confer immunity to only specific pathotypes and may be rapidly overcome. Identification of novel clubroot resistance (CR) from germplasm sources is necessary. In this study, Bap246 was tested by being crossed with different highly susceptible B. rapa materials and showed recessive resistance to clubroot. An F2 population derived from Bap246 × Bac1344 was used to locate the resistance Quantitative Trait Loci (QTL) by Bulk Segregant Analysis Sequencing (BSA-Seq) and QTL mapping methods. Two QTL on chromosomes A01 (4.67-6.06 Mb) and A08 (10.42-11.43 Mb) were found and named Cr4Ba1.1 and Cr4Ba8.1, respectively. Fifteen and eleven SNP/InDel markers were used to narrow the target regions in the larger F2 population to 4.67-5.17 Mb (A01) and 10.70-10.84 Mb (A08), with 85 and 19 candidate genes, respectively. The phenotypic variation explained (PVE) of the two QTL were 30.97% and 8.65%, respectively. Combined with gene annotation, mutation site analysis, and real-time quantitative polymerase chain reaction (qRT-PCR) analysis, one candidate gene in A08 was identified, namely Bra020861. And an insertion and deletion (InDel) marker (co-segregated) named Crr1-196 was developed based on the gene sequence. Bra013275, Bra013299, Bra013336, Bra013339, Bra013341, and Bra013357 in A01 were the candidate genes that may confer clubroot resistance in Chinese cabbage. The resistance resource and the developed marker will be helpful in Brassica breeding programs.

Pericarp color is a prominent agronomic trait that exerts a significant impact on consumer and breeder preferences. Genetic analysis has revealed that the pericarp color of bitter gourd is a quantitative trait. However, the underlying mechanism for this trait in bitter gourd remains largely unknown. In the present study, we employed bulked segregant analysis (BSA) to identify the candidate genes responsible for bitter gourd pericarp color (specifically, dark green versus white) within F2 segregation populations resulting from the crossing of B07 (dark green pericarp) and A06 (white pericarp). Through genomic variation, genetic mapping, and expression analysis, we identified a candidate gene named McPRR2, which was a homolog of Arabidopsis pseudo response regulator 2 (APRR2) encoded by LOC111023472. Sequence alignment of the candidate gene between the two parental lines revealed a 15-bp nucleotide insertion in the coding region of LOC111023472, leading to a premature stop codon and potentially causing a loss-of-function mutation. qRT-PCR analysis demonstrated that the expression of McPRR2 was significantly higher in B07 compared to A06, and it was primarily expressed in the immature fruit pericarp. Moreover, overexpression of McPRR2 in tomato could enhance the green color of immature fruit pericarp by increasing the chlorophyll content. Consequently, McPRR2 emerged as a strong candidate gene regulating the bitter gourd pericarp color by influencing chlorophyll accumulation. Finally, we developed a molecular marker linked to pericarp color, enabling the identification of genotypes in breeding populations. These findings provided valuable insights into the genetic improvement of bitter gourd pericarp color.

UNASSIGNED: Branching angle is an essential trait in determining the planting density of rapeseed (Brassica napus L.) and hence the yield per unit area. However, the mechanism of branching angle formation in rapeseed is not well understood.
UNASSIGNED: In this study, two rapeseed germplasm with extreme branching angles were used to construct an F2 segregating population; then bulked segregant analysis sequencing (BSA-seq) and quantitative trait loci (QTL) mapping were utilized to localize branching anglerelated loci and combined with transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qPCR) for candidate gene mining.
UNASSIGNED: A branching angle-associated quantitative trait loci (QTL) was mapped on chromosome C3 (C3: 1.54-2.65 Mb) by combining BSA-seq as well as traditional QTL mapping. A total of 54 genes had SNP/Indel variants within the QTL interval were identified. Further, RNA-seq of the two parents revealed that 12 of the 54 genes were differentially expressed between the two parents. Finally, we further validated the differentially expressed genes using qPCR and found that six of them presented consistent differential expression in all small branching angle samples and large branching angles, and thus were considered as candidate genes related to branching angles in rapeseed. Our results introduce new candidate genes for the regulation of branching angle formation in rapeseed, and provide an important reference for the subsequent exploration of its formation mechanism.