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Background The Western mosquitofish, Gambusia affinis, is a model for sex chromosome organization and evolution of female heterogamety. We previously identified a G. affinis female-specific marker, orthologous to the aminomethyl transferase (amt) gene of the related platyfish (Xiphophorus maculatus). Here, we have analyzed the structure and differentiation of the G. affinis W-chromosome, using a cytogenomics and bioinformatics approach. Results The long arm of the G. affinis W-chromosome (Wq) is highly enriched in dispersed repetitive sequences, but neither heterochromatic nor epigenetically silenced by hypermethylation. In line with this, Wq sequences are highly transcribed, including an active nucleolus organizing region (NOR). Female-specific SNPs and evolutionary young transposable elements were highly enriched and dispersed along the W-chromosome long arm, suggesting constrained recombination. Wq copy number expanded elements also include female-specific transcribed sequences from the amt locus with homology to TE. Collectively, the G. affinis W-chromosome is actively differentiating by sexspecific copy number expansion of transcribed TE-related elements, but not (yet) by extensive sequence divergence or gene decay. Conclusions The G. affinis W-chromosome exhibits characteristic genomic properties of an evolutionary young sex chromosome. Strikingly, the observed sex-specific changes in the genomic landscape are confined to the W long arm, which is separated from the rest of the W-chromosome by a neocentromere acquired during sex chromosome evolution and may thus have become functionally insulated. In contrast, W short arm sequences were apparently shielded from repeat-driven differentiation, retained Z-chromosome like genomic features, and may have preserved pseudoautosomal properties.

Salinity critically limits rice metabolism, growth, and productivity worldwide. Improvement of the salt resistance of locally grown high-yielding cultivars is a slow process. The objective of this study was to develop a new salt-tolerant rice germplasm using speed-breeding. Here, we precisely introgressed the hst1 gene, transferring salinity tolerance from “Kaijin” into high-yielding “Yukinko-mai” (WT) rice through single nucleotide polymorphism (SNP) marker-assisted selection. Using a biotron speed-breeding technique, we developed a BC3F3 population, named “YNU31-2-4”, in six generations and 17 months. High-resolution genotyping by whole-genome sequencing revealed that the BC3F2 genome had 93.5% similarity to the WT and fixed only 2.7% of donor parent alleles. Functional annotation of BC3F2 variants along with field assessment data indicated that “YNU31-2-4” plants carrying the hst1 gene had similar agronomic traits to the WT under normal growth condition. “YNU31-2-4” seedlings subjected to salt stress (125 mM NaCl) had a significantly higher survival rate and increased shoot and root biomasses than the WT. At the tissue level, quantitative and electron probe microanalyzer studies indicated that “YNU31-2-4” seedlings avoided Na+ accumulation in shoots under salt stress. The “YNU31-2-4” plants showed an improved phenotype with significantly higher net CO2 assimilation and lower yield decline than WT under salt stress at the reproductive stage. “YNU31-2-4” is a potential candidate for a new rice cultivar that is highly tolerant to salt stress at the seedling and reproductive stages, and which might maintain yields under a changing global climate.

Background Cadmium (Cd) is a widespread toxic heavy metal pollutant in agricultural soil, and Cd accumulation in rice grains is a major intake source of Cd for Asian populations that adversely affect human health. However, the molecular mechanism underlying Cd uptake, translocation and accumulation has not been fully understood in rice plants. Results In this study, a mutant displaying extremely low Cd accumulation ( lcd1 ) in rice plant and grain was generated by EMS mutagenesis from indica rice cultivar 9311 seeds. The candidate SNPs associated with low Cd accumulation phenotype in the lcd1 mutant were identified by MutMap and the transcriptome changes between lcd1 and WT under Cd exposure were analyzed by RNA-seq. The lcd1 mutant had lower Cd uptake and accumulation in rice root and shoot, as well as less growth inhibition compared with WT in the presence of 5 μM Cd. Genetic analysis showed that lcd1 was a single locus recessive mutation. The SNP responsible for low Cd accumulation in the lcd1 mutant located at position 8,887,787 on chromosome 7, corresponding to the seventh exon of OsNRAMP5 . This SNP led to a Pro236Leu amino acid substitution in the highly conserved region of OsNRAMP5 in the lcd1 mutant. A total of 1208 genes were differentially expressed between lcd1 and WT roots under Cd exposure, and DEGs were enriched in transmembrane transport process GO term. Increased OsHMA3 expression probably adds to the effect of OsNRAMP5 mutation to account for the significant decreases in Cd accumulation in rice plant and grain of the lcd1 mutant. Conclusions An extremely low Cd mutant lcd1 was isolated and identified using MutMap and RNA-seq. A Pro236Leu amino acid substitution in the highly conserved region of OsNRAMP5 is likely responsible for low Cd accumulation in the lcd1 mutant. This work provides more insight into the mechanism of Cd uptake and accumulation in rice, and will be helpful for developing low Cd accumulation rice by marker-assisted breeding.

Background SWEET is a newly identified family of sugar transporters. Although SWEET transporters have been characterized by using Arabidopsis and rice, very little knowledge of sucrose accumulation in the stem region is available, as these model plants accumulate little sucrose in their stems. To elucidate the expression of key SWEET genes involved in sucrose accumulation of sorghum, we performed transcriptome profiling by RNA-seq, categorization using phylogenetic trees, analysis of chromosomal synteny, and comparison of amino acid sequences between SIL-05 (a sweet sorghum) and BTx623 (a grain sorghum). Results We identified 23 SWEET genes in the sorghum genome. In the leaf, SbSWEET8-1 was highly expressed and was grouped in the same clade as AtSWEET11 and AtSWEET12 that play a role in the efflux of photosynthesized sucrose. The key genes in sucrose synthesis (SPS3) and that in another step of sugar transport (SbSUT1 and SbSUT2) were also highly expressed, suggesting that sucrose is newly synthesized and actively exported from the leaf. In the stem, SbSWEET4-3 was uniquely highly expressed. SbSWEET4-1, SbSWEET4-2, and SbSWEET4-3 were categorized into the same clade, but their tissue specificities were different, suggesting that SbSWEET4-3 is a sugar transporter with specific roles in the stem. We found a putative SWEET4-3 ortholog in the corresponding region of the maize chromosome, but not the rice chromosome, suggesting that SbSWEET4-3 was copied after the branching of sorghum and maize from rice. In the panicle from the heading through to 36 days afterward, SbSWEET2-1 and SbSWEET7-1 were expressed and grouped in the same clade as rice OsSWEET11/Xa13 that is essential for seed development. SbSWEET9-3 was highly expressed in the panicle only just after heading and was grouped into the same clade as AtSWEET8/RPG1 that is essential for pollen viability. Five of 23 SWEET genes had SNPs that caused nonsynonymous amino acid substitutions between SIL-05 and BTx623. Conclusions We determined the key SWEET genes for technological improvement of sorghum in the production of biofuels: SbSWEET8-1 for efflux of sucrose from the leaf; SbSWEET4-3 for unloading sucrose from the phloem in the stem; SbSWEET2-1 and SbSWEET7-1 for seed development; SbSWEET9-3 for pollen nutrition.

Introduction: Physcomitrium patens (Hedw.) Mitten (previously known as Physcomitrella patens ) was collected by H.L.K. Whitehouse in Gransden Wood (Huntingdonshire, United Kingdom) in 1962 and distributed across the globe starting in 1974. Hence, the Gransden accession has been cultured in vitro in laboratories for half a century. Today, there are more than 13 different pedigrees derived from the original accession. Additionally, accessions from other sites worldwide were collected during the last decades.Methods and Results: In this study, 250 high throughput RNA sequencing (RNA-seq) samples and 25 gDNA samples were used to detect single nucleotide polymorphisms (SNPs). Analyses were performed using five different P. patens accessions and 13 different Gransden pedigrees. SNPs were overlaid with metadata and known phenotypic variations. Unique SNPs defining Gransden pedigrees and accessions were identified and experimentally confirmed. They can be successfully employed for PCR-based identification.Conclusion: We show independent mutations in different Gransden laboratory pedigrees, demonstrating that somatic mutations occur and accumulate during in vitro culture. The frequency of such mutations is similar to those observed in naturally occurring populations. We present evidence that vegetative propagation leads to accumulation of deleterious mutations, and that sexual reproduction purges those. Unique SNP sets for five different P. patens accessions were isolated and can be used to determine individual accessions as well as Gransden pedigrees. Based on that, laboratory methods to easily determine P. patens accessions and Gransden pedigrees are presented.

Background Japanese apricot ( Prunus mume Sieb. et Zucc.) is popular for both ornamental and processing value, fruit color affects the processing quality, and red pigmentation is the most obvious phenotype associated with fruit color variation in Japanese apricot, mutations in structural genes in the anthocyanin pathway can disrupt the red pigmentation, while the formation mechanism of the red color trait in Japanese apricot is still unclear.  Results One SNP marker (PmuSNP_27) located within PmUFGT3 gene coding region was found highly polymorphic among 44 different fruit skin color cultivars and relative to anthocyanin biosynthesis in Japanese apricot. Meantime, critical mutations were identified in two alleles of PmUFGT3 in the green-skinned type is inactivated by seven nonsense mutations in the coding region, which leads to seven amino acid substitution, resulting in an inactive UFGT enzyme. Overexpression of the PmUFGT3 allele from red-skinned Japanese apricot in green-skinned fruit lines resulted in greater anthocyanin accumulation in fruit skin. Expression of same allele in an Arabidopsis T-DNA mutant deficient in anthocyanidin activity the accumulation of anthocyanins. In addition, using site-directed mutagenesis, we created a single-base substitution mutation (G to T) of PmUFGT3 isolated from green-skinned cultivar , which caused an E to D amino acid substitution and restored the function of the inactive allele of PmUFGT3 from a green-skinned individual. Conclusion This study confirms the function of PmUFGT3, and provides insight into the mechanism underlying fruit color determination in Japanese apricot, and possible approaches towards genetic engineering of fruit color.

Summary Vitamin A is a crucial yet scarce vitamin essential for maintaining normal metabolism and bodily functions in humans and can only be obtained from food. Carotenoids represent a diverse group of functional pigments that act as precursors for vitamins, hormones, aroma volatiles and antioxidants. As a vital vegetable in the world, elevated carotenoid levels in cucumber fruit produce yellow flesh, enhancing both visual appeal and nutritional value. However, the genetic mechanisms and regulatory networks governing yellow flesh in cucumbers remain inadequately characterized. In this study, we employed map‐based cloning to identify a Carotenoid Cleavage Dioxygenase 1 (CsCCD1) as a key genetic factor influencing yellow flesh in cucumbers. A causal single nucleotide polymorphism (SNP) in the eighth intron of CsCCD1 led to aberrant splicing, resulting in a truncated transcript. The truncated protein has significantly decreased enzyme activity and increased carotenoid accumulation in the fruit. CRISPR/Cas9‐generated CsCCD1 knockout mutants exhibited yellow flesh and significantly higher carotenoid content compared to wild‐type cucumbers. Metabolic profiling indicated a marked accumulation of β‐cryptoxanthin in the flesh of these knockout mutants. The intronic SNP was shown to perfectly segregate with yellow flesh in 159 diverse cucumber germplasms, particularly within the semi‐wild ecotype Xishuangbanna, known for its substantial carotenoid accumulation. Furthermore, transient overexpression of CsCCD1 in yellow‐fleshed Xishuangbanna cucumbers restored a white flesh phenotype, underscoring the critical role of CsCCD1 in determining flesh colour in both cultivated and semi‐wild cucumbers. These findings lay a theoretical foundation for breeding high‐nutrient yellow‐fleshed cucumber varieties.

Background Sweetpotato ( Ipomoea batatas (L.) Lam.) is an essential root crop with several nutritional benefits, including high dietary fiber content. While fiber contributes positively to human health by reducing the risk of metabolic and gastrointestinal diseases, excessive fiber accumulation can negatively impact texture and consumer preference. Despite its importance, the genetic mechanisms underlying fiber content in sweetpotato remain largely unexplored. Therefore, this study aimed to identify the genomic regions and candidate genes associated with fiber content through a genome-wide association study (GWAS). Results Significant phenotypic variation in fiber content were observed among 140 sweetpotato genotypes. The GWAS analysis identified seven significant single nucleotide polymorphisms (SNPs), with Iba_chr07a_20294133 and Iba_chr12a_38616338 consistently detected across the FarmCPU and BLINK models. Notably, three SNPs (Iba_chr01a_17621178, Iba_chr10a_773882, and Iba_chr12a_38616338) showed significant phenotypic differentiation between homozygous alleles, making them promising candidates for marker development. Candidate gene analysis identified four genes with significantly upregulated expression in high-fiber genotypes: IbANT1 (adenine nucleotide transporter BT1), IbCYP86B1 (cytochrome P450 86B1), IbSCR3 (scarecrow-like protein 3), and IbFER (FERONIA receptor-like kinase). These genes are involved in suberin biosynthesis, cell wall remodeling, and metabolic regulation, suggesting their crucial roles in fiber accumulation. Conclusion This study provides novel insights into the genetic regulation of fiber content in sweetpotato. The identification of significant SNPs and candidate genes offers valuable resources for breeding programs targeting fiber optimization. Further validation is essential for the effective application of these SNPs and genes into marker-assisted selection strategies.

Pea ( Pisum sativum L.) is an important high-protein crop that is grown in large areas in many temperate regions of the world. However, the molecular markers of the high content of seed storage proteins in pea remain unknown. The aim of our study was to sequence the genes of transcription factors ABI3 and FUS3 and analyze their expression levels in two groups of pea cultivars and lines, contrastingly differing in seed protein content, in the conditions of the Pre-Ural forest-steppe zone in Russia. By analyzing the seed protein content of 110 cultivars and lines, 12 samples with high protein content (23.5–26.1%) and 10 samples with low protein content (18.0–19.7%) were selected. Three nucleotide substitutions were identified in the ABI3 gene that may be associated with protein accumulation in seeds: 1047 bp (T/C), 1194 bp (C/T), and 1334 bp (A/T), but only the last one resulted in an amino acid substitution in the predicted protein molecule. Three substitutions were also identified in the FUS3 gene that may be associated with protein accumulation in seeds: 669 bp (T/G), 681 bp (T/C), and 996 bp (A/C), but none of these resulted in amino acid substitutions. The expression levels of ABI3 (PsABI3-5, PsABI3-6 isoforms) and FUS3 genes at all stages of seed development depended on the genotype. No correlation between the expression levels of these genes and protein content in seeds could be detected.

Hawk tea (Litsea coreana var. sinensis), derived from the tender shoots or leaves, rich in flavonoids can promote healthcare for humans. The primary flavonoid are kaempferol‐3‐O‐β‐D‐glucoside, kaempferol‐3‐O‐β‐D‐galactoside, quercetin‐3‐O‐β‐D‐glucoside, and quercetin‐3‐O‐β‐D‐galactoside. The existence of an association between leaf phenotype and flavonoid content, along with the underlying mechanisms of flavonoid biosynthesis, remains incompletely understood. In this study, 109 samples were analyzed to determine the correlation and genetic variability in leaf phenotype and flavonoid content. Furthermore, a transcriptome‐wide association study identified candidate loci implicated in the biosynthesis of four key flavonoids. The study revealed that genetic variability in leaf traits and flavonoid concentrations is predominantly attributed to interpopulation differences. Flavonoid accumulation was significantly correlated with tree DBH, indicative of age‐related traits. Transcriptome‐wide association analysis identified 84 significant SNPs associated with flavonoid content, with only 13 located within gene regions. The majority of these genes are implicated in metabolic processes and secondary metabolite biosynthesis. Notably, structural genes within these regions are directly involved in pathways known to regulate flavonoid metabolism, exerting a pivotal influence on flavonoid biosynthesis. These results revealed the physiological basis for the regulation of flavonoid content, as well as the molecular mechanisms for the biosynthesis of flavonoids in hawk tea. It also lays theoretical groundwork for subsequent explorations into the genetic determinants influencing flavonoid accumulation of hawk tea. A total of 84 significant SNPs related to flavonoid content were identified through transcriptome‐wide association analysis, and 13 SNPs located within gene regions were identified as structural genes primarily implicated in metabolic processes and the biosynthesis of secondary metabolites.