园艺作物种质资源与遗传育种Horticulture — Genetics · Breeding · Germplasm resources
Early flowering promotes early maturity, production, and the capacity to counteract biotic and abiotic stresses, making it an important agronomic trait in zucchini. The present study demonstrated that the zucchini inbred line ‘19’ consistently flowered early, taking significantly fewer days to bloom the first female flower (DFF) than the inbred line ‘113’. Genetic analysis revealed that DFF, an inheritable quantitative trait, is controlled by multiple genes. Based on the strategy of quantitative trait locus (QTL) sequencing (QTL-seq) combined with linkage analysis, three QTLs for DFF were identified on chromosomes 4, 11, and 20. This study used additional F2 populations grown under different environmental conditions for QTL mapping analysis of DFF with insertion/deletion (InDel) markers to validate these results. Using the composite interval mapping (CIM) method of R/qtl software, we only identified one major locus under all environmental conditions, located in a 117-kb candidate region on chromosome 20. Based on gene annotation, gene sequence alignment, and qRT-PCR analysis, we found that the Cp4.1LG20g08050 gene encoding a RING finger protein may be a candidate gene for the opposite regulation of early flowering in zucchini. In summary, these results lay a foundation for a better understanding of early flowering and improving early flowering-based breeding strategies in zucchini.
Chinese cabbage is an economically important Brassica vegetable worldwide, and clubroot, which is caused by the soil-borne protist plant pathogen Plasmodiophora brassicae is regarded as a destructive disease to Brassica crops. Previous studies on the gene transcripts related to Chinese cabbage resistance to clubroot mainly employed RNA-seq technology, although it cannot provide accurate transcript assembly and structural information. In this study, PacBio RS II SMRT sequencing was used to generate full-length transcriptomes of mixed roots at 0, 2, 5, 8, 13, and 22 days after P. brassicae infection in the clubroot-resistant line DH40R. Overall, 39 376 high-quality isoforms and 26 270 open reading frames (ORFs) were identified from the SMRT sequencing data. Additionally, 426 annotated long noncoding RNAs (lncRNAs), 56 transcription factor (TF) families, 1 883 genes with poly(A) sites and 1 691 alternative splicing (AS) events were identified. Furthermore, 1 201 of the genes had at least one AS event in DH40R. A comparison with RNA-seq data revealed six differentially expressed AS genes (one for disease resistance and five for defensive response) that are potentially involved in P. brassicae resistance. The results of this study provide valuable resources for basic research on clubroot resistance in Chinese cabbage.
Various genetic and biochemical characteristics exist in tea plant cultivars, and they largely determine production suitability and tea quality. Here, we performed transcriptomic and metabolomic analyses of young shoots of seven tea cultivars and identified major regulatory transcription factors (TFs) for the characteristic metabolites in different cultivars based on weighted gene co-expression network analysis (WGCNA). Phenotypically, we found that ‘Tieguanyin’ (TGY) and ‘Fujian Shuixian’ (FJSX), which are suitable for oolong tea, had higher catechin contents. The metabolites of ‘Jinxuan’ (JX) were more prominent, especially the contents of phenolic acids, flavonoids, terpenes, and tannins, which were higher than those of the other six cultivars. Moreover, ‘Fudingdabai’ (FDDB), which is suitable for white tea, was rich in amino acids, linolenic acid, and saccharides. At the molecular level, hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HCT) (CsTGY12G0001876, and CsTGY06G0003042) led to the accumulation of chlorogenic acid in TGY. The main reason for the higher l-ascorbic acid content in FJSX was the high expression levels of L-galactono-1,4-lactone hydrogenase (GalLDH) (CsTGY13G0000389) and Myo-inositol oxygenase (MIOX) (CsTGY14G0001769, and CsTGY14G0001770), which were regulated by WRKY (CsTGY11G0001197). Furthermore, FDDB, ‘Longjing 43’ (LJ43), ‘Shuchazao’ (SCZ) and ‘Baihaozao’ (BHZ) had higher free fatty acid contents, among which MYB (CsTGY14G0002344) may be a hub gene for the regulation of palmitoleic acid accumulation. More importantly, we found that the shoots of TGY were green with purple, mainly due to the accumulation of anthocyanins and the downregulation of the Mg-protoporphyrin IX nonomethyl ester cyclase (MPEC) (CsTGY10G0001989) gene that affects chlorophyll synthesis. These results will provide a theoretical reference for tea cultivar breeding and suitability.
Flesh firmness (FF) is an important and complex trait for melon breeders and consumers. However, the genetic mechanism underlying FF is unclear. Here, a soft fruit melon (P5) and a hard fruit melon (P10) were crossed to generate F2, and the FF and fruit-related traits were recorded for two years. By performing quantitative trait locus (QTL) specific-locus amplified fragment (SLAF) (QTL-SLAF) sequencing and molecular marker-linkage analysis, 112 844 SLAF markers were identified, and 5 919 SNPs were used to construct a genetic linkage map with a total genetic distance of 1 356.49 cM. Ten FF- and fruit-related QTLs were identified. Consistent QTLs were detected for fruit length (FL) and fruit diameter (FD) in both years, and QTLs for single fruit weight (SFW) were detected on two separate chromosomes in both years. For FF, the consistent major locus (ff2.1) was located in a 0.17-Mb candidate region on chromosome 2. Using 429 F2 individuals derived from a cross between P5 and P10, we refined the ff2.1 locus to a 28.3-kb region harboring three functional genes. These results provide not only a new candidate QTL for melon FF breeding but also a theoretical foundation for research on the mechanism underlying melon gene function.
Isolation and functional analysis of SrMYB1, a direct transcriptional repressor of SrUGT76G1 in Stevia rebaudiana
SrUGT76G1, the most well-studied diterpene glycosyltransferase in Stevia rebaudiana, is key to the biosynthesis of economically important steviol glycosides (SGs). However, the molecular regulatory mechanism of SrUGT76G1 has rarely been explored. In this study, we identified a MYB transcription factor, SrMYB1, using a yeast one-hybrid screening assay. SrMYB1 belongs to the typical R2R3-type MYB protein and is specifically localized in the nucleus with strong transactivation activity. The transcript of SrMYB1 is predominantly accumulated in flowers, but is also present at a lower level in leaves. Yeast one-hybrid and electrophoretic mobility shift assays verified that SrMYB1 binds directly to the MYB binding sites in the F4-3 fragment (+50–(–141)) of the SrUGT76G1 promoter. Furthermore, we found that SrMYB1 could significantly repress the expression of SrUGT76G1 in both epidermal cells of tobacco leaves and stevia callus. Taken together, our results demonstrate that SrMYB1 is an essential upstream regulator of SrUGT76G1 and provide novel insight into the regulatory network for the SGs metabolic pathway in S. rebaudiana.
The tea plant [Camellia sinensis (L.) O. Kuntze] is an industrial crop in China. The Anhui Province has a long history of tea cultivation and has a large resource of tea germplasm with abundant genetic diversity. To reduce the cost of conservation and utilization of germplasm resources, a core collection needs to be constructed. To this end, 573 representative tea accessions were collected from six major tea-producing areas in Anhui Province. Based on 60 pairs of simple sequence repeat (SSR) markers, phylogenetic relationships, population structure and principal coordinate analysis (PCoA) were conducted. Phylogenetic analysis indicated that the 573 tea individuals clustered into five groups were related to geographical location and were consistent with the results of the PCoA. Finally, we constructed a core collection consisting of 115 tea individuals, accounting for 20% of the whole collection. The 115 core collections were considered to have a 90.9% retention rate for the observed number of alleles (Na), and Shannon’s information index (I) of the core and whole collections were highly consistent. Of these, 39 individuals were preserved in the Huangshan area, accounting for 33.9% of the core collection, while only 10 individuals were reserved in the Jinzhai County, accounting for 8.9% of the core set. PCoA of the accessions in the tea plant core collection exhibited a pattern nearly identical to that of the accessions in the entire collection, further supporting the broad representation of the core germplasm in Anhui Province. The results demonstrated that the core collection could represent the genetic diversity of the original collection. Our present work is valuable for the high-efficiency conservation and utilization of tea plant germplasms in Anhui Province
Tea is one of the most popular non-alcoholic beverages in the world, and free amino acids, especially theanine, make a major contribution to the umami taste of tea. However, the genetic basis of the variation in amino acid content in tea plants remains largely unknown. Here, we measured the free amino acid content in fresh leaves of 174 tea accessions over two years using a targeted metabolomics approach and obtained genotype data via RNA sequencing. Genome-wide association studies were conducted to investigate loci affecting the content of free amino acids. A total of 69 quantitative trait loci (–log10(P-value)>5) were identified. Functional annotation revealed that branched-chain amino acid aminotransferase, glutamine synthetase, nitrate transporter, and glutamate decarboxylase might be important for amino acid metabolism. Two significant loci, glutamine synthetase (Glu1, P=3.71×10–4; Arg1, P=4.61×10–5) and branched-chain amino acid aminotransferase (Val1, P=4.67×10–5; I_Leu1, P=3.56×10–6), were identified, respectively. Based on the genotyping result, two alleles of CsGS (CsGS-L and CsGS-H) and CsBCAT (CsBCAT-L and CsBCAT-H) were selected to perform function verification. Overexpression of CsGS-L and CsGS-H enhanced the contents of glutamate and arginine in transgenic plants, and overexpression of CsBCAT-L and CsBCAT-H promoted the accumulation of valine, isoleucine and leucine. Enzyme activity assay uncovered that SNP1054 is important for CsGS catalyzing glutamate into glutamine. Furthermore, CsGS-L and CsGS-H differentially regulated the accumulation of glutamine, and CsBCAT-L and CsBCAT-H differentially regulated the accumulation of branched-chain amino acids. In summary, the findings in our study would provide new insights into the genetic basis of amino acids contents variation in tea plants and facilitate the identification of elite genes to enhance amino acids content.
Molecular marker-assisted selection (MAS) can significantly accelerate and improve the efficiency of the breeding process in seedless grape cultivars. In this study, we developed the KASP_VviAGL11 and VviAGL11_410 markers based on a single nucleotide polymorphism (SNP) site (Chr18: 26889437 (A/C)) of the VviAGL11 gene, and compared them with previously reported SSR markers p3_VvAGL11 and 5U_VviAGL11 by testing 101 cultivars and 81 F1 hybrid progenies. The results showed that both of the proposed markers obtained 100% accuracy rates in detecting allele A, which was closely associated with the seedless trait in grapes, while p3_VvAGL11 and 5U_VviAGL11 had lower accuracy rates due to their tendency to produce false positives. After careful evaluation of the technical advantages and disadvantages associated with these markers, we concluded that KASP_VviAGL11 was superior in terms of simplicity, cost-effectiveness, efficiency, and accuracy. Thus, we optimized the process of molecular MAS for seedless grapes, focusing on the KASP_VviAGL11 marker as a central component, to provide key technical support for the development of new seedless grape cultivars.
Genome wide association analysis identifies candidate genes for fruit quality and yield in Actinidia eriantha
Quality and yield are the primary concerns in kiwifruit breeding, but research on the genetic mechanisms of fruit size, shape, and ascorbic acid (ASA) content is currently very limited, which restricts the development of kiwifruit molecular breeding. In this study, we obtained a total of 8.88 million highly reliable single nucleotide polymorphism (SNP) markers from 140 individuals from the natural hybrid offspring of Actinidia eriantha cv. ‘White’ using whole genome resequencing technology. A genome-wide association study was conducted on eight key agronomic traits, including single fruit weight, fruit shape, ASA content, and the number of inflorescences per branch. A total of 59 genetic loci containing potential functional genes were located, and candidate genes related to single fruit weight, fruit length, ASA content, number of inflorescences per branch and other traits were identified within the candidate interval, such as AeWUSCHEL, AeCDK1 (cell cycle dependent kinase), AeAO1 (ascorbic oxidase) and AeCO1 (CONSTANS-like 4). After constructing an RNAi vector for AeAO1 and injecting it into the fruit of cv. ‘Midao 31’ to interfere with the expression of the AeAO1 gene, the results showed that the activity of ascorbic oxidase in the fruit of ‘Midao 31’ significantly decreased, while the content of ASA significantly increased. This study provides valuable insights into the genetic basis of variation in A. eriantha fruit traits, which may benefit molecular marker-assisted breeding efforts.