Candidate Gene Identification and Transcriptome Analysis of Tomato male sterile-30 and Functional Marker Development for ms-30 and Its Alleles, ms-33, 7B-1, and stamenless-2.

Male sterility is a valuable trait for hybrid seed production in tomato (Solanum lycopersicum). The mutants male sterile-30 (ms-30) and ms-33 of tomato exhibit twisted stamens, exposed stigmas, and complete male sterility, thus holding potential for application in hybrid seed production. In this study, the ms-30 and ms-33 loci were fine-mapped to 53.3 kb and 111.2 kb intervals, respectively. Tomato PISTILLATA (TPI, syn. SlGLO2), a B-class MADS-box transcription factor gene, was identified as the most likely candidate gene for both loci. TPI is also the candidate gene of tomato male sterile mutant 7B-1 and sl-2. Allelism tests revealed that ms-30, ms-33, 7B-1, and sl-2 were allelic. Sequencing analysis showed sequence alterations in the TPI gene in all these mutants, with ms-30 exhibiting a transversion (G to T) that resulted in a missense mutation (S to I); ms-33 showing a transition (A to T) that led to alternative splicing, resulting in a loss of 46 amino acids in protein; and 7B-1 and sl-2 mutants showing the insertion of an approximately 4.8 kb retrotransposon. On the basis of these sequence alterations, a Kompetitive Allele Specific PCR marker, a sequencing marker, and an Insertion/Deletion marker were developed. Phenotypic analysis of the TPI gene-edited mutants and allelism tests indicated that the gene TPI is responsible for ms-30 and its alleles. Transcriptome analysis of ms-30 and quantitative RT-PCR revealed some differentially expressed genes associated with stamen and carpel development. These findings will aid in the marker-assisted selection for ms-30 and its alleles in tomato breeding and support the functional analysis of the TPI gene.

Tomato protein Rx4 mediates the hypersensitive response to Xanthomonas euvesicatoria pv. perforans race T3.

Bacterial spot, which is caused by several Xanthomonas species, is an economically important disease in tomato (Solanum lycopersicum). Great efforts have been made for the identification of resistant sources and the genetic analysis of resistance. However, the development of resistant commercial varieties is slow due to the existence of multiple species of the pathogen and a poor understanding of the resistance mechanism in tomato. The current study revealed that the Rx4 gene encodes a nucleotide-binding leucine-rich repeat protein in the wild tomato species Solanum pimpinellifolium and specifically recognizes and confers a hypersensitive response (HR) to Xanthomonas euvesicatoria pv. perforans race T3 expressing the AvrXv3 avirulence protein. Complementation of the Rx4 gene in the susceptible tomato line Ohio 88119 using a transgenic approach resulted in HR, whereas knockout of the gene through CRISPR/Cas9 editing in resistant lines Hawaii 7981 and PI 128216 led to non-HR to race T3. Transcription of Rx4 was not induced by the presence of race T3. Furthermore, the Rx4 protein did not show physical interaction with AvrXv3 but interacted with SGT1-1 and RAR1. Virus-induced gene silencing of SGT1-1 and RAR1 in the resistant line PI128216 suppressed the HR to race T3. Taken together, our study confirms Rx4 is the gene conferring the HR to bacterial spot race T3 and reveals the potential roles of SGT1-1 and RAR1 as signals in the Rx4-mediated HR. This discovery represents a step forward in our understanding of the mechanism of resistance to bacterial spot in tomato and may have important implications for understanding plant-bacterial interactions.

Mapping and characterization of the Rx3 gene for resistance to Xanthomonas euvesicatoria pv. euvesicatoria race T1 in tomato.

KEY MESSAGE: Rx3 encodes a typical CC-NBS-LRR resistance protein and confers the resistance to Xanthomonas euvesicatoria pv. euvesicatoria race T1 causing bacterial spot in tomato. Bacterial spot caused by at least four species of Xanthomonas is an epidemic disease severely affecting tomato production worldwide. The use of resistant cultivars is an economical and effective approach to control the disease. An unimproved tomato breeding line Hawaii 7988 has been considered as the most reliable source for resistance to X. euvesicatoria pv. euvesicatoria race T1, and the Rx3 locus located at a 4.53-Mb region on chromosome 5 (SL4.0) is the major locus for resistance to race T1 in this line. In the current study, the Rx3 locus was firstly located to a 1.05-Mb region based on comparisons of marker polymorphisms between the susceptible line Ohio 88119 and resistant lines Hawaii 7998, Ohio 9834 and FG02-7530. Using recombinant inbred lines (F5:6, F6:7, and F7:8) derived from a cross between Ohio 88119 and Ohio 9834, the Rx3 locus was finally mapped to a 64.3-kb interval between markers MG-Rx3-4 and MG-Rx3-A6. The Solyc05g053980 gene, designated as Rx3, encoding a coiled-coil nucleotide-binding leucine-rich repeat protein was considered as the candidate for the Rx3 locus. Expression of the gene could be induced by the infection of race T1 strain. Knockout of the Solyc05g053980 gene through CRISPR/Cas9 editing system in the resistant line FG02-7530 decreased resistance to race T1 strain. These results provide a close step for understanding the resistance mechanism to race T1 in Hawaii 7998 and guide tomato breeders accordingly to improve bacterial spot disease resistance in tomato.

Mapping of CaPP2C35 involved in the formation of light-green immature pepper (Capsicum annuum L.) fruits via GWAS and BSA.

KEY MESSAGE: Genome-wide association study, bulked segregant analysis, and genetic analysis delimited the LG locus controlling light-green immature pepper fruits into a 35.07 kbp region on chromosome 10. A strong candidate gene, CaPP2C35, was identified in this region. In pepper (Capsicum annuum L.), the common colors of immature fruits are yellowish white, milky yellow, green, purple, and purplish black. Genes related to dark green, white, and purple immature fruits have been cloned; however, only a few studies have investigated light-green immature fruits. Here, we performed a genetic study using light-green (17C827) and green (17C658) immature fruits. The light-green color of immature fruits was controlled by a single locus-dominant genetic trait compared with the green color of immature fruits. We also performed a genome-wide association study and bulked segregant analysis of immature-fruit color and mapped the LG locus to a 35.07 kbp region on chromosome 10. Only one gene, Capana10g001710, was found in this region. A G-A substitution occurred at the 313th base of the Capana10g001710 coding sequence in 17C827, resulting in the conversion of the α-helix of its encoded PP2C35 protein into a ß-fold. The expression of Capana10g001710 (termed CaPP2C35) in 17C827 was significantly higher than in 17C658. Silencing CaPP2C35 in 17C827 resulted in an increase in chlorophyll content in the exocarp and the appearance of green stripes on the surface of the fruit. These results indicate that CaPP2C35 may be involved in the formation of light-green immature fruits by regulating the accumulation of chlorophyll content in the exocarp. Thus, these findings lay the foundation for further studies and genetic improvement of immature-fruit color in pepper.

A high-continuity and annotated tomato reference genome.

BACKGROUND: Genetic and functional genomics studies require a high-quality genome assembly. Tomato (Solanum lycopersicum), an important horticultural crop, is an ideal model species for the study of fruit development. RESULTS: Here, we assembled an updated reference genome of S. lycopersicum cv. Heinz 1706 that was 799.09 Mb in length, containing 34,384 predicted protein-coding genes and 65.66% repetitive sequences. By comparing the genomes of S. lycopersicum and S. pimpinellifolium LA2093, we found a large number of genomic fragments probably associated with human selection, which may have had crucial roles in the domestication of tomato. We also used a recombinant inbred line (RIL) population to generate a high-density genetic map with high resolution and accuracy. Using these resources, we identified a number of candidate genes that were likely to be related to important agronomic traits in tomato. CONCLUSION: Our results offer opportunities for understanding the evolution of the tomato genome and will facilitate the study of genetic mechanisms in tomato biology.

Transcription of lncRNA ACoS-AS1 is essential to trans-splicing between SlPsy1 and ACoS-AS1 that causes yellow fruit in tomato.

Phytoene synthase (PSY) has been considered as an important regulatory enzyme in carotenoids biosynthesis pathway. Previous study finds that the yellow fruit in Solanum lycopersicum var. cerasiforme accession PI 114490 is caused by loss-of-function of SlPSY1 due to trans-splicing between SlPsy1 and an unknown gene transcribed from neighbour opposite strand DNA of SlPsy1. The genomic DNA sequences of SlPsy1 between red and yellow-fruited tomato lines have one single-nucleotide polymorphism (SNP) in the fourth intron and one SSR in the intergenic region. In the current study, the cause of trans-splicing event was further investigated. The data showed that the previously defined unknown gene was a putative long non-coding RNA ACoS-AS1 with three variants in many yellow-fruited tomato lines. The intronic SNP and intergenic SSR were tightly associated with trans-splicing event SlPsy1-ACoS-AS1. However, transgenic tomato lines carrying the genomic DNA of SlPsy1 from PI 114490 did not generate transcripts of ACoS-AS1and SlPsy1-ACoS-AS1 suggesting that only the intronic SNP could not cause the trans-splicing event. Over-expression of SlPsy1-ACoS-AS1 in red-fruited tomato line M82 did not have any phenotype change while over-expression of wild type SlPsy1 resulted in altered leaf colour. Sub-cellular localization analysis showed that SlPSY1-ACoS-AS1 could not enter plastids where SlPSY1 has its enzyme activity. Mutation of ACoS-AS1 in PI 114490 generated by CRISPR/Cas9 techniques resulted in red fruits implying that ACoS-AS1 was essential to trans-splicing event SlPsy1-ACoS-AS1. The results obtained here will extend knowledge to understand the mechanism of trans-splicing event SlPsy1-ACoS-AS1 and provide additional information for the regulation of carotenoids biosynthesis.

Fine mapping and molecular marker development of the Sm gene conferring resistance to gray leaf spot (Stemphylium spp.) in tomato.

KEY MESSAGE: The tomato gray leaf spot resistance gene Sm was fine-mapped in a 185-kb region through a map-based cloning strategy and genome-wide association study; a candidate gene was proved to be involved in Sm-mediated resistance through transient gene silencing. Gray leaf spot, caused by Stemphylium spp., is a warm weather foliar disease in tomato (Solanum lycopersicum L). Resistance against gray leaf spot is conferred by a single incompletely dominant gene (Sm) located on chromosome 11. This study aimed to map and identify molecular marker tightly linked to the Sm gene for the use of marker-assisted selection in breeding. Using an F2 population derived from a cross between the resistant line '9706' and the susceptible line 'Heinz 1706', the Sm gene was mapped to a 185-kb interval between two markers, InDel343 and InDel-FT-32 on chromosome 11, which was consistent with the result of a genome-wide association study using 289 diverse accessions. An ORF predicted in this region was proved to be involved in Sm-mediated resistance through transient gene silencing and seems to be a good candidate of the Sm locus. To clone the Sm gene, a bacterial artificial chromosome (BAC) library was screened and one BAC clone B80B15 containing the predicted ORF was identified. The analysis of sequence and structure characteristics demonstrated that the candidate gene was not a typical type resistance gene. Additionally, a co-dominant marker Sm-InDel, which produced a 122-bp or 140-bp fragment for resistant or susceptible alleles, respectively, was developed. This marker was validated in 289 germplasm and could be used in marker-assisted selection for gray leaf spot resistance.

Genome-Wide Correlation of 36 Agronomic Traits in the 287 Pepper (Capsicum) Accessions Obtained from the SLAF-seq-Based GWAS.

There are many agronomic traits of pepper (Capsicum L.) with abundant phenotypes that can benefit pepper growth. Using specific-locus amplified fragment sequencing (SLAF-seq), a genome-wide association study (GWAS) of 36 agronomic traits was carried out for 287 representative pepper accessions. To ensure the accuracy and reliability of the GWAS results, we analyzed the genetic diversity, distribution of labels (SLAF tags and single nucleotide polymorphisms (SNPs)) and population differentiation and determined the optimal statistical model. In our study, 1487 SNPs were highly significantly associated with 26 agronomic traits, and 2126 candidate genes were detected in the 100-kb region up- and down-stream near these SNPs. Furthermore, 13 major association peaks were identified for 11 key agronomic traits. Then we examined the correlations among the 36 agronomic traits and analyzed SNP distribution and found 37 SNP polymerization regions (total size: 264.69 Mbp) that could be selected areas in pepper breeding. We found that the stronger the correlation between the two traits, the greater the possibility of them being in more than one polymerization region, suggesting that they may be linked or that one pleiotropic gene controls them. These results provide a theoretical foundation for future multi-trait pyramid breeding of pepper. Finally, we found that the GWAS signals were highly consistent with those from the nuclear restorer-of-fertility (Rf) gene for cytoplasmic male sterility (CMS), verifying their reliability. We further identified Capana06g002967 and Capana06g002969 as Rf candidate genes by functional annotation and expression analysis, which provided a reference for the study of cytoplasmic male sterility in Capsicum.

Identification of candidate genes underlying genic male-sterile msc-1 locus via genome resequencing in Capsicum annuum L.

KEY MESSAGE: Based on genome resequencing, a strong candidate gene Capana02g002096 was identified in this study. Capana02g002096 encodes a homolog of AtDYT1 which is a bHLH transcription factor and involves in the early tapetal development. Genic male-sterile line is an efficient tool for commercial hybrid seed production in pepper; however, so far, only few genes controlling this trait have been cloned. A spontaneous genic male-sterile mutant, msc-1, had been identified and widely used in China, of which the male-sterile trait was proved to be controlled by a single recessive locus. For cloning the gene(s) underlying the msc-1 locus, genome resequencing and comparison analyses were performed between male-sterile and male-fertile lines. According to the genomic variations and genes' annotations, Capana02g002096 was selected as a candidate gene underlying the msc-1 locus. Capana02g002096 encodes a homolog of AtDYT1, which is a bHLH transcription factor and involves in the early tapetal development. Moreover, a 7-bp deletion was identified in the exon of Capana02g002096, which led to a premature stop codon and may cause a loss-of-function mutation. Further genotyping in the 16C1369AB population containing 1110 plants, a F2 population consisting of 510 plants and 46 inbreed lines revealed that the male-sterile phenotype was co-segregated with the 7-bp deletion. Additionally, real-time PCR analysis revealed that Capana02g002096 was an anther-specific gene and repression of the gene's expression through VIGS led to male-sterile phenotype. Therefore, based on the evidence at genetic, genomic, transcriptional and posttranscriptional levels, Capana02g002096 was considered as a strong candidate gene underlying the msc-1 locus in pepper and was renamed Msc-1.

The Aborted Microspores (AMS)-Like Gene Is Required for Anther and Microspore Development in Pepper (Capsicum annuum L.).

Pepper (Capsicum annuum L.) is an economically important vegetable crop worldwide. Although many genes associated with anther and pollen development have been identified, little is known about the mechanism of pollen abortion in pepper. Here, we identified and isolated two putative aborted microspore (AMS) isoforms from pepper flowers: CaAMS1 and CaAMS2. Sequence analysis showed that CaAMS2 was generated by retention of the fourth intron in CaAMS1 pre-mRNA. CaAMS1 encodes a putative protein with a basic helix-loop-helix (bHLH) domain belonging to the MYC subfamily of bHLH transcription factors, and it is localized to the nucleus. Truncated CaAMS2-1 and CaAMS2-2 are produced by alternative splicing. Quantitative real-time PCR analysis showed that CaAMS (referred to CaAMS1 and CaAMS2-2) was preferentially expressed in stamens and its expression level gradually decreases with flower development. RNA in situ hybridization analysis showed that CaAMS is strongly expressed in the tapetum at the tetrad and uninucleate stages. Downregulation of CaAMS led to partial shortened filaments, shriveled, indehiscent stamens and abortive pollens in pepper flowers. Several genes involved in pollen exine formation were downregulated in defective CaAMS-silenced anthers. Thus, CaAMS seems to play an important role in pepper tapetum and pollen development by regulating a complex genetic network.

Association Analysis for Bacterial Spot Resistance in a Directionally Selected Complex Breeding Population of Tomato.

Bacterial spot of tomato is caused by at least four species of Xanthomonas with multiple physiological races. We developed a complex breeding population for simultaneous discovery of marker-trait linkage, validation of existing quantitative trait loci (QTL), and pyramiding of resistance. Six advanced accessions with resistance from distinct sources were crossed in all combinations and their F1 hybrids were intercrossed. Over 1,100 segregating progeny were evaluated in the field following inoculation with X. euvesicatoria race T1 strains. We selected 5% of the most resistant and 5% of the most susceptible progeny for evaluation as plots in two subsequent replicated field trials inoculated with T1 and T3 (X. perforans) strains. The estimated heritability of T1 resistance was 0.32. In order to detect previously reported resistance genes, as well as novel QTL, we explored methods to correct for population structure and analysis based on single markers or haplotypes. Both single-point and haplotype analyses identified strong associations in the genomic regions known to carry Rx-3 (chromosome 5) and Rx-4/Xv3 (chromosome 11). Accounting for kinship and structure generally improved the fit of statistical models. Detection of known loci was improved by adding kinship or a combination of kinship and structure using a Q matrix from model-based clustering. Additional QTL were detected on chromosomes 1, 4, 6, and 7 for T1 resistance and chromosomes 2, 4, and 6 for T3 resistance (P < 0.01). Haplotype analysis improved our ability to trace the origin of positive alleles. These results demonstrate that both known and novel associations can be identified using complex breeding populations that have experienced directional selection.

A chimeric transcript containing Psy1 and a potential mRNA is associated with yellow flesh color in tomato accession PI 114490.

Carotenoid content is the primary determinant of fruit color that affects nutritional value and appearance in tomato. Phytoene synthase (PSY) is the key regulatory enzyme in the carotenoid biosynthesis pathway. Absent function of PSY1 in tomato fruit results in yellow flesh phenotype. We, here, report that two different transcripts, a wild-type (Psy1) and a chimeric mRNA (Psy1/Unknown), exist in a yellow-fruited tomato accession PI 114490. Psy1/Unknown is generated by joining exons from two different genes, Psy1 and an unknown gene, transcribed using both complementary DNA strands. The Psy1 shows low expression in the fruit of PI 114490, while the expression of Psy1/Unknown in the fruit of PI 114490 shows the same pattern as Psy1 in red fruit. The PSY1/Unknown has a lower function than PSY1 in a bacterial expression system. Coincidence of one single-nucleotide polymorphism (SNP) in the fourth intron and one simple sequence repeat (SSR) with 19 AT repeats in the downstream sequence of Psy1 gene with Psy1/Unknown in a set of yellow-fruited tomato lines indicates that Psy1/Unknown might be caused by the SNP and/or SSR. One possible explanation of these observations is trans-splicing. Severely reduced Psy1 transcript caused by Psy1/Unknown results in low accumulation of carotenoid and yellow flesh in PI 114490.

Acyl-CoA synthetase 1 plays an important role on pollen development and male fertility in tomato.

The development of pollen is critical to male reproduction in flowering plants. Acyl-CoA synthetase (ACOS) genes play conserved functions in regulating pollen development in various plants. Our previous work found that knockout of the SlACOS1 gene in tomato might decrease fruit setting. The current study further revealed that SlACOS1 was important to pollen development and male fertility. The SlACOS1 gene was preferentially expressed in the stamen of the flower with the highest expression at the tetrad stage of anther development. Mutation of the SlACOS1 gene by the CRISPR/Cas9-editing system reduced pollen number and viability as well as fruit setting. The tapetum layer exhibited premature degradation and the pollen showed abnormal development appearing irregular, shriveled, or anucleate in Slacos1 mutants at the tetrad stage. The fatty acid metabolism in anthers was significantly impacted by mutation of the SlACOS1 gene. Furthermore, targeted fatty acids profiling using GC-MS found that contents of most fatty acids except C18:1 and C18:2 were reduced. Yeast complementation assay demonstrated that the substrate preferences of SlACOS1 were C16:0 and C18:0 fatty acids. Male fertility of Slacos1 mutant could be slightly restored by applying exogenous palmitic acid, a type of C16:0 fatty acid. Taken together, SlACOS1 played important roles on pollen development and male fertility by regulating the fatty acid metabolism and the development of tapetum and tetrad. Our findings will facilitate unraveling the mechanism of pollen development and male fertility in tomato.

Optimized carbonization of coffee shell via response surface methodology: A circular economy approach for environmental remediation.

The disposal of coffee shell waste on farmland, is a common practice that can causing the environmental and waste valuable resources. Carbonization has been identified as an effective method for transforming coffee shells into useful products that mitigate environmental pollution. Through the response surface methodology, the carbonization conditions of the coffee shells were optimized and its potential as a biochar-based slow-release urea fertilizer was explored. Experiments were conducted on coffee shell performance under varying carbonization conditions such as temperature (600-1000 °C), time (1-5 h), and heating rate (5-20 °C/min). The results indicated that the ideal urea adsorption was 56.3 mg/g, achieved under carbonization conditions of 2.83 h, 809 °C, and 15.3 °C/min. The optimal nutrient release rate within seven days was 45.4% under carbonization conditions of 3.19 h, 813 °C, and 15.0 °C/min. The infrared spectroscopy analysis indicates that carbonization conditions influenced the absorption peak intensity of coffee shell biochar, while the functional group types remain unchanged. The biochar exhibits diverse functional groups and abundant pores, making it a promising candidate for use as a biochar-based fertilizer material. Overall, the findings demonstrate an effective waste management approach that significantly reduces environmental pollutants while remediating pollution.

Self-powered and speed-adjustable sensor for abyssal ocean current measurements based on triboelectric nanogenerators.

The monitoring of currents in the abyssal ocean is an essential foundation of deep-sea research. The state-of-the-art current meter has limitations such as the requirement of a power supply for signal transduction, low pressure resistance, and a narrow measurement range. Here, we report a fully integrated, self-powered, highly sensitive deep-sea current measurement system in which the ultra-sensitive triboelectric nanogenerator harvests ocean current energy for the self-powered sensing of tiny current motions down to 0.02 m/s. Through an unconventional magnetic coupling structure, the system withstands immense hydrostatic pressure exceeding 45 MPa. A variable-spacing structure broadens the measuring range to 0.02-6.69 m/s, which is 67% wider than that of commercial alternatives. The system successfully operates at a depth of 4531 m in the South China Sea, demonstrating the record-deep operations of triboelectric nanogenerator-based sensors in deep-sea environments. Our results show promise for sustainable ocean current monitoring with higher spatiotemporal resolution.

Southern Tibetan rifting since late Miocene enabled by basal shear of the underthrusting Indian lithosphere.

Syncontractional extension is prominent in present-day Tibet, but its origin remains vigorously debated. Several deep-seated geodynamic processes (e.g., Indian underthrusting, horizontal flow, and mantle upwelling) have been linked to Tibetan rifting. Indian underthrusting is a good candidate because it can well explain why surface rifts are more prominent south of the Bangong-Nujiang suture; however, how Indian underthrusting causes extension is not well understood and lacks observational constraints. Seismic anisotropy, measured by exploiting the birefringence effect of shear waves, can be indicative of the deformation styles within the crust. Here, we unveil the dominant convergence-parallel alignment of anisotropic fabrics in the deep crust of the southern Tibetan rifts using seismic recordings collected from our recently deployed and existing seismic stations. This finding suggests that the strong north-directed shearing exerted by the underthrusting Indian plate is key to enabling present-day extension in southern Tibet.

Fine mapping and analysis of a candidate gene in tomato accession PI128216 conferring hypersensitive resistance to bacterial spot race T3.

Bacterial spot caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans and X. gardneri is one of the most destructive diseases in tomatoes (Solanum lycopersicum L.) growing in tropical and subtropical regions. Exploring resistance genes from diverse germplasm and incorporating them into cultivated varieties are critical for controlling this disease. The S. pimpinellifolium accession PI128216 was reported to carry the Rx4 gene on chromosome 11 conferring hypersensitivity and field resistance to race T3. To facilitate the use of marker-assisted selection in breeding and map-based cloning of the gene, an F(2) population derived from a cross between the susceptible variety OH88119 and the resistant accession PI128216 was created for fine mapping of the Rx4 gene. Using 18 markers developed through various approaches, we mapped the gene to a 45.1-kb region between two markers pcc17 and pcc14 on chromosome 11. A NBS-LRR class of resistance gene was identified as the candidate for the Rx4 gene based on annotation results from the International Tomato Annotation Group. Comparison of the genomic DNA sequences of the Rx4 alleles in PI128216 and OH88119 revealed a 6-bp insertion/deletion (InDel) and eight SNPs. The InDel marker was successfully used to distinguish resistance and susceptibility in 12 tomato lines. These results will facilitate cloning the Rx4 gene and provide a useful tool for marker-assisted selection of this gene in tomato breeding programs.

Does Non-Food Cultivation of Cropland Increase Farmers' Income?

The production of cash crops is often regarded as an effective way to increase farmers' income. This study evaluates the impact of non-food cultivation of cropland on farmers' income by using the least-squares (OLS) model in Zhejiang Province, eastern China. Farmers are further divided into different groups according to their income levels to analyze the different impacts of non-food cultivation on their household income. The result shows that non-food cultivation has a significant negative effect on farmers' income, with a more pronounced effect on farmers with a relatively low income. Accordingly, the increase in the proportion of cash crops that are grown does not increase the income of farmers in Zhejiang; instead, this harms their income. Therefore, farmers in Zhejiang should not rely on the cultivation of cash crops for their prosperity but must focus on participating in non-farm employment to increase their household income.

Spatiotemporal Pattern of Urban-Rural Integration Development and Its Driving Mechanism Analysis in Hangzhou Bay Urban Agglomeration.

The quantitative analysis of the urban-rural integration development (URID) level and its driving factors is of great significance for the new-type urbanization of urban agglomerations. This study constructed a multidimensional framework in the perspective of a population-space-economy-society-ecology framework to measure the URID level from 2000 to 2020 and further explored the driving mechanism of the URID changes by a geographical detector model in the Hangzhou Bay urban agglomeration (HBUA). The results showed that the land-use change in the HBUA from 2000 to 2020 showed a typical characteristic of the transition between cultivated and construction land. The URID level in the HBUA improved from 0.294 in 2000 to 0.563 in 2020, and the year 2005 may have been the inflection point of URID in the HBUA. The URID level showed a significant spatial aggregation with high values. Hangzhou, Jiaxing, and Ningbo were hot spots since 2015, and the cold spots were Huzhou and Shaoxing. The population and spatial integration had more important impacts on URID levels in 2000, 2005, and 2020, while economic and social integration had more significant impacts on URID levels in 2010 and 2015. This study provided a deeper understanding of the evolution of URID in an urban agglomeration and could be used as a reference for decision makers.