STAT3, an oncogene, contributes to insensitivity of chemotherapy and radiotherapy in tumor, reduces the clinical efficacy. Meanwhile, STAT3β, a STAT3 splicing isoform, is related to the inhibition of tumor growth and chemosensitivity. STAT3 may become a potential target to overcome the chemo(radio)resistance, which benefit for developing novel drugs targeting STAT3 or alternative splicing regulators.Chemoradiotherapy is one of the most effective and extensively used strategies for cancer treatment. Signal transducer and activator of transcription 3 (STAT3) regulates vital biological processes, such as cell proliferation and cell growth. It is constitutively activated in various cancers and limits the application of chemoradiotherapy. Accumulating evidence suggests that STAT3 regulates resistance to chemotherapy and radiotherapy and thereby impairs therapeutic efficacy by mediating its feedback loop and several target genes. The alternative splicing product STAT3β is often identified as a dominant-negative regulator, but it enhances sensitivity to chemotherapy and offers a new and challenging approach to reverse therapeutic resistance. We focus here on exploring the role of STAT3 in resistance to receptor tyrosine kinase (RTK) inhibitors and radiotherapy, outlining the potential of targeting STAT3 to overcome chemo(radio)resistance for improving clinical outcomes, and evaluating the importance of STAT3β as a potential therapeutic approach to overcomes chemo(radio)resistance. In this review, we discuss some new insights into the effect of STAT3 and its subtype STAT3β on chemoradiotherapy sensitivity, and we explore how these insights influence clinical treatment and drug development for cancer. Chemoradiotherapy is the main treatment for various solid malignancies through the induction of tumor DNA damage, and it can be used alone or in combination with surgery. Because of the genetic and epigenetic heterogeneity of cancer, the response to chemoradiotherapy is variable, and accompanied by therapeutic toxicities and resistances that limit the effectiveness of chemoradiotherapy ,2,3,4. ISignal transducer and activator of transcription (STAT) proteins are a family of cytoplasmic transcription factors and were discovered by James E. Darnell while investigating interferon signal transduction . Seven m2-4GAA) elements in target genes to modulate gene transcription [STAT3 is identified as an acute-phase response factor that is activated in response to IL-6, growth factors and hormones ,15,16,17cription ,19. STATcription . STAT3β cription . Constitcription ,28,29,30cription ,32,33. Herein, this review focuses on the mechanism of STAT3 in resistance to chemo(radio)therapy and improvement in the therapeutic effect of chemoradiotherapy (CRT)-resistant tumors by targeting STAT3.Mammalian cells use various regulatory mechanisms, involving feedback loops and crosstalk between signaling pathways, to adjust to perturbations in cellular homeostasis . These mDrug-sensitive, oncogene-addicted cancer cells secrete factors that contribute to drug resistance via feedback mechanisms. For example, the secretion of IL-6 in turn stimulates the STAT3 pathway and mediates resistance to PI3K inhibitors . IL-11, In addition, STAT3 on Tyr705 correlates with Burkitt lymphoma (BL) chemoresistance. Zhong’s group revealed that STAT3 confers chemoresistance to cells by mediating the antioxidant feedback . In respRadiotherapy, alone or in combination with surgery and chemotherapy, is an indispensable link in comprehensive cancer treatment. Radiotherapy causes DNA damage directly or indirectly through generation of ROS and induces various forms of cancer cell death, including apoptosis, autography, mitotic catastrophe, necrosis and senescence. Radioresistance of tumor cells is one of the main biological factors that affect clinical outcomes ,53,54,55The development of radioresistance following radiation therapy coincides with the radiation-induced appearance of pSTAT3, epithelial–mesenchymal transition (EMT) and self-renewal. IL6/STAT3/TWIST signaling pathway activation mediates esophageal squamous carcinoma EMT, whereas inactivation of STAT3 by an IL-6 inhibitor or siRNA-mediated downregulation of Twist enhances cancer cell apoptosis and reverses radiation-induced EMT . The STATogether, these findings provide convincing evidence that STAT3 is activated by radiation and contributes to the cancer phenotype. Both STAT3 Y705 and S727 are potential targets for overcoming clinical radioresistance. Interrupting the STAT3 pathway by using STAT3 inhibitors, RNA interference and short hairpin RNA could sensitize radioresistant cancer cells and improve clinical outcomes.Activated STAT3 binds to DNA sequences (GAS element) in target genes and usually correlates with the enhanced expression of anti-apoptotic proteins, prevention of cell cycle arrest and promotion of cell proliferation . A numbeBcl-2, Bcl-xL and Mcl-1 are anti-apoptotic members of the Bcl-2 family and frequently dysregulated in various cancers. STAT3 has been shown to significantly enhance Bcl-2 and Bcl-xL promoter activity. Chromatin immunoprecipitation analysis indicates that activated STAT3 directly binds to the promoters of Bcl-2, Bcl-xL and Mcl-1 genes ,98. HajiThe STAT3/Mcl-1 signaling axis is a target for overcoming resistance to BH3 mimetic ATB-737 (Bcl-2 inhibitor). Sorafenib, a multiple tyrosine kinase inhibitor, has been found to target Mcl-1 and synergize with ATB-737 to induce apoptosis in glioma cells . HoweverExposure of cells to IR (irradiation) can also increase Bcl-2 expression levels, which are associated with radioresistance . In anotAnisomeles indica, could overcome resistance to temozolomide  in glioblastoma. It reduced Slug, Vimentin, N-cadherin and β-catenin and also disrupted STAT3 signaling [Indeed, STAT3 is also important for initiating the epithelial-mesenchymal transition (EMT) program, which is associated with cancer therapy resistance . Snail, ignaling . Moreoveignaling . STAT3 upregulates the expression of Snail, contributes to temozolomide resistance in GBM and is associated with recurrent GBM tumors . AnotherTwist basic helix loop helix transcription factor 1 (Twist1), a regulator of EMT, is upregulated in cisplatin-resistant ovarian cancer cells via STAT3 activation . InhibitSurvivin is an inhibitor of the apoptosis protein family, and its aberrant expression correlates with a poor prognosis and contributes to chemo(radio)resistance . STAT3 iSTAT3/survivin signaling regulates a poor response to radiotherapy in HER2-positive breast cancer , ESCC 55 and lunCyclin D1 peaks during mid-G1 when growth factor-deprived cells re-enter the cell cycle. Previous reports demonstrate that cyclin D1 confers chemo- and radioresistance to several tumor cells ,129,130.Cyclin D1 can be stimulated by low-dose ionizing radiation (LDIR) in human keratinocytes. Inhibition of cyclin D1 using siRNA can inhibit the LDIR-associated anti-apoptotic response and eliminate LDIR-induced adaptive radioresistance . RadioreActivation of STAT3 is important for tumor-induced immunosuppression, which regulates the expression of immunosuppressive molecules . Yu’s st+ CD25+ regulatory T cells are an important population of immunosuppressive cells. Treg cells from 12B1 tumor-bearing animals suppressed the function of DCs in a TGF-β- and IL-10-dependent manner and were associated with the activation of STAT3 [+ T cells have a lower type 2 T helper cell (Th2) immune response and considerably increase the expression level of IL-10 and TGF-β for Th3-like differentiation via the enhanced recruitment of STAT3 to their promoters [IL-10 and TGF-β have been demonstrated to be involved in the suppression of the anti-tumor immune response of Treg cells . Studiesof STAT3 . Suppresromoters . STAT3 has been identified on the promoter of vascular endothelial growth factor (VEGF) and was found to promote the growth and metastasis of human pancreatic cancer cells . ChronicIn various cells and tissues, the ratio of STAT3α to STAT3β ranges from 3:1 to 10:1 at the mRNA level, and from 1:3 to 10:1 at the protein level. Biethahn and colleagues found that STAT3β was expressed at a high level in CD34+ cells by Western blot analysis ,157,158.Given a truncated C-terminal transactivation domain, transcriptional activation by STAT3β probably involves cooperation with other transcription factors, such as c-Jun and STRA13 ,162,163.STAT3β has gained attention as a potent tumor suppressor and a favorable prognostic factor in ESCC with or without chemoradiotherapy, and it strongly correlates with longer overall survival and recurrence-free survival . RecentlAs mentioned earlier, STAT3β is a truncated form of STAT3α due to alternative splicing and could be of value if one could control STAT3 alternative splicing. Nevo-Caspi and colleagues show that the level of ADAR editing enzymes increases upon deferoxamine treatment in lymphoblastoid cells, elevating the level of editing in Alu sequences residing in STAT3 introns . They fuFew studies have focused on switching STAT3α to STAT3β, which contributes to the inhibition of cancer cell growth . As mentSTAT3 has been reported in chemoresistance and radioresistance. In contrast, STAT3β plays an essential tumor-suppressive role in cancer ,175. PhoEarly clinical studies demonstrated the promise of therapy that targets STAT3. However, no inhibitors that directly target STAT3 have been approved by the FDA for clinical application. In the following section, we focus on inhibitors that directly target STAT3 through its N-terminal domain, DNA-binding domain, linker domain and SH2 domain .The N-terminal domain is essential for DNA binding, nuclear translation and protein–protein interactions, and a crystal structure is available for designing STAT3 NTD inhibitors . A strucBlocking STAT3 by targeting the DBD domain could affect DNA binding ability and has the potential to inhibit STAT3-dependent gene transcription. Galiellalactone a, a natuNiclosamide, an FDA-approved anthelmintic drug, as well as other small-molecule inhibitors, inhibits STAT3 activity. In combination with radiation, it can overcome the radioresistance of lung cancer xenografts . Even thStructure-based drug design and computational docking have been used for screening small-molecule inhibitors that target the DNA-binding domain of STAT3. The DBD-binding drug inS3-54 e selectiResearchers have found a small-molecule inhibitor of the STAT3 linker domain. Tatsuya et al. identified the benzoquinone derivative BPMB as a STAT3 inhibitor. BPMB could selectively inhibit proliferation in constitutively-activated STAT3 breast cancer cells. From an analysis by matrix-assisted laser desorption/ionization-mass spectrometry, Cys550 was found to be an important residue for crosslinking between STAT3 and BPMB. Cys550 could be a drug target for the development of irreversible STAT3 inhibitors . FurtherPhosphorylation of tyrosine residues in the SH2 domain of STAT3 is essential for receptor recognition as well as dimerization. In a search to identify small molecule inhibitors of STAT3, an SH2 domain-binding phosphopeptide PY*LKTK (Y* represents phosphotyrosine) was identified and formed inactive STAT3·PY*LKTK complexes. It has significant in vivo activity and suppresses v-Src transformation by blocking STAT3 signaling . Since tHigh-throughput screening of chemical libraries and computational docking studies have been used to discover inhibitors of STAT3. Stattic (6-nitro-benzo(b)thiophene-1,1-dioxide 1) was found to bind to the STAT3 SH2 domain. Another study demonstrated that Stattic enhanced radiosensitivity ,203. STAO-tosyl [S3I-201  and OPB-51602, potent small molecule inhibitors of STAT3, were found to strongly inhibit STAT3 phosphorylation and lead to effective antitumor responses ,220,221.Aberrant STAT3 activation is related to cell malignant transformation, tumor proliferation, differentiation and anti-apoptosis . Thus, SThe peptide (DTRYLEQLHQLY) and ST3-H2A2 (LDTRYLEQLHKLY) provide an additional strategy in the discovery of anti-STAT3 therapeutic agents as a novel anticancer therapy. However, the mechanisms of the peptides’ interaction with the N-terminal domain of STAT3 still need to be investigated. The ”undruggable” nature of a DBD has been proven druggable in the case of STAT3. Inhibitors targeting the DBD have been in a minor development stage and will face challenges. In recent years, silibinin has been shown to have unique characteristics whereby it binds directly with high affinity to both the SH2 domain and DBD of STAT3, making it a promising lead candidate for designing novel and effective STAT3 inhibitors . AlthougAt present, the main focus is on STAT3 inhibitors that bind to the tyrosine phosphorylation site of the SH2 domain of STAT3. The general structure of the SH2 domain and the mode of phosphopeptide recognition are conserved. The site is rich in positively charged residues, which requires a high negative charge of small molecules . TherefoIn summary, small-molecule STAT3 inhibitors have antitumor activities and inhibit STAT3 phosphorylation but are yet to be approved for clinical cancer therapy. Inhibitors that target the SH2 domain of STAT3, such as STA-21, OPB-31121 and OPB-51602, have completed Phase I/II studies. These compounds are expected to progress in further clinical trials in the future and pave a new avenue for cancer therapy. The inhibitors targeting STAT3 may induce unexpected side-effects because of the repression of STAT3β, and regulators and drugs for the regulation of STAT3 alternative splicing remain to be investigated.Chemoradiotherapy represents an important treatment for cancers. However, the intrinsic and acquired resistances of drug or radiation treatment limit its effectiveness in the clinic. Current evidence indicates that STAT3 is an oncogene, is constitutively activated in a variety of cancers, and regulates resistance to chemotherapy and radiotherapy. Signal transduction pathways influencing radiosensitivity mainly upregulate anti-apoptotic and cell cycle genes, induce EMT or modulate DNA repair . TargetiConversely, constitutively active STAT3 inhibits cell proliferation and invasion in PTEN-deficient cancer cells ,232. TheInterestingly, the ratio of STAT3α/STAT3β impacts the fate of a tumor. Splicing mediators, oligonucleotides and morpholinos can switch STAT3α to STAT3β in several cancer cells. Hopefully, in the next few years, several drugs that induce the expression of STAT3β by affecting alternative splicing regulators and increase chemo- and radiotherapeutic efficiency will be successfully applied in the treatment of cancer patients.


Eupallasella percnurus). This fish has low genetic variation and is at risk of extinction in the western edge of its range, which includes Poland. Fish from one Polish population were captured . Fin clips were taken, and DNA was extracted. Polymorphic microsatellites (13) were used to prepare genetic profiles, assess genetic variation in the fish and estimate genetic diversity in their progeny. Alleles were scored using an automatic capillary sequencer. The four and eight best variants of spawning pairs, and the optimal sets for group volitional breeding  were identified using Genassemblage 2.0. In the sets of 8 and 16 fish for group breeding, the mean heterozygosity, the number of alleles, and the share of “weak” heterozygotes  were better than the mean values for the progeny of all potential breeding pairs. For group volitional breeding, one set of four males and four females, and numerous sets of eight males and eight females would enable transmission of all 33 alleles identified in the potential broodstock and an expected progeny heterozygosity of 0.441 and 0.414, respectively. These expected heterozygosity values are higher than those in the broodstock. For practical purposes, the larger sets would be preferable for avoiding a future inbreeding and genetic drift.In conservation of captively propagated species, conserving genetic diversity is important. Here, we present an example of the use of Genassemblage 2.0 software in conserving the genetic variation of the lake minnow ( Conserving genetic diversity is important in conservation of captively propagated species. Unfortunately, genetic variation within captively propagated populations may decrease6 due to (1) obtaining large groups of juveniles from a few parental individuals, (2) pairing individuals without knowledge of the genetic differences between them, (3) possible inbreeding events7, or (4) domestication of the broodstock8. A decrease in population genetic variation may reduce that population’s viability and its potential to adapt to environmental stresses3. Guidelines have been developed to inform broodstock collection, mating, rearing of young, and outplanting9.Maintaining high genetic variation, as measured by high heterozygosity and allelic diversity, is crucial for keeping a population viable and able to adapt to environmental changesEupallasella percnurus , a cyprinid fish. Its populations occur in the northern hemisphere from Poland to the Pacific coast10. In the western part of its range, this species is in critical danger of extinction due to the specific nature of its habitats, which are small and shallow water bodies that exist for only several decades11. In Poland, the lake minnow is under strict protection, involving active conservation measures12. One of the most common measures used so far is outplanting of juvenile fish produced in aquaculture facilities to selected water bodies to establish new populations13.An example of a species that is at risk of experiencing reduced genetic variation is the lake minnow, 14. To use the information in these genetic profiles for managing genetic diversity, bioinformatic tools are needed15. To meet this need, Genassemblage 2.0 software was constructed. Its installer, a detailed user guide and examples of input files can be downloaded free of charge from the author's website: https://pracownicy.uwm.edu.pl/d.kaczmarczyk/main_page.htm.One way of addressing the problem of potential loss of genetic variation in such populations is by preparing genetic profiles of individuals by screening highly polymorphic fragments of DNA such as microsatellites. With these profiles, individuals that are as different as possible can be identified and assembled in to breeding pairsIn this paper, we present an example of the use of Genassemblage 2.0 in conservation of the genetic variation of the lake minnow. The software was used to find the four best combinations of spawning pairs and to find the optimal set of individuals for group spawning.16. The integrity of the DNA samples was visually inspected after their electrophoresis in a 1.5% agarose gel stained with ethidium bromide, and DNA yields were quantified by spectrophotometric analysis15.23 males and 25 females from one Polish population  were caught using baited traps. Fin clips were taken, and DNA was extracted from all fin samples using a Genomic Mini AX Tissue SPIN DNA Extraction and Purification Kit . The extraction procedure was performed following the manufacturer’s recommendationsCa3, Ca4 and Ca1217, Z9878, Z10362 and Z1341918 (primer sequences were taken from GeneBank https://www.ncbi.nlm.nih.gov); and Eupe1, Eupe2, Eupe4, Eupe5, Eupe6, Eupe7 and Eupe9 were amplified using primer sequences described by Kaczmarczyk and Gadomski19 and deposited in GeneBank (https://www.ncbi.nlm.nih.gov). The primer sequences, repeat motifs, their accession numbers and details of PCR amplification were given by Kaczmarczyk and Wolnicki16. The forward primer of each primer pair was 5′-end labeled with fluorescent dyes . The lengths of the amplified DNA fragments were determined using an Applied Biosystems 3130 Genetic Analyzer against GS400LIZ size standards. Allele determination was performed using GeneMapper 3.0 software (Applied Biosystems) according to the manufacturer’s recommendations.To prepare genetic profiles and assess genetic variation in the fish sampled from the Guzy population and in groups of their progeny, 13 microsatellites were used as genetic markers. Loci Ho), expected heterozygosity (He) and the number of alleles (ar), ) at a given locus and across loci were calculated for both groups of fish using MSA software20.Observed heterozygosity . The coefficients used for calculating the v index were a relative index of heterozygosity (iH) 0.45, a share of weak heterozygotes (iwh) 0.1, and a relative index of allelic number (iar) 0.45; the number of breeding pairs was set at four and then at eight.The best possible breeding pairs for controlled mating were identified based on the heterozygosity, share of “weak heterozygous genotypes” Find best set of individuals for group spawning was used. One set consisted of four males and four females, and the other, of eight males and eight females. The number of alleles was selected as the primary indicator. The calculation performed by this module is based on the assumption that each spawner contributes equally to the progeny.To find the optimal set for volitional group-spawning, the module These studies were approved by the Local Committee for the Experiments on Animals in Olsztyn, Poland (permission No. 15/2015) issued on 25.03.2015. All procedures were performed in accordance to relevant guidelines.Ho was 0.386, He was 0.375, and the share of weak heterozygotes was 0.208.In the group of broodstock candidates, 33 alleles were identified across all microsatellite loci. The With this group of male and female candidates, it was possible to assemble breeding pairs in 575 different combinations. The progeny of each potential pair would have inherited from 17 to 26 (mean of 21) alleles. This is lower than the 33 alleles that were identified in the parental group. Across the investigated markers, heterozygosity ranged from 0.192 to 0.519 (mean 0.378), but only in 211 pairs (36.7%) was the observed heterozygosity higher than that in the parental group as a whole. The average share of weak heterozygotes in groups of progeny was 0.365, ranging from 0.167 to 0.500. The value of this indicator was less than or equal to that in the parental group in only 65 progeny groups (11.3%).v index, a set of four pairs were selected as the optimal pairs for breeding: male Gu01 and female Gu06, male Gu05 and female Gu07, male Gu18 and female Gu14, and male Gu38 and female Gu30 . If any molecular marker is polymorphic and inherited according to Mendelian laws, and numbers can be assigned its their alleles, then it can be used. Genassemblage software does not have a limit at the number of molecular markers that can be used. In this study, we used the polymorphism of microsatellite fragments as a marker of genetic differences between individuals. Markers other than microsatellite fragments such as single nucleotide polymorphism can be used, if numerical values are assigned to their alleles.Identification of a group of pairs or sets of group-spawning individuals that are optimal for conservation of genetic variation can be time-consuming and difficult. Manually finding the best of them is problematic because of the complexity of the calculations and the huge number of potential pair variants or set compositions. For this reason, bioinformatic tools can be very helpful. Genassemblage 2.0 is a versatile tool for artificial breeding of conserved species such as the lake minnow. It can be used to identify the best breeding set for either individual or group breeding. This is merely an example of how the software works  existing populations with juveniles originating from aquaculture facilities16.In the group of potential spawners from the Guzy population, we found that the values of genetic variation indicators, such as heterozygosity, allelic diversity and share of weak heterozygotes, indicate that genetic variation in the group of potential parents is low. This finding is similar to reports on other Polish populations of this species27. Consequently decrease of population fitness in the next generation or generations is more likely28. In the presented example, we found that using a set of breeding pairs chosen by Genassemblage 2.0 will provide a higher level of genetic variation in the progeny than the mean value obtained with the progeny of all potential breeding pairs. Identification of the best set of breeding pairs using the v index can increase heterozygosity and reduce the number of weak heterozygotes in progeny. Moreover, each individual included in the set of breeding pairs proposed by Genassemblage 2.0 appears in only one breeding pair, which reduces the risk of inbreeding in future generations.Our results indicate that random selection of fish for breeding is likely to result in lower genetic variation in the offspring than in the parents. It is known that using a small number of individuals for breeding without information about their genetic similarity and relationships (random selection) can accelerate a decrease in allelic diversityNe27, inbreeding in future generations and increased genetic drift in the population. It is difficult to indicate exactly how many pairs should be used for breeding, but larger numbers help to conserve within-population genetic variation9. Consequently, the example that was presented with eight breeding pairs would be more appropriate for conservation of genetic variation than the set of four pairs, even if the four pairs had slightly better genetic variation indicators. Generally speaking, if enough males or females are available and the same individual is not used more than once, it recommended to mate more pairs than four and use Genassemblage 2.0 software to help find the best ones. Genassemblage 2.0 software can be helpful for volitional breeding. If we can assume that the contribution of each spawner to the progeny is equal or very similar, we can find a set of individuals that will transmit all the alleles identified in a group of potential parents to their progeny. In this situation, it is even possible to obtain progeny with higher heterozygosity than in the parents’ generation. Moreover, the software can indicate alternative sets for volitional breeding that provide a level of genetic diversity similar to that in the best set. This can be useful when it is not possible to use the best set. It should be taken into account that, in volitional mass spawning of some fish species, the parental contribution to the progeny can be highly skewed35. However, this has not been demonstrated with the lake minnow, and our results suggest that using larger sets of males and females in volitional breeding could be more suitable than using separate pairs. Again, it should be noted that, although the average predicted heterozygosity would be slightly lower with eight males and eight females than with four of each, it would be better to use the larger set for the reasons given in the preceding paragraph.It should be noted that, in the example presented above, four breeding pairs were chosen for illustrative purposes only. In a real-world application, it would be better to choose more individuals. Using only a few individuals for breeding is associated with a higher risk of a decrease in 36. In comparison, Genassemblage 2.0 has expanded functionality and can be used for more diverse tasks, such as group breeding. Moreover, the calculation of the v index, which was implemented in Genassemblage 2.0, enables comparison and evaluation of each breeding pair and selection of the best one. Genassemblage 2.0 has a modular interface, expanded functionality and a more user-friendly design, which makes it even more useful and easy-to-use in conservation of genetic variation. Importantly, the use of Genassemblage 2.0 software is not limited to fish species. It can be used to manage the genetic variation of most species for both conservation and commercial purposes.The previous version of the software (Genassemblage 1.0) accurately predicted genetic variation in the progeny of breeding pairs


Early references to Ca de Rater and Ca de Bestiar endangered autochthonous breeds were witnesses to their exceptional ratting/pet and shepherding/guard skills for centuries. Studbooks or associations promote an increase in the number of effectives and their genealogies. Genetic diversity parameters were evaluated along the history of definition of both dog breeds. Guard or hunting purposes condition an increased registration of genealogical information. Hunting animals have more complete genealogies and need more time to select breeding animals. Male guard dogs are preferred over females due to their suitability for guarding. Selection for performance acts as a diversity promoter and breeding policy driver. The uses or purposes for which certain breeds were selected condition the genetic diversity evolution of endangered breeds, even if these share the same geographic isolation conditions.Despite the undefinition of the origins of Ca de Rater (CR) and Ca de Bestiar (CB) dogs, references to these endangered autochthonous breeds highlighted their ratting/pet and shepherding/guard skills for centuries. Genealogical historical records were traced back to founders. Founder number in the reference population , historical and reference maximum generations traced , and historical average number of complete generations (1.04 for both breeds) were determined. Structure assessment revealed the existence of subpopulations regarding criteria such as breeders (75 and 17), breeder location (32 and eight), owners (368 and 198), and owner location (73 and 51) for CR and CB, respectively. Average inbreeding (F) within breed subpopulations ranged from 0.27–1.20% for CB breeders and the rest of subpopulation criteria for both breeds, respectively, except for CB owners and owner location. F ranged from 0.27–1.41% for CB historical population and CR current population, respectively. The study of genetic diversity revealed a relatively similar genetic background between subpopulations. Average coancestry between and within breeds suggested a similar evolutionary process. However, Mann–Whitney U test determined significant differences for diversity parameters  between breeds and their functionalities. Conclusively, functionality in dog breeds may determine the genetic diversity evolution of endangered breeds, even when these share the same geographic isolation conditions. An analysis of the historical records of the Majorcan Ca de Rater (CR) suggests rather undefined origins . The firThe breed definition may have parallelly occurred with the Majorcan repopulations of Valencian towns such as Taberna  in the 17th century, who potentially introduced a Majorcan type of dog which fitted the morphology and purposes of a rat terrier . ReciproGradually, CR spread across many other Spanish regions . The infCa de Bestiar (CB) origins are undefined . The firDespite the common traditional and popular recognition of both breeds, it would not be until the 1970s when the recovery and orderly breeding of these populations began. During the 1980s, the Board of Native breeds of Majorca implemented programs to promote breed recovery. The Club of Ca de Bestiar was unofficially established in 1978, although the breed standard was not drafted until 1980. The standard established the characteristics and was used as a valid reference for determining both the morphological and the phaneroptical adscription of animals to the population and to provide advice on reproduction, which laid the basis for the breed’s recovery. The official recognition of the Club of Ca de Bestiar did not occur until 2001, when Royal Decree 558/2001 was published. On the other hand, the Spanish Club of Ca de Rater Mallorquí was set in 1990 but was not officially constituted until 2001 . The standard of the breed was officially recognized in 2004 (APA Order/807/2004). Since then, both breeds have been exposed to a high risk of loss of genetic diversity resulting from a long process of breeding during which foreign breeds contributed to the loss of their genetic identity from the 1950s . This isInitial stages of breed morphofunctional standardization may promote the cross between close relatives to find and fix the characteristics or qualities that define a breed. This initial mass selection is carried out without considering the possible harmful effects derived from the loss of simultaneous genetic diversity and the increase in other parameters such as inbreeding. Concerns on the potential effects of inbreeding and reduced diversity on health, functionality, and welfare in dog breeds have led to a call for improved genetic management practices . FunctioDog breeds have traditionally been classified depending on their use, which tended to result in some genetically unrelated breeds being grouped together, particularly when dogs of widely different geographical origin were considered . As a reWhen selecting for an enhanced performance, breeding practices may less likely lead to pronounced changes in basic form and function and, hence, in the underlying diversity. Contrastingly, when selecting for conformation, breeding practices may lead to significant changes in the appearance and performance skills of many breeds, sometimes distorted to extremes . AlthougFor these reasons, the objectives of this work were as follows: (1) to study the evaluation of the integrity of the historical pedigree record of CR and CB, controlling the repercussions of the ancestors and founders; (2) to evaluate the current population structure, genetic variability, and the connections between genetic and demographic parameters, measuring the existing genetic flow and quantifying the risk of loss of genetic diversity, evaluating the degree of threat to which each breed is exposed to suggest effective conservation and selection strategies; (3) to analyze whether functional criteria and idiosyncrasies related to functional breeding may have conditioned genetic diversity and population structure throughout the process of isolation and definition of each breed, even when they shared a common territorial niche. This model can act as a tool to assess the degree of endangerment to which dog breeds may be exposed, which may help implementing effective conservation strategies and selection practices that could be extensible to other populations of dogs and other endangered small populations.The pedigree records used in this study were provided by the Ca de Rater Mallorquí Club (for CR) and by the Associació de Criadors i Propietaris del Ca Pastor Mallorqui—Club del Ca de Bestiar (for CB). The complete pedigree file includes 1810 animals  born between January 1987 and September 2017 for the historical population of CR. Contrastingly, for CB, the historical pedigree file includes 385 animals  born between June 1994 and April 2018. Genealogical information of each dog was traced back to its ancestors and analyzed. Molecular breed assignment analyses were performed to check for breed assignment percentage (breed purity) and to parallelly detect the potential introduction of other breeds to ensure the reliability of our results using the International Society of Animal Genetics (ISAG) Dog core short tandem repeat (STR) panel with 22 markers . The CanDemographic and genetic diversity parameters were evaluated using ENDOG (v4.8) software  and CFC The maximum number of offspring per sires and dams and mean offspring number per male or female were computed by evaluating the trends described by birth number during the period that the study comprised. Pedigree completeness index (PCI) was assessed through the maximum, complete, and equivalent number of generations traced . The stuGeneration length  and the Genetic diversity was evaluated through the parameters described below.Individual inbreeding coefficient (F) was computed as described by Luo , the avez et al. . Mean ΔRz et al. . RegressThe individual rate of coancestry  was computed as described by Lacy [af) necessary to explain the complete genetic diversity was computed as described by Boichard et al. [gf) was computed as defined by Lacy [j was computed as its expected genetic contribution independent from the contributions of the other ancestors [The effective number of founders  was computed as described in Lacy [GD and GD * indicates the GD loss accumulated since the population foundation [Nef) was computed following the premises in Caballero and Toro [The mean effective population size . n ≤ 1000 samples) was performed to study data distribution using the Shapiro–Francia normality routine of the Stata Version 15.0 software. Levene’s test was performed to determine the homogeneity of variance across groups using the explore procedure of the descriptive statistics package in SPSS Statistics, Version 25.0, IBM Corp. [p < 0.05), Mann–Whitney U and independent median t-tests were performed to detect potential differences in population statistics and diversity parameters between CR and CB breeds, respectively , average relatedness coefficient (ΔR), number of maximum generations, number of complete generations, equivalent number of generations, individual increase in mean inbreeding , and genetic conservation index (GCI)). Mann–Whitney U and independent median t-tests were performed using the independent samples procedure of the nonparametric tests task of SPSS Statistics for Windows, Version 25.0, IBM Corp. [The Shapiro–Francia W’ test  for unequal variances with unequal sample sizes.According to Gibbons and Chakraborti , the estCa de Rater Mallorquí Club and Associació de Criadors i Propietaris del Ca Pastor Mallorqui—Club del Ca de Bestiar gave their informed consent for the use of pedigree data before the study was performed. As biological samples were not taken, further permission was not necessary. The study was conducted in accordance with the Declaration of Helsinki. The Spanish Ministry of Economy and Competitivity through Royal Decree Law 53/2013 and its credited entity, the Ethics Committee of Animal Experimentation from the University of Córdoba, permitted the application of the protocols presented in this study as cited in the fifth section of its second article, as the animals assessed were used for credited zootechnical use. This national Decree follows European Union Directive 2010/63/UE, from 22 September 2010.Two historical birth peaks occurred for both breeds: from 2002 to 2003 and from 2007 to 2008 in CR; in 2007 and in 2009 in CB. However, a drastic reduction in the number of births of the CR breed was observed from 2008 onward. Such a drastic reduction was not observed for the CB breed. The average historical number of births was 58 and 16 and the years for which the highest number of births were registered were 2007, and 2007 and 2009 (163 and 37 births) for CR and CB, respectively. However, after 2011 there was a progressive decrease in the number of births in the CR breed which never reported the minimum levels reached during the 1987–1997 period. Contrastingly, despite CB presenting remarkable fluctuations in the number of births across years, the highest values found for 2007 and 2009 (37) were never reached again in the history of the breed. For CB, the trends were maintained in time and the values never fell below those for the period from 1994–2001. The historical number of complete generations in the last decade was 1.04 ± 0.79 and 1.04 ± 0.62, for CR and CB, respectively. The historical number of equivalent generations was 1.50 ± 1.09 and 0.89 ± 0.99 for CR and CB, respectively. The completeness index of the pedigree experienced a mean increase per generation of 3.646% and 1.558% when the historical and current populations for CR and CB were compared, respectively. The minimum index of completeness of the pedigree was reached for the fifth generation (percentage of great-great-grandparents known) of the historical population for both breeds, with CR reporting a 1% higher value than CB. In contrast, the maximum completeness index was reached for the first generation (known parents) for both breeds with CR reporting almost 34% higher values than CB. The summary of the results for pedigree completeness index-related parameters between the historical and current populations for both breeds are reported in The average age of the males and females in reproduction was very similar (~14 years) for the historical and reference populations of both breeds. Generation length was 3.94 and 3.93 years and 3.09 and 4.04 for the historical and reference populations of CR and CB, respectively. The lowest values reported for generation interval were 3.76, 3.63, and 2.70 for the gametic routes of sire to son and dam to son in the historical populations and sire to daughter in the current populations of the CR and CB, respectively . Table SInbreeding coefficients for the historical and current populations were 1.15% and 1.41% and 0.27% and 0.34% for CR and CB, respectively. Despite these relatively low coefficients, highly inbred animals were recorded in the historical and current populations. The maximum percentage of inbreeding (26.41%) was reported for the historical and current populations of CR. CB reached half the value reported for CR in both populations (13.38%). The percentage of inbred animals was 1–2-fold higher in the historical and current populations of CR compared to CB . NonrandThe average coancestry in the historical and reference populations was 1.24% and 1.41%, respectively, for CR, while it was 0.86% for the historical and reference populations of CB.The results for the analysis of probabilities of gene origin, ancestral contributions, and the loss of genetic diversity are shown in Genetic diversity was around 99% in both breeds. The lowest value was reported for CR, although the differences with CB were not remarkable (98.76%), which was the population for which genetic diversity loss was consequently slightly greater. The loss of genetic diversity due to genetic drift was 0.5% and 0.3% in CR and CB populations, respectively. A value of 1.24% and 0.9% was reported for the genetic diversity loss that could be attributed to bottlenecks and genetic drift for the CR and CB reference populations .The average relatedness (kinship) coefficient was 24.09% and 1.73% in the CR and CB reference populations. For the CR, considering the marginal genetic contributions of ancestors, a single ancestor (identification number 138) explained 9.25% to 14.13% of the genetic pool of the historical population and 8.97% to 13.55% of that of the current population. Contrastingly, for CB, marginal genetic contributions of a single ancestor (identification number 202) explained 9.77% to 15.89% of the genetic pool of the historical population and 4.42% to 8.37% of that of the current population. Additionally, 15 and 31 individuals explained 50% of the gene pool of the current and historical populations of CR and CB, respectively.Results for effective sizes calculated through the individual inbreeding rate and individual coancestry rate are reported in A total of 67,161 and 19,306 Nei’s genetic distances were considered when the stratification criterion was the owner for CR and CB, respectively. The average Nei’s genetic distance was 0.103 and 0.274 for CR and CB owners, respectively. Contrastingly, for breeder, breeder location, and owner location this parameter was 0.074 and 0.038, 0.021 and 0.042, and 0.025 and 0.072 for CR and CB, respectively. The number of equivalent subpopulations for all population sets was 0.370 and 0.075 for CR and CB, respectively . The aveIT) was −0.001 and −0.006 for all subpopulations and criteria considered. The coefficient of inbreeding of an individual with respect to the subpopulation (FIS) varied from −0.390 for the subpopulations of the CB breed when the owner criterion was followed to a maximum of −0.023 for the subpopulations of CR when the owner location criterion was considered (ST), i.e., the effect of the subpopulations compared to the total population, reached a maximum value of 0.276 for the owner subdivision or stratification criterion in CB and a minimum of 0.021 for the owner location subdivision or stratification criterion in CR.Mean coancestry in the metapopulation and autocoancestry reported values of 0.013 and 0.506 for all population subdivisions  for CR, while these values were 0.009 and 0.501, respectively, for all population subdivisions  for CB. The analysis of population structure through Wright’s F statistics  reportednsidered . The corThe analysis of the structure of owners and breeders revealed that none of them could be considered as the nucleus of the population. We found that 100% of owners used foreign sires in both breeds, and none of them could be considered totally isolated. In total, 173 owners used the maximum percentage of own parents (66.67%) for CB, while 315 owners used the maximum percentage of own parents (25.00%) for CR.In total, 29 pairs of owners of CRs presented the longest genetic distance, which was 0.547. On the other hand, 236 pairs of owners of CBs were distanced by the longest genetic distance (0.508). t-tests  which are only around 10 to 15 years old, even if a great breeding tradition of these breeds can be found in the archipelago prior to the recognition of the breed . This maMaximum, complete, and equivalent generations in CB were significantly lower than the values reported for CR . The meaBreeder dendrograms lay the historical basis of owner dendrograms; hence, breeder pack structures can be inferred to build the skeleton of the owner pack network . Both brGeneration intervals resembled the values found by Leroy et al.  for BassIn general, mean generation intervals were slightly longer for male offspring (via both sire and dam) than for female offspring, with the highest value being reported for the gametic pathway of sire to son for the current population of CB, which may derive from the higher number of male dogs than female dogs whose age at the birth of their offspring was above the mean. A certain preference for dogs of a particular sex has been reported and may be based on a perceived dimorphic superiority in some desired traits, which may condition the time taken by breeders to determine the validity of a certain animal as a mating animal. For instance, males have normally been reported to be bolder, higher driven, and more aggressive than females, who tend to learn faster, be somehow less territorial, and more biddable. Differences in boldness and shyness are acknowledged predictors of performance in working dogs, with bolder animals being the better performers . BoldnesThe policies of breeders denote the use of certain males or females which may be more popular among the individuals in the population. Contextually, having a low replacement rate could contribute to generation elongation which has been reported for other endangered dog or equine breeds . Both spA slightly higher number of females than males selected for breeding was reported in both breeds. However, female to male ratio inverted in CB, which suggests a certain interest for breeders and owners in the males of the CB breed, which could be presumably linked to their application as guard or shepherding animals. In nature, for males, territorial defense versus access to female success ratio may be balanced by ameliorative reproductive success, whereas females have fewer direct advantages from this ratio; hence, the energy invested in the defense may be detracted by the functions related to the sex-specific behaviors linked to reproduction . The samMating between dogs that are related beyond second cousins (F < 0.0156) has been reported in small territorially isolated populations such as the ones in our study as suggested by Marin et al. . HoweverIn our study, inbreeding levels showed an increasing trend which stabilized around 14 or eight maximum generations for CR and CB, respectively . This coAll diversity parameters differed highly statistically between guard/shepherding dogs and ratting/hunting/pet dogs , with thn et al.  suggesteProvided the historical territorial isolation to which CR and CB have been exposed, a solid interconnection between available genetic resources through the implementation of appropriate breeding practices may have been attempted, as suggested by nonrandom mating results in CR, which could be said to be in genetic equilibrium. This may suggest that this population will be balanced as soon as no external influence such as selection can promote the increase in mutation rates. The negative values for CB may imply that mating among the animals may not have been performed seeking particular phenotypical, phaneroptic, or functional characteristics. These values contrast the ones obtained for Ca Mè by Marin et al. , whose pThe number of equivalent subpopulations below 2 indicates a high structuration of both populations  whose values were lower than those found for Ca Mè . AccordiST value of zero suggests no population structuring or subdivision, i.e., complete panmixia or random mating. The concept of panmixia is opposed to the concept of assortative or nonrandom mating  and, therefore, that the two populations examined do not share any genetic diversity. At a breed level, FST values are always expected to be below 0.05, as this may be the lower limit for species differentiation. Nevertheless, computing FST values can report very important information about the relationships among lower-scale genetic subdivisions of a population, such as breeds or varieties, or those linked to specific features such as coat color or even functionality.A value of one for FST are comparatively interpreted with FIS values. At a breed level (FST below 0.05 context), when owner, breeder, and their locations are considered as criteria of population subdivision, negative values of FIS may address the existence of a certain disequilibrium in breeding policies acting in favor of an unexpected mating rate of unrelated animals under a model of random mating. This imbalance may derive from the fact that breeders and owners may tend to mate animals that have desirable aesthetic qualities or higher performance in different skills as a way to improve the resulting puppies through the complementarity between features or abilities displayed by both parents, which was also supported by the results of the Mann–Whitney U test .The ratio of fy et al. . The stuy et al.  showed tThese results evidence that founding genotypes are still representative in current populations of CB and CR. The values found for both breeds also suggest that, although bottlenecks may have occurred, they have not had an effect. Bottlenecks need not lead to or save a population of a breed from extinction. A loss of diversity as a function of bottlenecks occurring in populations could manifest itself from the two options described. Thus, simultaneously with this loss of diversity, deleterious mutations could have been both eliminated and fixed in the population, which could have led both to the sanitation of populations and to an increase in the threat of extinction to which breeds are exposed .In this context, according to Broeckx , once reOn occasions, sublining can derive from the reproductive policies historically followed by breeders as suggested by Marin et al.  and MariThe consolidation of genetic management structures such as studbooks or associations promotes an increase in the number of effectives and their genealogical information in endangered breeds. Guard or hunting purposes may condition an increased registration of genealogical information provided the value of ancestors may be considered an evidence of the potentially higher value of individuals. Hunting animals need longer time intervals until animals are selected for breeding, and genealogical information in hunting animals may be more complete than in pet, ratting, or guard/shepherding breeds. Male guard dog generation intervals suggest that they may be preferred over female dogs which may be based on their special suitability to develop guarding tasks. Selection for performance may act as a diversity promoter. Breed functionality and, hence, its social context may condition whether breeding policies focus on the selection against undesirable features or the promotion of desirable ones. Choosing one alternative or the other may depend on whether performance, independently of the task that the dogs are required to develop, is among the selection criteria of the breeds or not.


Meretrix petechialis collected from China was analyzed using sequence-related amplified polymorphism (SRAP) markers. Eight primer pairs produced 132 polymorphic loci with an average of 16.5 loci per primer pair. A population from Jiangsu had the highest percentage of polymorphic loci at 27.27%, suggesting that these resources had a rich genetic diversity. The Nei’s gene diversity of the nine populations ranged from 0.0647 to 0.0793; a population from Shandong was the lowest and a population from North Korea the highest. The Shannon’s information index was between 0.1023 and 0.1202, with the lowest in the Shandong population and the highest in the Jiangsu population. The Nei’s unbiased genetic distance between the nine populations was 0.0243–0.0570 and the genetic similarity was 0.9446–0.9760; the genetic distance between Guangxi and Shandong populations was the furthest (0.0570) and the genetic distance between Shandong and Jiangsu populations was the closest (0.0243). Nei’s gene diversity analysis indicated that the genetic variance was mainly found within individual geographical populations, and the analysis of molecular variance revealed low but significant genetic differentiation among local and regional populations. The limited gene flow (Nm = 0.555) was inferred as a major reason for the extent of genetic differentiation in M. petechialis. The results obtained here indicated that M. petechialis have high degree of genetic diversity and the potential of further breeding with excellent germplasm resources. This study provides a scientific basis for the protection of germplasm resources and the breeding of M. petechialis.Genetic variation in nine stocks of  Meretrix petechialis is a marine shellfish species of economic and medicinal value that is mainly distributed and cultured in coastal China. In recent years, the scale of breeding in M. petechialis has resulted in an increasing demand for seedlings and the artificial seeds transplantation  markers , Guangxi (GX), Panjin (PJ), Shandong (SD), Dandong (DD), Hainan (HN), Dalian (DL), North Korea (NK) and Jiangsu (JS)  . The add2O to make up the volume of the PCR reaction to 25 μL. The reaction conditions were: 5 min at 94 °C, 5 cycles, 1 min at 94 °C, an annealing temperature of 35 °C for 1 min, an extension temperature of 72 °C, and an extension time of 1 min, 1 min at 94 °C, an annealing temperature of 50 °C for 1 min, extension at 72 °C for 1 min, a total of 35 cycles; extension at 72 °C for 10 min and stored at 4 °C. After the PCR was completed, it was detected by 6% denatured polyacrylamide gel electrophoresis. To ensure the accuracy of denatured polyacrylamide gel electrophoresis, a control was used for each test.Primers for Me7 and Em15 were based on published SRAP sequence , and 105M. petechialis groups was constructed according to the genetic distance with MEGA 7.0  value of the primer is calculated using PIC software. POPGENE32  was usedMEGA 7.0 . The totMEGA 7.0 . The totMEGA 7.0 .K populations based on their multilocus genotype. The estimated K value was set to 1–9 then STRUCTURE was used with 10,000 iterations and a burn-in period of 10,000. All runs were repeated 10 times at each K, the most appropriate value of K was identified with calculated ΔK by using Structure Harvester ver.0.6.92 (http://taylor0.biology.ucla.edu/structureHarvester/)  . Then, tPP 1.1.2 , and thePP 1.1.2  for clusM. petechialis, 105 pairs of SRAP primer combinations were screened, and 30 pairs of primer combinations with clear, stable and few bands were selected, of which eight pairs of primer combinations  can obtain stable, reproducible and polymorphic amplification maps. The polymorphic loci amplified by different primer combinations are different. The number of polymorphic loci detected by each pair of primers is 12–23, and the average number of loci per pair of primers is 16.5. The fragment size range of 116–250 bp has a high polymorphism, which indicates that SRAP can be applied to study genetic diversity in M. petechialis.Using the genomic DNA of nine populations of Me5/Em14 polymorphic stripe number was the highest (23). The Me3/Em4 primer’s polymorphic frequency was the highest for 53.85% and the Me6/Em5 was the lowest at 29.27% (mean = 42.86%). The PIC value of the Me5/Em14 primer was the highest (0.90), the PIC value of the Me4/Em5 primer was the lowest (0.78) and the average PIC value of eight pairs of primers was 0.86  to 0.0793 (NK) and the Shannon’s information index was between 0.1023 and 0.1202, with the SD population being the lowest and the JS population being the highest   , inter-sA (RAPD) , amplifiA (RAPD) . In addiA (RAPD) , 2016. Te genome  and possM. petechialis. The results show that 132 polymorphic bands were detected by eight pairs of SRAP primers and the number of polymorphic alleles with an average amplification of each pair of primers was 16.5. Compared with the results of the study that analyzed the germplasm resources in three geographical groups of M. petechialis from Guangxi Beihai (GX), Jiangsu Nantong (JS) and Liaoning Dalian (LN) by SRAP markers  was inferred as a major reason for the extent of genetic differentiation in M. petechialis germplasm resources, its genetic background has become unstable. The choice of breeding is the main method to improve the beneficial traits of M. petechialis. At the same time, genetic structure analysis of M. petechialis can be carried out by developing and applying molecular techniques to provide a scientific basis for the genetic breeding of M. petechialis. The results obtained here indicated that M. petechialis have high degree of genetic diversity and the potential of further breeding with excellent germplasm resources. The GX population might be more suitable as the base population for selective breeding because its genetic improvement potential is the highest.In recent years, due to the continuous reduction of M. petechialis were analyzed by SRAP molecular marker to estimate the genetic differentiation and population structure. Nei’s gene diversity analysis indicated that the genetic variance was mainly found within individual geographical populations and the AMOVA revealed low but significant genetic differentiation among local and regional populations. The limited gene flow was inferred as a major reason for the extent of genetic differentiation in M. petechialis. The GX population might be more suitable as the base population for selective breeding because its genetic improvement potential is the highest.In the present study, different geographical populations of 10.7717/peerj.8723/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.8723/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.8723/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.8723/supp-4Supplemental Information 4Click here for additional data file.


To detect possible non-linear and interaction effect between predictive variables, a kernel-based regularized least squares (KRLS) model was used for empirical analysis. The results show that there was considerable heterogeneity between cities regarding marginal effect of GC on ΔPM2.5, which could potentially be explained by different seasons, latitude, urban maintenance expenditure (UE), real GDP per capita (PG), and population density (PD). Also described in this study, in cities with high UE, the growth of GC, PG, and PD always remain a positive impact on mitigation of haze pollution. This shows that government expenditure on urban maintenance can reduce or mitigate the environmental pollution from economic development. In addition, the influence of other urban elements on air quality had also been analyzed so that different combinations of mitigation policies are proposed for different regions in this study to meet the mitigation targets.In China, severe haze is a major public health concern affecting residents' health and well-being. This study used hourly air quality monitoring data from 285 cities in China to analyze the effect of green coverage (GC) and other economic variables on the incremental PM With the rapid urbanization and economic development, urban greening has become an urban livability standard and important symbol of residents' well-being . How to 2.5 removal by trees from 10 American cities, substantial health improvements and economic value produced by urban trees have been found  are needed to reflect the improvement of haze pollution by urban greening. Third, studies at different spatial scales have revealed that the effect of green cover on PM2.5 concentrations is not a simple linear relationship, which influenced by many other factors such as regional development patterns , government expenditure (for urban maintenance), etc. Finally, to capture the compounding effects of urban physical parameters on PM2.5, the kernel-based regularized least squares (KRLS) model was adopted to consider possible interactions among variables.This study set out to explore the contribution of green cover and other elements to reduce haze pollution, and further to evaluate the roles of these elements in terms of both singular and interacting behaviors. Those results would be helpful to formulate effective strategies for improving the urban atmosphere environment. To that end, we use ground-level PMAs of late, numerous methods were reported to characterize the effect of air quality factors varying along different spatial distribution and conditional distribution such as local linear method , geograp2.5 data adopted in this study have covered 285 cities in China , published hourly by China National Environmental Monitoring CenterThe ground-level PM2.5 contribution of transportation to average mass concentration can be 25–50%   and time zones, the start time and duration of the increase of particulate matter were different, complex formulas were therefore needed to ensure that the maintenance duration and intensity of the increase in PM2.5 concentrations from mobile source are accounted for  is a loss function that measures the estimated error at each observation, R(f) is a regular term that measures the complexity of the model, and λ ∈ R is a control parameter determining the tradeoff between model fit and complexity. H is the “hypothesis space” formed by all possible functions.Where V, and the Tikhonov regularization method is used to construct complexity penalty R(f), optimization of objective function in Equation (8) can be expressed as:To solve the minimization problem of objective function, the least square method is used to measure the model's variance loss 2, referring to the method of Hainmueller and Hazlett ]. It should be noted that for each data set in K, the process of generating the independent variable weight c is similar to the linear solution of the mathematical expectation of the dependent variable in the similarity of the sample observations in the fixed function window width, so any independent variable has its corresponding weight c.Equation (11) reveals that when the kernel function window width σX =  is a data set composed of N D-dimensional independent variables, according to Equation (8), any given sample j can be calculated to correspond to the d-dimensional independent variable partial derivative of the objective function:Assuming that By calculating the point-by-point partial derivative of the d-dimensional independent variable, the average marginal influence of the d-dimensional independent variable on the dependent variable can be obtained2.5 concentration increment in 285 cities are illustrated in 2.5 was significantly higher in winter than leaf-period, which demonstrates the necessity of building different models to capture spatiotemporal trends.Evident seasonal variations of ΔPM2.5 and ΔPM2.5 in all 285 sample cities, which were categorized into five based on Jenks natural breaks. As shown in 2.5 were distributed mainly in north China and the regions with low ΔPM2.5 were distributed mainly in south China, with areas above 32° N experiencing a higher ΔPM2.5 in summer than in winter. We can see that a far greater impact on ΔPM2.5 is due to changes in season but not spatial spillover effect.2.5 concentrations were largely consistent and showed a bimodal pattern. In summer and winter, no increase in PM2.5 concentration was detected in four cities during peak hours . Among them, Zhangjiakou and Changsha belong to Hebei, one of the provinces with the most severe haze pollution in China, not ruling out the possibility that the nighttime factory emissions wiped out the PM2.5 increase that should had occurred in the morning. Since it needs to use the logarithmic form of observed value for model calculation, we used 0.01  instead for cities without PM2.5 increase.What needs illustration is that, in most cities, the diurnal variations of PMA logarithmic version of Hansen's threshold model was estimated using different threshold variables. 2.5 by using threshold regressions. As the results show, green coverage in cities with low population size had a positive effect on PM2.5 at 1% significance level, with a coefficient of −0.344, while no significant correlation was found in the cities of medium and higher population size (Urban resident population >220.18 million). In models with population density as threshold variable, cities with medium population density size was negatively correlated with GC at 1% significance level with a coefficient of −0.443. This means that, for countries with low population (column I) or medium population density size (column V), a 1% increase in green coverage caused a 0.3~0.4% reduction in ΔPM2.5 concentrations. Despite the significant negative correlation observed in both column I and column V between GC and ΔPM2.5, cities in two columns are non-coplanar in space , while the same but weak impact exists in medium-sized cities (220.18 < Urban resident population < = 640.22). Moreover, there was a significant relationship between dummy variable N/S and ΔPM2.5.Population density was positively correlated with ΔPM2.5 are not simply linear. Urban resident population threshold is likely evolved from different stages of urban development and economic scale; population density threshold is likely derived from land-sea gradients and latitudinal position. In this context, several sub-objectives were set up and validated by KRLS model to provide reference for urban planners, i.e.,The preceding analyses indicated that the effects of green cover and other factors on ΔPM2.5 at different levels of socio-economic variables.To investigate the heterogeneity in the marginal effects of greening on ΔPMWhat effect does government's strong/weak support for environmental protection have on haze pollution control?2.5 and other economic variables?Can we find trajectories of drivers like EKC theory in relationships between ΔPM2.5 varies with data points. Thus, when the marginal effects are heterogeneous, KRLS results may be informative.Traditional OLS regression was conducted along with the KRLS analysis to compare the two statistical approaches . Unlike 2.5 in winter may decrease, a further understanding of the interaction between GC and ΔPM2.5 is required. The positive correlation between N/S and CR was detected in both statistical models, suggesting that it is reasonable to investigate the effect of urban green cover on air quality in the north and south of Qinling-Huaihe line, which would however require a different isolation protocol.2.5. The significant negative correlation between DE and ΔPM2.5 was detected in both statistical models, suggesting that relatively compact urban landscape may be an effective strategy to relieve morning air pollution from human economic activity. PG values also exhibited weakly negative correlation with the ΔPM2.5, revealed that economic development is environmentally sound while the mechanism remains to be analyzed. Additionally, a significant positive effect of EM on ΔPM2.5 was found during the winter, revealed the need of strict control on pollution emissions in winter.We also found relationships between economic variable (DE & PG) and ΔPMThe KRLS model was adopted in this study to examine the changes in marginal effects of GC with variation of other variables. This study focused not only on the effects of predictors on haze pollution but also on its heterogenicity varied with latitude and season. In addition, we have also introduced urban maintenance expenditure as control variable, focusing on urban development patterns hidden behind these variables and corresponding mitigation measures.To evaluate statistically significant interaction effects, additional analyses were conducted by regressing pointwise derivatives of a given independent variable on other predictors, one at a time .2.5 was relatively high. Therefore, how the influence of background PM2.5 concentration on GC coefficient varies with NS is shown in 2.5 has no significant effect on the coefficient of GC in the southern cities (latitude < 32°N), where levels of pollutants are relatively low. However, for cities located in the north with relatively high background PM2.5 concentrations, the increase of GC will lead to the increase of haze concentration, which is especially obvious in winter. But the conclusion is not universal, as is shown in 2.5. It can be easily inferred from the 2.5 in northern cities but can effectively improve urban haze pollution in southern, with that effect in developed cities more remarkable. Finally, the bottom graphs 2.5 in southern cities but increased haze pollution in northern cities, the effect fades following the addition of population density such that the marginal effect of PD approached 0 in big cities. In winter  in reducing haze pollution.Urban maintenance expenditure is a new indicator of the importance to environmental protection; it is well-suited to examining ΔPM from GC , with an from GC  declaredincrease , such thntration . As a ma2.5 were different between northern and southern cities, which was very rarely discussed before  after reaching the threshold. For northern cities located on the plains with high population density and haze severe pollution, PM2.5 concentrations are more dominated by pollution sources    since exollutant . In addi2.5 emissions has been discussed in numerous studies .Second, the deposition effect of urban green on air quality might be too small to be detected in a single-year cross-sectional regression analysis, as the results of this study confirmed that other factors such as real GDP per capita, urban density, and air humidity have a more significant effect on ΔPM2.5 in the urban areas, we decided to point to this observation in the discussion, as this provides an interesting starting point for future research. In addition, background PM2.5 concentration was an important factor affecting the marginal benefit of greening, it's a pity we have not explored this process specifically, which may inform design of future research studies that further explore these relationships  and spatial variation of incremental PM2.5 concentrations in 285 cities in China has been explored. We use the threshold model to make an exploratory analysis on the influence factors of incremental PM2.5 concentrations during peak hours (ΔPM2.5). For comprehensive results, a KRLS model was used to further explore the non-linearity, interaction, and heterogeneity among parameters. The main findings were as follows:This study investigated the influence of urban greening and other urban elements on incremental concentration of PM2.5. Additionally, the elasticity and significance of each independent variable to the dependent variable may vary across other independent variables level and will also change with season and latitude. To this end, we divide Chinese cities into three categories and adopt different measures to different regions according to local conditions and specific drivers. The results and implications are presented below.In addition to green coverage, the estimation results suggest that population density, real GDP per capita, government environmental investment are essential driving factors affecting ΔPMClass I: Southern cities with low government environmental investment. In this kind of city, green cover and economic growth show strong effect on mitigating haze pollution at their respective high level. It was found that excessive population agglomeration can also exacerbate haze pollution. The green coverage per capita needs to be further improved for better air quality. On the one hand, the government should increase the expenditure of greening and air sector, on the other hand, for the densest urban areas, government should reduce the density of economic activities to allocate green resources rationally.2.5 concentration, haze pollution was enhanced by green coverage. Contribution of urban density to PM2.5 concentration shows positive and negative effects at low and high levels, respectively. That's to say, more intensive development may have reduced vehicle travel distances and toxic air emissions in big cities. Therefore, the control and supervision of pollution sources is crucial for controlling haze pollution. At the early stages of urban development, mitigation interventions related to urban patterns have the greatest potential, reasonable urban planning should be made to reduce environmental stress for follow-up human activity intensive areas . For cities with high population density and smog pollution, a plausible spatial pattern is needed to reduce the burden of commuting, avoiding excessive population concentration, while offsetting the negative environmental impact of economic activities by improving the efficiency of land resource utilization and establishing more stringent policies to limit the discharge of pollutants from production and life.Class II: Northern cities with low government environmental investment. In this kind of city, population density and green coverage per capita were the main influencing factors of haze during the peak period. For cities in the central plains of China, with high background PMClass III: Cities with high government environmental investment. Compared with other categories, it's a healthy urban development model that the increase of GC always effectively reduces haze concentration during peak period. In addition, the increase in population density and economic level also expanded the marginal effect of green coverage to reduce air pollution. Altogether, government environmental expenditure was observed to be a powerful means to reduce haze pollution during peak period in every stage of urban development and every region.http://data.cnki.netPublicly available datasets were analyzed in this study. This data can be found here: http://www.gtarsc.com/http://www.cnemc.cn/sssj/https://www.ceicdata.com/zh-hansftp://ftp.ncdc.noaa.gov/pub/data/noaa/isd-lite/.SW: designed the manuscript, structured and wrote the manuscript, data processed and map designed, indicator calculation, and result analysis. SC: methodology for KRLS model. XQ: funding acquisition, validation, conceptualization, and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sunflower germplasm collections are valuable resources for broadening the genetic base of commercial hybrids and ameliorate the risk of climate events. Nowadays, the most studied worldwide sunflower pre-breeding collections belong to INTA (Argentina), INRA (France), and USDA-UBC (United States of America–Canada). In this work, we assess the amount and distribution of genetic diversity (GD) available within and between these collections to estimate the distribution pattern of global diversity. A mixed genotyping strategy was implemented, by combining proprietary genotyping-by-sequencing data with public whole-genome-sequencing data, to generate an integrative 11,834-common single nucleotide polymorphism matrix including the three breeding collections. In general, the GD estimates obtained were moderate. An analysis of molecular variance provided evidence of population structure between breeding collections. However, the optimal number of subpopulations, studied via discriminant analysis of principal components (K = 12), the bayesian STRUCTURE algorithm (K = 6) and distance-based methods (K = 9) remains unclear, since no single unifying characteristic is apparent for any of the inferred groups. Different overall patterns of linkage disequilibrium (LD) were observed across chromosomes, with Chr10, Chr17, Chr5, and Chr2 showing the highest LD. This work represents the largest and most comprehensive inter-breeding collection analysis of genomic diversity for cultivated sunflower conducted to date. Helianthus annuus spp. macrocarpus) is one of the most important oilseed crops, with a global production value estimated at USD 20 billion per year (FAO 2016). Its early domestication occurred in the interior mid-latitudes of eastern North America ca. 4000 years ago, but it became an oil crop only when it reached Russia late in the XVIIIth century. The foundational efforts of Pustovoit at VNIIMK to develop high yielding, open-pollinated varieties with high oil content are considered the main genetic base of modern sunflower breeding [Helianthus petiolaris and cultivated sunflower [Sunflower , the Instituto Nacional de Tecnología Agropecuaria (INTA) has pioneered sunflower breeding and has become one of the most prolific sunflower breeders in the country [Argentina has a long tradition in sunflower breeding. Since 1931, and by exploiting the diversity of a broad range of foreign genetic resources in combination with introgressions of wild  country ,5,6.Today, the major sunflower producing countries are Ukraine, Russia, the European Union, and Argentina. According to Vear , in spitOver the last years, these institutions made a significant contribution towards the development and characterization of proprietary breeding and pre-breeding collections ,12,13,14With the publication of the first sunflower reference genome , a largeIn this work, we implement for the first time a double digest RAD seq approach  to genotData generation: The pre-breeding collection of INTA, composed of 135 sunflower inbred lines preserved at the Active Germplasm Bank of INTA Manfredi (AGB-IM), was genotyped using a double digest restriction-site associated DNA sequencing (ddRADseq) protocol adapted from Peterson et al. . Two rarprocess_radtags routine implemented in Stacks . Only biallelic positions were kept for further analysis. Finally, the Populations command was used to generate the final VCF file.Variant calling: Raw Illumina reads were de-multiplexed and quality-checked using the s (v1.42 ). After s . Singlesoftware , as descsoftware . AdditioddRADseq data exploration and VCF filtering: Filters related to the percentage of missing data (80%), number of SNPs per sequenced region or tag (no more than 4 SNPs/tag), and minor allele frequency (MAF >0.05) were applied to the VCF matrix using R and custom scripts. Plots were performed using the R package “ggplot2” .Circa 10 TB of low-coverage (~10×) whole genome sequencing (WGS) data were retrieved from the European Nucleotide Archive . This cohttps://github.com/gamerino/SNPsRFImputation). After imputation, given that the methodology discards those SNPs that cannot be accurately imputed, an intersection of the three matrices was done in order to obtain the same set of SNPs for all populations. The variants called in this work for the three sunflower populations have been submitted to Ensembl Plants , where The Variant Effect Prediction (VEP) tool , availabMeasures of genetic diversity, including unbiased expected heterozygosity (He), observed heterozygosity (Ho), allele frequency and minor allele frequency (MAF) were estimated between and within populations, using the R packages “PopPR”  and “AdeThe extent of differentiation between INTA, INRA, and USDA-UBC was investigated via analysis of molecular variance (AMOVA), using the R Package “PopPR” . StatistThe Bayesian approach implemented in STRUCTURE ,31 was ufind.clusters’, evaluating a range from 1 to 40. The optimal number of clusters was chosen on the basis of the lowest associated Bayesian information criterion (BIC).Genetic relationships among accession were also examined by applying discriminant analysis of principal components . The fuIn addition, a relatedness analysis using Identity-By-Descent (IBD) measures between all the accessions was performed using the R package “SNPRelate” .In order to compare with previous work ,8,9,11, A total of ten (10) public sunflower inbred lines that were present in more than one breeding collection were compared using the percentage of shared alleles to assess potential discrepancies among collections.2 vs. physical distance (bp) per chromosome were plotted using the R package “ggplot2” [The extent of LD was estimated using the R package “Synbreed”  with a gggplot2” . The y ~A total of 126 M reads were produced across six pooled batches with 24 inline barcodes along with a 6-bases TruSeq indexing system to tag each pool. After de-multiplexing, about 125 M reads were available for downstream analyses. Along with the removal of barcode sequences, reads were all clipped to a fixed length of 110 bp in order to (i) remove low-quality bases at the 3′-ends and (ii) maintain a consistent length given the variable length of the barcodes. This processing was necessary to prevent any incompatibility issues with downstream analysis software. An average of 930,000 reads per sample was generated , and ~97% of them mapped against the reference genome [This final matrix was used as input for variant calling under the “GENOTYPE_GIVEN_ALLELES” option in GATK for the USDA-UBC and INRA data.From the low-coverage (~10x) WGS data retrieved from ENA, an average of 1.25% (ranging from 0% to 5.99%) of the USDA-UBC reads and 2.47% (ranging from 0.29% to 7.53%) of the INRA reads were discarded after trimming. Of these, 96.69% (68.60–98.44%) of USDA-UBC data and 97.27% (94.53–99.38%) of INRA data mapped against the reference genome. From the initial 34,488 SNP list used as input for variant calling, 22,207 SNPs in the USDA-UBC population and 20,481 SNPs in the INRA population passed the filters specified for variant calling and were genotyped in at least one accession.The imputation algorithm uses correlation and LD to select the predictors from the list of SNPs fully genotyped in each population. For the INTA data, a total of 1697 SNPs were genotyped in all accessions, while 842 and 7789 were fully genotyped in the USDA-UBC and INRA breeding collections, respectively. After imputation, a total of 20,750, 18,525, and 18,925 SNPs were obtained for INTA, USDA-UBC and INRA populations, respectively.Intersecting the imputed matrices rendered a total of 11,834 SNPs in common between the three breeding collections. The markers showed a uniform distribution in all the sunflower chromosomes , varyingThe predicted consequences of all the genotyped SNPs, classified in 16 terms defined by the Sequence Ontology , as wellThe GD values expected heterozygosity (He), observed heterozygosity (Ho) and minor allele frequency (MAF) were estimated for the full panel of accessions, as well as for each breeding collection. The results are presented in Regardless of the population size (n), the He and Ho values were comparable between breeding collections. A total of 18 SNPs  had low frequency (MAF <0.05) in both INRA and USDA-UBC collections, but had MAF >0.05 in INTA accessions. A graphical representation of individual heterozygosis between and within populations is presented in p < 0.01, 999 permutations).The analysis of molecular variance (AMOVA) using 11,834 SNPs revealed significant genetic differentiation of populations, with variation among breeding collections, representing 4.58% of the total genetic variance  and the 6 groups retrieved from STRUCTURE or the 12 groups retrieved from DAPC, some clusters were consistently enriched with accessions from a single origin . AccordiDistance matrices based on IBD were constructed for all pairs of individuals. Distances varied from 0.000 to 0.389, with an average of 0.172. The function cut-tree identified 9 groups . The denA total of 389 out of 482 accessions were classified as HA/RHA, while the remaining 93 accessions for which no information was available, were kept as N/A. The distinction between HA and RHA was displayed on the second axis of the PCA, although an overlapping zone can be seen . The firThe list of duplicated inbred lines, along with their inter-collection identity estimates, is presented in 2, were obtained for the full panel of accessions, as well as for each of the breeding collections. More than 4.8 M pairwise comparisons were done per breeding population.The patterns of LD decay, measured as r2 ranging from 0.19 to 0.22), independently of the set of accessions under analysis, followed by Chr17 (mean r2 ranging from 0.11 to 0.16), Chr5 (mean r2 ranging from 0.12 to 0.15), and Chr2 (mean r2 ranging from 0.11 to 0.14). The INRA breeding population also showed high LD at Chr15 (mean r2: 0.11). On the other hand, Chr14 showed the lowest LD (mean r2 ranging from 0.04 to 0.05). Visual inspection of the heatmap showed specific non-recombining regions  and chromosomes with reduced recombination .The results of the LD analysis are summarized in Germplasm collections are valuable resources for crop improvement. However, to fully unlock their potential, it is critical to have detailed information about the amount and the distribution of the genetic diversity available within collections. Through the integration of genomic data for three of the most studied sunflower breeding collections, INTA, INRA and USDA-UBC, this work represents the largest and most comprehensive analysis of genetic diversity, population structure and linkage disequilibrium for cultivated sunflower conducted to date.Reduced representation sequencing methods, as genotyping by sequencing (GBS) and double digest RAD seq (ddRADseq) provide a high number of polymorphic loci at a relatively low cost. A few GBS approaches were reported recently for sunflower ,40,41,42The genotyping strategy implemented here combined proprietary ddRADseq with public WGS data to obtain an integrative SNP-matrix, including individuals from different breeding programs. This strategy allows not only to augment the number of individuals/populations under study but also to give significance to the big amount of publicly available NGS data, contributing to open data science and better use of the available resources. Although this is a powerful tool for both population and evolutionary genomics, there is no clear consensus on how to perform these analyses. Moreover, the use of a SNP-list to call variants in specific positions, instead of doing whole genome variant calling, reduces the computer processing time significantly, with mapping against the reference genome being the most computationally demanding step of the methodology. On the other hand, the main drawback of this combined genotyping strategy is that it restricts the queried SNPs to those polymorphic in the firstly genotyped dataset . However, this did not seem to impact significantly in our results, where each population showed genetic diversity levels similar to those reported in previous work . In theAn initial characterization of the SNP-matrix showed that the markers are uniformly distributed across sunflower chromosomes, being the number of SNPs in accordance with chromosome length . This validates the performance of the ddRADseq assay, which is expected to generate evenly distributed markers across the genome . MoreoveIn general, the genetic diversity estimates obtained within the global and the individual breeding collections were moderate, with comparable levels of expected heterozygosity, independently of the population size. As expected when working with inbred lines, the observed heterozygosity values were low in each breeding collection. Differences in allele frequencies between breeding collections were apparent, not only when observing the minor allele frequency values but also when inspecting the allele frequency plots. The He values obtained here (~0.452) are higher than those reported by Mandel et al.  using a Our analysis of population structure revealed that differences among breeding programs explained only a small proportion of total genetic variation. However, although none of the clustering methods used here showed a direct correspondence with the origin of accessions, some groups were consistently recovered. This is the case for STRUCTURE Groups 6 and 3, which are composed of USDA-UBC accessions, and STRUCTURE Group 4, which mainly consists of INTA inbred lines. These distinct groups of accessions are of particular interest when planning the incorporation of new genetic backgrounds to each breeding collection. Among them, STRUCTURE group 4 contains a mixture of Argentinean HA and RHA inbred lines, bred for traits of agronomic importance such as drought stress tolerance and rust and sclerotinia head rot resistance ,45.Previous population studies based on these sunflower collections  estimates as a function of a physical distance (bp) in sunflower. Whole genome LD estimates reported until now were based on genetic distance , Chr10 showed the highest LD values, followed by Chr17, Chr5 and Chr2. Moreover, specific intra population LD patterns were observed, as high LD in Chr15, in INRA accessions. Mandel et al.  and NambThis work summarizes the most comprehensive characterization of sunflower genetic diversity encompassing breeding collections from INTA, INRA and USDA-UBC. Even though genetic differences were detected between breeding origins, they only explain 4.58% of the total variability. This fact added to the moderate genetic diversity estimates obtained here and similarities between LD patterns, suggest some homogeneity among international breeding materials, and a narrow genetic base of current sunflower breeding. In this regard, gene banks and crop wild relatives collections hold a substantial amount of genetic diversity for many agronomically important traits that can be exploited in order to expand the breeding genetic base and to cope with the changing environmental challenges for the crop.