Progress Towards Genetics and Breeding for Minor Genes Based Resistance to Ug99 and Other Rusts in CIMMYT High-Yielding Spring Wheat

Wheat rusts continue to cause significant losses worldwide despite major efforts given to their genetic control. This is due to frequent evolution and selection of virulence in pathogen overcoming the deployed race-specific resistance genes. Although the life of effective race-specific resistance genes can be prolonged by using gene combinations, an alternative approach being implemented at CIMMYT is to deploy varieties that posses adult plant resistance （APR） based on combinations of minor, slow rusting genes. When present alone, the APR genes do not confer adequate resistance especially under high disease pressure; however, combinations of 4 or 5 minor genes usually result in ＂near-immunity＂ or a high level of resistance. Although only a few APR genes are catalogued, various APR QTLs are now known and could lead to further characterization of additional genes. Four characterized genes have pleiotropic effects in conferring partial APR to all 3 rusts and powdery mildew, thus simplifying the task of breeding wheat varieties that are resistant to multiple diseases. Significant progress was made recently in developing high-yielding wheat germplasm that possesses high levels of APR to all three rusts by implementing a Mexico- Kenya shuttle breeding scheme. Parents with APR to Ug99 were hybridized with high-yielding parents that had adequate to high levels of APR to leaf rust and yellow rust. Segregating populations and advanced lines from these crosses were selected under high rust pressures in Mexico （leaf rust and yellow rust） and Kenya （Ug99 stem rust and yellow rust） to identify high- yielding progenies that possess high to adequate APR to all three rusts. International distribution of these high-yielding wheats is underway through CIMMYT intemational yield trials and screening nurseries. It is expected that several wheat varieties with APR to three rusts will be released and grown in various countries in the near-future that will allow determining the durability of resistance.     

Inheritance of Powdery Mildew Resistance in Cucumber （Cucumis sativus L.） and Development of an AFLP Marker for Resistance Detection

Cucumber powdery mildew is one of the most destructive diseases of cucumber throughout the world. In the present study, inheritance of powdery mildew resistance in three crosses, and linkage of resistance with amplified fragment length polymorphism （AFLP） markers are studied to formulate efficient strategies for breeding cultivars resistant to powdery mildew. The joint analysis of multiple generations and AFLP technique has been applied in this study. The best model is the one with two major genes, additive, dominant, and epistatic effects, plus polygenes with additive, dominant, and epistatic effects （E-l-0 model）. The heritabilities of the major genes varied from 64.26% to 97.82%, and susceptibility was incompletely dominant for the two major genes in the three crosses studied. The additive effects of the two major genes and the dominant effect of the second major gene were high, and the epistatic effect of the additive-dominant between the two major genes was the highest in cross I . In cross II, the absolute value of the additive effect, dominant effect, and potential ratio of the first major gene were far higher than those of the second major gene, and the epistatic effect of the additive-additive was the highest. The genetic parameters of the two major genes in cross III were similar to those in cross II. Correlation and regression analyses showed that marker E25/M63-103 was linked to a susceptible gene controlling powdery mildew resistance. The marker could account for 19.98% of the phenotypic variation. When the marker was tested on a diverse set of 29 cucumber lines, the correlation between phenotype and genotype was not significant, which suggested cultivar specialty of gene expression or different methods of resistance to powdery mildew. The target DNA fragment was 103 bp in length, and only a small part was found to be homologous to DNA in the other species evaluated, which indicated that it was unique to the cucumber genome.     

QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli

The Italian wheat cv.Strampelli displays high resistance to powdery mildew caused by Blumeria graminis f.sp.tritici.The objective of this study was to map quantitative trait loci(QTLs) for resistance to powdery mildew in a population of 249 F 2:3 lines from Strampelli/Huixianhong.Adult plant powdery mildew tests were conducted over 2 yr in Beijing and 1 yr in Anyang and simple sequence repeat(SSR) markers were used for genotyping.QTLs Qpm.caas-3BS,Qpm.caas-5BL.1,and Qpm.caas-7DS were consistent across environments whereas,Qpm.caas-2BS.1 found in two environments,explained 0.4-1.6,5.5-6.9,27.1-34.5,and 1.0-3.5% of the phenotypic variation respectively.Qpm.caas-7DS corresponded to the genomic location of Pm38/Lr34/Yr18.Qpm.caas-4BL was identified in Anyang 2010 and Beijing 2011,accounting for 1.9-3.5% of phenotypic variation.Qpm.caas-2BS.1 and Qpm.caas-5BL.1 contributed by Strampelli and Qpm.caas-3BS by Huixianhong,seem to be new QTL for powdery mildew resistance.Qpm.caas-4BL,Qpm.caas-5BL.3,and Qpm.caas-7DS contributed by Strampelli appeared to be in the same genomic regions as those mapped previously for stripe rust resistance in the same population,indicating that these loci conferred resistance to both stripe rust and powdery mildew.Strampelli could be a valuable genetic resource for improving durable resistance to both powdery mildew and stripe rust in wheat.     

Pathotypes and Genetic Diversity of Blumeria graminis f. sp. hordei in the Winter Barley Regions in China

Barley powdery mildew caused by Blumeria graminis f. sp. hordei （Bgh） is one of the most destructive foliar diseases of barley in the winter barley region of China. The evaluation and assessment of the virulence and diversity of Bgh populations help to determine effective sources of resistance to the pathogen. 515 isolates were collected from seven populations of Bgh on cultivated barley in seven geographically distant locations in 2006. Their virulence was determined by inoculation onto 26 differential host lines. All of the isolates belonged to 58 pathotypes and 13 of which included 81% of these isolates. The most abundant pathotype was pathotype 0047 （18.3%）, the second most abundant was pathotype 0045 （11.8%） and the third most abundant was pathotype 0057 （7.8%）. Most of virulent genes investigated in this study showed similar frequencies in the seven different areas. These indicate that the seven locations may be in a uniform epidemiological region and barley cultivars in these areas may have similar genetic background. Diversities within these populations and distances between these populations measured by KOIND package were different. Correlations were not found between the genetic distance and the geographical distances between different locations. This suggested that long distance spread and local epidemics existed in the major winter barley growing regions in China.     

Molecular Screening and Resistance Evaluation of American Wheat Cultivars to Chinese Stripe Rust Races

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the major diseases of wheat in China. In order to asses the resistance levels and existing Yr genes among 59 wheat cultivars （lines） from the Pacific Northwest （PNW） of the United States, to provide resistance resources for genetic improvement of wheat stripe rust resistance in China, 59 wheat cultivars （lines） from PNW of the United States were infected by 3 mixed races of predominant Chinese stripe rust races CRY31, CRY32, and CRY33 to evaluate their resistance at seedling and adult plant stages, and screened with molecular markers tightly linked to currently effective all-stage resistance genes YrlO, Yrl5 and adult plant resistance genes Yrl8, Yr39. Of 59 American cultivars （lines）, five cultivars （lines）, Expresso, 02W50076, ACS52610, WA008012, and WA00801833, had all-stage resistance, showing resistance to mixed races of CRY31, CRY32, and CRY33 at both seedling and adult plant stages. 33 cultivars （lines） had adult plant resistance, only showing resistance to stripe rust at adult stage. Based on the molecular screening, none of the 59 PNW cultivars （lines） had the polymorphic bands of linked markers to YrlO. There were 12, 33 and 29 cultivars （lines） which bad polymorphic bands of linked markers to Yr15, Yr18 and Yr39, accounting for 20, 55 and 49% of the 59 PNW cultivars （lines）, respectively. All these results suggested that Yr15, Yr18 and Yr39 were widespread among PNW cultivars （cultivars） and could be utilized in Chinese wheat stripe rust resistance breeding.     

Reactive oxygen species are involved in cell death in wheat roots against powdery mildew

Inoculation of wheat(Triticum aestivum L.) leaves with wheat powdery mildew fungus(Blumeria graminis f. sp. tritici) induces the cell death in adventitious roots. Reactive oxygen species(ROS) play a key role in respond to biotic stress in plants. To study the involvement of ROS and the degree of cell death in the wheat roots following inoculation, ROS levels and microstructure of root cells were analyzed in two wheat cultivars that are susceptible(Huamai 8) and resistant(Shenmai 8) to powdery mildew fungus. At 18 d after powdery mildew fungus inoculation, only Huamai 8 displayed the leaf lesions, while root cell death occurred in both varieties. Huamai 8 had a high level of ROS accumulation, which is associated with increased root cell degradation, while in Shenmai 8, there was little ROS accumulation correlating with slight root cell degradation. The molecular study about the expression levels of ROS scavenging genes(MnSOD and CAT) in wheat roots showed that these genes expression decreased after the leaves of wheat was inoculated. The difference between Huamai 8 and Shenmai 8 on subcellular localization of H2 O2 and O2–· was corresponded with the different down-regulation of the genes encoding for superoxide dismutase and catalase in two wheat cultivars. These results suggested that ROS were involved in the process by which powdery mildew fungus induced cell death in wheat roots.     

Messages from Powdery Mildew DNA： How the Interplay with a Host Moulds Pathogen Genomes

The genomes of the barley, Arabidopsis and pea powdery mildew are significantly larger than those of related fungi. This is due to an extraordinary expansion of retro-trasposons that are evident as repetitive elements in the sequence. The protein coding genes are fewer than expected due to an overall reduction in the size of gene families, a reduction in the number of paralogs and because of the loss of certain metabolic pathways. Many of these changes have also been observed in the genomes of other taxonomically unrelated obligate biotrophic pathogens. The only group of genes that bucks the trend of gene loss, are those encoding small secreted proteins that bear the hall marks of effectors.     

Transgenic Expression of a Functional Fragment of Harpin Protein Hpa1 in Wheat Represses English Grain Aphid Infestation

The harpin protein Hpa1 produced by the rice bacterial blight pathogen promotes plant growth and induces plant resistance to pathogens and insect pests. The region of 10-42 residues （Hpa110-42） in the Hpa1 sequence is critical as the isolated Hpa110-42 fragment is 1.3-7.5-fold more effective than the full length in inducing plant growth and resistance. Here we report that transgenic expression of Hpa110-42 in wheat induces resistance to English grain aphid, a dominant species of wheat aphids. Hpa110-42-induced resistance is effective to inhibit the aphid behavior in plant preference at the initial colonization stage and repress aphid performances in the reproduction, nymph growth, and instar development on transgenic plants. The resistance characters are correlated with enhanced expression of defense-regulatory genes （EIN2, PP2-A, and GSL10） and consistent with induced expression of defense response genes （Hel, PDF1.2, PR-1b, and PR-2b）. As a result, aphid infestations are alleviated in transgenic plants. The level of Hpa110-42-induced resistance in regard to repression of aphid infestations is equivalent to the effect of chemical control provided by an insecticide. These results suggested that the defensive role of Hpa110-42 can be integrated into breeding germplasm of the agriculturally signiifcant crop with a great potential of the agricultural application.     

Development of an STS Marker Linked to Powdery Mildew Resistance Genes PmLK906 and Pm4a by Gene Chip Hybridization

Wheat （Triticum aestivum L.） line Lankao 90（6） carries a recessive powdery mildew resistance gene temporarily named PmLK906 on chromosome 2AL. Near PmLK906 there is another known powdery mildew resistance gene locus Pm4. To track the two powdery mildew resistance genes in wheat breeding program by marker assisted selection （MAS）, a linked molecular marker was developed in this study. Wheat gene chip hybridization combined with bulked segregant analysis （BSA） was used to develop an sequence-tagged sites （STS） marker for PmLK906 and Pm4. A new 2 125 bp full-length cDNA clone （GenBank accession no. EU082094） similar to csAtPR5 ofAegilops tauschii was isolated from Lankao 90（6） 21-12, and temporarily named TaAetPR5. Specific products could be amplified from cultivars or lines possessing Pm4a, Pm4b and PmLK906 with primers p9-7pl and p9-7p2 derived from TaAetPR5. TaAetPR5 was linked to PmLK906 at a genetic distance of 7.62 cM, and cosegregated with Pm4a. The p9-7p1 and p9-7p2 could be used as an STS marker for these resistance genes in wheat breeding. Because this marker was cosegregated with Pm4a, it can be used in map-based cloning of the alleles at Pm4 locus also.     

Sequence Polymorphisms and Phylogenetic Relationships of hina Gene in Wild Barley from Tibet, China

The hina gene encodes a HINA protein in seeds of barley （Hordeum vulgare）, which was known to affect the grain hardness. 171 hina gene sequences from Tibetan wild barley accessions and worldwide were characterized. Across 1 452 nucleotides of 171 hina genes, 152 SNPs were detected, giving an average frequency of one SNP per 9.5 bases. There were 93 singleton variable sites （the nucleotide polymorphism only observed in a single accession）, 59 polymorphic sites （the polymorphisms found in two or more accessions） and 8 indels. A total of 18 haplotypes were defined, and most of the barley accessions shared one gene haplotype. H. spontaneum had a wider haplotype distribution. Through the analysis of median-joining network of the 18 haplotypes, 4 haplotype groups were found, which were testified by neighbor-joining tree based on the complete sequence alignment. Extremely low level of hina gene diversity was observed in Tibetan wild barley accessions, indicating that Tibet is unlikely a center of origin for cultivated barley.     

Seedling and adult plant resistance to leaf rust in 46 Chinese bread wheat landraces and 39 wheat lines with known Lr genes

Wheat leaf rust,caused by Puccinia triticina(Pt),is an important foliar disease that has an important influence on wheat yield.The most economic,safe and effective way to control the disease is growing resistant cultivars.In the present study,a total of 46 wheat landraces and 34 wheat lines with known Lr(leaf rust resistance)genes were inoculated with 16Pt pathotypes for postulating seedling resistance gene(s)in the greenhouse.These cultivars and five wheat differential lines with adult plant resistance(APR)genes(Lr12,Lr22b,Lr34,Lr35 and Lr37)were also evaluated for identification of slow rusting resistance in the field trials in Baoding,Hebei Province of China in the 2014–2015 and 2015–2016 cropping seasons.Furthermore,10 functional molecular markers closely linked to 10 known Lr genes were used to detect all the wheat genotypes.Results showed that most of the landraces were susceptible to most of the Pt pathotypes at seedling stage.Nonetheless,Lr1 was detected only in Hongtangliangmai.The field experimental test of the two environments showed that 38 landraces showed slow rusting resistance.Seven cultivars possessed Lr34 but none of the landraces contained Lr37 and Lr46.Lr genes namely,Lr9,Lr19,Lr24,Lr28,Lr29,Lr47,Lr51 and Lr53 were effective at the whole plant stage.Lr18,Lr36 and Lr45 had lost resistance to part of pathotypes at the seedling stage but showed high resistance at the adult plant stage.Lr34 as a slowing rusting gene showed good resistance in the field.Four race-specific APR genes Lr12,Lr13,Lr35 and Lr37 conferred good resistance in the field experiments.Seven race-specific genes,Lr2b,Lr2c,Lr11,Lr16,Lr26,Lr33 and LrB had lost resistance.The 38 landraces showed slow rusting resistance to wheat leaf rust can be used as resistance resources for wheat resistance breeding in China.     

The EST Analysis of A Suppressive Subtraction cDNA Library of Chinese Wild Vitis pseudoreticulata Inoculated with Uncinula necator

Uncinula necator is a worldwide serious fungus disease causing annual heavy lost on grapevine production. In order to get more informations on defense related EST sequences and help breeding program, we constructed and characterized a suppression subtractive hybridization cDNA library with artificially inoculated leaves and control Chinese wild Vitis pseudoreticulata clone Baihe-35-1, which is highly resistant to powdery mildew. In the library, the length of 58 EST fragments known as putative functions varied from 130 to 800 bp, and 60% of the ESTs exhibited high similarity to known sequences in database of GenBank with BLASTX analysis. These genes were involved in stress/defense response, detoxification, signal transduction, disease defense, and etc., and 14 ESTs remained unknown or hypothetical proteins, which may be new genes. The experiment provided an important basis for studying the disease-resistance mechanism and obtaining the genes for the aim of improving grapevine powdery mildew resistance.     

Inheritance and Molecular Mapping of Stripe Rust Resistance Gene Yr88375 in Chinese Wheat Line Zhongliang 88375

Stripe rust is one of the most important diseases of wheat worldwide. Inheritance of stripe rust resistance and mapping of resistance gene with simple sequence repeat （SSR） markers are studied to formulate efficient strategies for breeding cultivars resistant to stripe rust. Zhongliang 88375, a common wheat line, is highly resistant to all three rusts of wheat in China. The gene conferring rust disease was deduced originating from Elytrigia intermedium. Genetic analysis of Zhongliang 88375 indicated that the resistance to PST race CYR31 was controlled by a single dominant gene, temporarily designated as Yr88375. To molecular map Yr88375, a F2 segregating population consisting of 163 individuals was constructed on the basis of the hybridization between Zhongliang 88375 and a susceptible wheat line Mingxian 169; 320 SSR primer pairs were used for analyzing the genetic linkage relation. Six SSR markers, Xgwm335, Xwmc289, Xwmc810, Xgdmll6, Xbarc59, and Xwmc783, are linked to Yr88375 as they were all located on chromosome 5BL Yr88375 was also located on that chromosome arm, closely linked to Xgdmll6 and Xwmc810 with genetic distances of 3.1 and 3.9 cM, respectively. The furthest marker Xwmc783 was 13.5 cM to Yr88375. Hence, pedigree analysis of Zhongliang 88375 combined with SSR markers supports the conclusion that the highly resistance gene Yr88375 derived from Elytrigia intermedium is a novel gene for resistance to stripe rust in wheat. It could play an important role in wheat breeding programs for stripe rust resistance.     

Messages from Powdery Mildew DNA:How the Interplay with a Host Moulds Pathogen Genomes

The genomes of the barley,Arabidopsis and pea powdery mildew are signif icantly larger than those of related fungi.This is due to an extraordinary expansion of retro-trasposons that are evident as repetitive elements in the sequence.The protein coding genes are fewer than expected due to an overall reduction in the size of gene families,a reduction in the number of paralogs and because of the loss of certain metabolic pathways.Many of these changes have also been observed in the genomes of other taxonomically unrelated obligate biotrophic pathogens.The only group of genes that bucks the trend of gene loss,are those encoding small secreted proteins that bear the hall marks of effectors.     

Characterization of the Promoter of a Homolog of Maize MADS-Box Gene m18

Maize （Zea mays L.） is one of the world’s major food crops, and often suffers from tremendous yield loss caused by abiotic stresses. The MADS-box genes are known to play versatile roles in plants, controlling plant responses to multiple abiotic stresses. However, understanding of regulation of their expressions by the conventional loss-of-function approach is very dififcult. So far, regulation of MADS-box gene expression is little known. The best approach to retrieve expression regulation of this category of genes is to characterize expression of their promoters. In this study, the promoter of a homolog （GenBank accession no. EC864166） of maize MADS-box gene m18 was cloned by way of genome-walking PCR, named Pro66. Predicative analysis indicated that Pro66 contains more than one TATA box and multiple cis-acting environmental conditions-responsive elements （ECREs）. Pro66 could drive expression of theβ-glucuronidase （GUS）-encoding gene in maize, and heterologous expression of GUS in red pepper stressed by water deifcit, salt, copper, iron deifciency, heat, cold, and grown under short and long photoperiods, echoing predicative ECREs. Conclusively, maize MADS-box gene m18 likely plays versatile functions in maize response to multiple abiotic stresses due to the promoter with multiple cis-acting elements. The complex arrangement of multiple cis-acting elements in the promoter features meticulously regulated expression of m18. The results give informative clues for heterologous utilisation of the promoters in monocot and dicot species. The copy of the ECREs and heterologous expression of the promoter in dicot species are also discussed.     

Research on Oxalate Oxidase and Its Genes in Plants

This paper introduces the discovery,composition and structure of oxalate oxidase,as well as illustrates the biological functions of this enzyme.With a comprehensive introduction upon previous researches upon gene cloning and heredity transformation of this enzyme,it indicates that heredity transformation can increase the content of oxalate oxidase within the plants and also enhance their resistance.The paper also points out the problems such as lack of gene resources and difficulty in the transformation of heterologous genes,and the focus in later researches should be laid upon the exploration of plant resources relative to this enzyme and selection of resistant species.     

NBS Profiling Identifies Potential Novel Locus from Solanum demissum That Confers Broad-Spectrum Resistance to Phytophthora infestans

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, is the most serious disease of potato worldwide. The adoption of varieties with resistance genes, especially broad-spectrum resistance genes, is the most efficient approach to control late blight. Solanum demissum is a well-known wild potato species from which 11 race-specific resistance genes have been identified, however, no broad-spectrum resistance genes like RB have been reported in this species. Here, we report a novel reisistance locus from S. demissum that potentially confer broad-spectrum resistance to late blight. A small segregating population of S. demissum were assessed for resistance to aggressive P. infestans isolates（race 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11）. This coupled with nucleotide binding site（NBS） profiling analyses, led to the identification of three fragments that linked to the potential candidate resistance gene（s）. Cloning and sequence analysis of these fragments suggested that the identified resistance gene locus is located in the region containing R2 resistance gene at chromosome 4. Based on the sequences of the cloned fragments, a co-segregating sequence characterized amplified region（SCAR） marker, RDSP, was developed. The newly identified marker RDSP will be useful for marker assisted breeding and further cloning of this potential resistance gene locus.     

Population Diversity of Puccinia graminis is Sustained Through Sexual Cycle on Alternate Hosts

A high degree of virulence diversity has been maintained in the population of Puccinia graminis f. sp. tritici （Pgt） in northwestern United States. Although Berberis vulgaris is present in the region and Pgt has been isolated from aecial infections on B. vulgaris, the population is too diverse to be explained by the limited presence of B. vulgaris alone. Since 2008, we have isolated P. graminis from aecial infections on fruits of Mahonia repens and Mahonia aquifolium from northwestern United States. These two native woody shrub species, widely distributed in western North America, were once classified as resistant to P. graminis based on artificial inoculations. By isolating P. graminis from aecia, we established that M. repens and M. aquifolium along with B. vulgaris （albeit infrequent） serve as the alternate hosts ofP. graminis in the region. The isolates of P. graminis from Mahonia of North America had diverse virulence patterns and most of the isolates could be differentiated on Morocco, Line E, Chinese Spring, Little Club, LMPG-6, Rusty, and other genotypes that are considered to be universally susceptible to most Pgt isolates. This discovery explained the persistence of virulence diversity of Pgt observed in isolates derived from uredinia on cereal crops in the region. In addition to cereal crops, uredinial stage of the P. graminis population is sustained by wild grasses, especially Elymus glaucus, a native grass sharing the same habitat with the rusted Mahonia spp. Although virulence to some important stem rust resistance genes was observed in some isolates derived from Mahonia of North America when tested against single stem rust resistance gene stocks, the overall virulence is very limited in these isolates. This is likely a result of limited selection pressure on the rust population. In contrast to northwestern United Sates, the Pgt population in east of the Rocky Mountains of North America has declined steadily with a single race, QFCSC, being predominant in the last decade. This decline is likely due to a combination of factors, of which a lack of sexual recombination in the region is perhaps the most important one.     

Regionalization of wheat powdery mildew oversummering in China based on digital elevation

Blumeria graminis f. sp. tritici, the pathogen that causes wheat powdery mildew, is one of the most important diseases affecting wheat production in China, and the oversummering is the key stage of wheat powdery mildew epidemic. The more oversummering regionalization of wheat powdery mildew has played an important role in disease prediction, prevention and control. In this study, we analyzed the correlation between oversummering data of wheat powdery mildew and the meteorological factors over the past years, and determined that temperature was the key meteorological factor influencing oversummering of wheat powdery mildew. The average temperature at which wheat powdery mildew growth was terminated(26.2°C) was used as the threshold temperature to regionalize the oversummering range of wheat powdery mildew. This regionalization was done using the GIS ordinary kriging method combined with the Digital Elevation model(DEM) of China. The results showed that annual probability of oversummering region based on Model 26.2 were consistent with the actual survey of the more summer wheat powdery mildew. Wheat powdery mildew oversummering regions in China mainly cover mountainous or high-altitude areas, and these regions form a narrow north-south oversummering zone. Oversummering regions of wheat powdery mildew is mainly concentrated in the high-altitude wheat growing areas, including northern and southern Yunnan, northwestern Guizhou, northern and southern Sichuan, northern and southern Chongqing, eastern and southern Gansu, southeastern Ningxia, northern and southern Shaanxi, central Shanxi, western Hubei, western Henan, northern and western Hebei, western Liaoning, eastern Tibet, eastern Qinghai, western Xinjiang and other regions of China.