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On infection, plant‐parasitic nematodes establish feeding sites in roots from which they take up carbohydrates among other nutrients. Knowledge on how carbohydrates are supplied to the nematodes’ feeding sites is limited. Here, gene expression analyses showed that RNA levels of OsSWEET11 to OsSWEET15 were extremely low in both Meloidogyne graminicola (Mg)‐caused galls and noninoculated roots. All the rice sucrose transporter genes, OsSUT1 to OsSUT5, were either down‐regulated in Mg‐caused galls compared with noninoculated rice roots or had very low transcript abundance. OsSUT1 was the only gene up‐regulated in galls, at 14 days postinoculation (dpi), after being highly down‐regulated at 3 and 7 dpi. OsSUT4 was down‐regulated at 3 dpi. No noticeable OsSUTs promoter activities were detected in Mg‐caused galls of pOsSUT1 to ‐5::GUS rice lines. Loading experiments with carboxyfluorescein diacetate (CFDA) demonstrated that symplastic connections exist between phloem and Mg‐caused giant cells (GCs). According to data from OsGNS5‐ and OsGSL2‐overexpressing rice plants that had decreased and increased callose deposition, respectively, callose negatively affected Mg parasitism and sucrose supply to Mg‐caused GCs. Our results suggest that plasmodesmata‐mediated sucrose transport plays a pivotal role in sucrose supply from rice root phloem to Mg‐caused GCs, and OsSWEET11 to ‐15 and OsSUTs are not major players in it, although further functional analysis is needed for OsSUT1 and OsSUT4., Plasmodesmata play a pivotal role in sucrose supply from root phloem to Meloidogyne graminicola‐caused giant cells in rice, and the functions of OsSUT1 and OsSUT4 in this process need further assessment.

Heterodera avenae and H. filipjevi are cereal cyst nematodes (CCNs) that infect cereals in 16 provinces of China. CCN populations from Xuchang, Tangyin, Qihe, and Juye were tested using 23 barley, oat, and wheat entries of the International Test Assortment for Defining Cereal Cyst Nematode Pathotypes. H. avenae populations from Tangyin, Qihe, and Juye were classified as pathotype Ha91, and H. filipjevi from Xuchang was classified as a new pathotype similar to pathotype West. Among 42 other winter wheat cultivars, 29 and 30 were differentially susceptible, 13 and 12 were differentially resistant to H. avenae and H. filipjevi, respectively. Three entries were resistant to both species, and three other entries were resistant to H. avenae and moderately resistant to H. filipjevi. Coating wheat seed with abamectin + isopycnic imidacloprid or methylene (bis) thiocyanate + thiamethoxam reduced the number of H. avenae and H. filipjevi cysts by 46 to 56%, increased wheat yield by 9 to 27%, and improved net income by 660 to 2,640 Chinese Yuan ha−1, respectively. Resistant wheat cultivars are scarce in China, and seed coating is considered the most suitable method for controlling CCNs in the North China Plain, where crop rotation cannot be practiced.

The root-knot nematode Meloidogyne graminicola is considered one of the most devastating pests in rice-producing areas, and nematicides are neither ecofriendly nor cost effective. More acceptable biological agents are required for controlling this destructive pathogen. In this study, the biocontrol potential of Aspergillus welwitschiae AW2017 was investigated in laboratory and greenhouse experiments. The in vitro ovicidal and larvicidal activities of A. welwitschiae metabolites were tested on M. graminicola in laboratory experiments. The effect of A. welwitschiae on the attraction of M. graminicola to rice and the infection of rice by M. graminicola was evaluated in a greenhouse. The bioagent AW2017 displayed good nematicidal potential via its ovicidal and larvicidal action. The best larvicidal activity was observed at a concentration of 5×AW2017, which caused an 86.2% mortality rate at 48 h post inoculation. The highest ovicidal activity was recorded at a concentration of 5×AW2017, which resulted in an approximately 47.3% reduction in egg hatching after 8 d compared to the control. Under greenhouse conditions, the application of A. welwitschiae significantly reduced the root galls and nematodes in rice roots compared to the control. At a concentration of 5×AW2017, juveniles and root galls in rice roots at 14 d post inoculation (dpi) were reduced by 24.5 and 40.5%, respectively. In addition, the attraction of M. graminicola to rice roots was significantly decreased in the AW2017 treatment, and the development of nematodes in the AW2017-treated plants was slightly delayed compared with that in the PDB-treated control plants. The results indicate that A. welwitschiae is a potential biological control agent against M. graminicola in rice.

Summary Despite the fact that venom allergen‐like proteins (VAPs) have been identified in many animal‐ and plant‐parasitic nematodes, studies on VAPs in Heterodera avenae, which is an important phytonematode, are still in their infancy. Here, we isolated, cloned and characterized two VAPs, named HaVAP1 and HaVAP2, from H. avenae. The two encoded proteins, HaVAP1 and HaVAP2, harbour an SCP‐like domain each, but share only 38% identity with each other. HaVAP1 and HaVAP2 are expressed in subventral and dorsal oesophageal glands, respectively. HaVAP1 is expressed mainly at the early stages, whereas HaVAP2 accumulates principally at the late stages. Both HaVAP1 and HaVAP2 are secreted when expressed in Nicotiana benthamiana leaves, but HaVAP1 is delivered into chloroplasts, whereas HaVAP2 is translocated to the nucleus without signal peptides. Knocking down HaVAP1 increased the virulence of H. avenae. In contrast, silencing of HaVAP2 hampered the parasitism of H. avenae. Both HaVAP1 and HaVAP2 suppressed the cell death induced by BAX in N. benthamiana leaves. Moreover, HaVAP2 physically interacted with a CYPRO4‐like protein (HvCLP) of Hordeum vulgare in the nucleus of the plant. It is reasonable to speculate that the changes in the transcript of HvCLP are associated with HaVAP2 during the parasitism of H. avenae. All results obtained in this study show that both HaVAP1 and HaVAP2 are involved in the parasitism of H. avenae, but they possess different functions, broadening our understanding of the parasitic mechanism of H. avenae.

Fusarium is usually thought to cause soybean root rot, which results in a large quantity of annual yield loss in soybean production, by its secretions including Fusarium toxins and cell wall degrading enzymes, but not by the conidia themselves that do not underlie any virulence so far. Here we report that the conidia of one Fusarium solani isolate are able to be virulent to soybean and make soybean seedlings wilted alone. We isolated them from the wilted plants in a soybean-production field and molecularly identified 17 Fusarium isolates through phylogenetic analysis. Of them, except for one isolate that showed diversity of virulence to different soybeans (virulent to one soybean whereas avirulent to another soybean), the others were all virulent to the two tested soybeans: both conidia cultures and secretions could make soybean seedlings wilted at 5 days post infection, and their virulence had dosage effects that only conidia cultures of at least 5×106 conidia mL–1 could show virulence to soybean; however, the sole conidia of the F. solani isolate #4 also exhibited virulence to soybean and could make soybean seedlings wilted. Finally, we developed the specific cleaved amplified polymorphic sequences (CAPS) markers to easily differentiate Fusarium isolates. The isolate #4 in this work will likely be used to investigate the new mechanism of virulence of Fusarium to soybean.

The root-knot nematode Meloidogyne graminicola, which is distributed worldwide, is considered a major constraint on rice production in Asia. The present study used the root gall index and number of nematodes inside the roots to evaluate resistance/susceptibility to M. graminicola in different subpopulations of Oryza sativa (aus, hybrid aus, indica, hybrid indica, temperate japonica, tropical japonica). Nematode development in highly resistant varieties was also evaluated. Analyses of randomly selected 35 varieties showed the number of M. graminicola nematodes inside the roots correlated very strongly (r=0.87, P≤0.05) with the nematode gall index, and the results from pot and field experiments revealed similar rankings of the varieties for resistance/susceptibility. Among the 136 tested varieties, temperate japonica displayed the highest gall index, followed by tropical japonica, indica, hybrid indica, aus, and hybrid aus. Zhonghua 11 (aus), Shenliangyou 1 (hybrid aus) and Cliangyou 4418 (hybrid indica) were highly resistant to M. graminicola under both pot and field conditions. Further examination of nematode development suggested that compared to susceptible rice, M. graminicola penetrated less often into highly resistant varieties and more frequently failed to develop into females. The promising varieties found in the present research might be useful for the breeding of hybrid rice in China and for the further development of practical nematode management measures.

Few molecular details of effectors of Heterodera avenae parasitism are known. We performed a high-throughput sequencing analysis of the H. avenae transcriptome at five developmental stages. A total of 82,549 unigenes were ultimately obtained, and 747 transcripts showed best hits to genes putatively encoding carbohydrate-active enzymes in plant-parasitic nematodes that play an important role in the invasion process. A total of 1,480 unigenes were homologous to known phytonematode effectors, and 63 putative novel effectors were identified in the H. avenae transcriptomes. Twenty-three unigenes were analyzed by qRT-PCR and confirmed to be highly expressed during at least one developmental stage. For in situ hybridization, 17 of the 22 tested putative effectors were specifically expressed and located in the subventral gland cells, and five putative novel effectors were specifically expressed in the dorsal gland. Furthermore, 115 transcripts were found to have putative lethal RNA interference (RNAi) phenotypes. Three target genes with lethal RNAi phenotypes and two of the four tested putative effectors were associated with a decrease in the number of cysts through in vitro RNAi technology. These transcriptomic data lay a foundation for further studies of interactions of H. avenae with cereal and H. avenae parasitic control.

Small grain cereals have served as the basis for staple foods, beverages, and animal feed for thousands of years. Wheat, barley, oats, rye, triticale, rice, and others are rich in calories, proteins, carbohydrates, vitamins, and minerals. These cereals supply 20% of the calories consumed by people worldwide and are therefore a primary source of energy for humans and play a vital role in global food and nutrition security. Global production of small grains increased linearly from 1960 to 2005, and then began to decline. Further decline in production is projected to continue through 2050 while global demand for these grains is projected to increase by 1% per annum. Currently, wheat, barley, and oat production exceeds consumption in developed countries, while in developing countries the consumption rate is higher than production. An increasing demand for meat and livestock products is likely to compound the demand for cereals in developing countries. Current production levels and trends will not be sufficient to fulfill the projected global demand generated by increased populations. For wheat, global production will need to be increased by 60% to fulfill the estimated demand in 2050. Until recently, global wheat production increased mostly in response to development of improved cultivars and farming practices and technologies. Production is now limited by biotic and abiotic constraints, including diseases, nematodes, insect pests, weeds, and climate. Among these constraints, plant-parasitic nematodes alone are estimated to reduce production of all world crops by 10%. Cereal cyst nematodes (CCNs) are among the most important nematode pests that limit production of small grain cereals. Heavily invaded young plants are stunted and their lower leaves are often chlorotic, forming pale green patches in the field. Mature plants are also stunted, have a reduced number of tillers, and the roots are shallow and have a “bushy-knotted” appearance. CCNs comprise a number of closely-related species and are found in most regions where cereals are produced.

WOS:000408078300014 Plant-parasitic nematodes are very common on cereal crops and cause economic losses via reduction in grain quality and quantity. During 2014, 83 soil samples were collected from wheat and barley fields in 21 districts of 13 provinces across five regions (Central Anatolia, Marmara, Aegean, Southeast Anatolia, and Black Sea Region) of Turkey. Cyst-forming nematodes were found in 66 samples (80%), and the internal transcribed spacer (ITS) sequencing and species-specific PCR identified the species in 64 samples as Heterodera filipjevi, Heterodera latipons, and Heterodera avenae. The predominant pathogenic cereal cyst nematode was H. filipjevi, which was found in all five regions surveyed. H. avenae was only detected in Southeast Anatolia whereas H. latipons was detected in Southeast Anatolia and Central Anatolia. ITS-rDNA phylogenetic analyses showed that H. avenae isolates from China clustered with H. australis, and Turkish isolates were closely related to European and USA isolates of this species. H. filipjevi from Turkey and China were clustered closely with those from the UK, Germany, Russia, and the USA. The density of many of these populations exceeded or approached the maximum threshold level for economic loss. To our knowledge, this is the first report of H. filipjevi in Diyarbakir, Edirne, and Kutahya provinces, and the first report of H. avenae in Diyarbakir Province. These results exhibit the most rigorous analysis to date on the occurrence and distribution of Heterodera spp. in Turkey's major wheat-producing areas, thus providing a basis for more specific resistance breeding, as well as other management practices.

Heterodera avenae (cereal cyst nematode, CCN) infects many cereal crops and causes serious yield losses worldwide. Interaction studies investigating H. avenae and its hosts are still in their infancy. In this study, a barley model plant, the Hordeum vulgare cultivar Golden Promise, was investigated for its potential as a candidate model host to study its interaction with H. avenae. CCN-infective juveniles were attracted by the root tips and gathered around the root elongation zones of Golden Promise on 0.7% water agar plates. The juveniles invaded the roots and developed successfully until maturation at 40 days after inoculation in sterile sand soil. The cryotomy and syncytium measurements indicated that the syncytia enlarged gradually throughout the development of the nematodes and caused the corresponding root regions to swell obviously. Quantitative real-time PCR analysis showed that the down-regulation of defence-related barley genes and up-regulation of development-related barley genes contribute to the understanding of compatible interaction between H. avenae and Golden Promise. Barley stripe mosaic virus (BSMV) virus-induced gene silencing (VIGS) can be used in the roots of Golden Promise. In conclusion, the Hordeum vulgare cultivar Golden Promise is a suitable candidate model host for interaction studies with Heterodera avenae. The studies presented above document the first CCN host that not only has published genome context but also be compatible to BSMV VIGS.

Cereal cyst nematodes are the most important plant-parasitic nematodes on cereal crops in wheat producing areas of the world. Heterodera filipjevi was first reported in China in 2010. In this study, species-specific sequence characterized amplified region–polymerase chain reaction (SCAR-PCR) assays for detection and identification of H. filipjevi from infected wheat roots and soil were developed. The species-specific primers were designed according to the randomly amplified polymorphic DNA (RAPD) markers amplified with random primer OPK16. A 646-bp specific fragment of sequence was generated, which characterized amplified regions in H. filipjevi. The detection limitation of the PCR assay was as low as 0.125 μl second-stage juvenile (J2) lysate, 3.9 × 10–3 μl adult female lysate, and 10–3 μl cyst lysate. The method was able to detect the various stages (J2, J3, J4, and female) of H. filipjevi, and a single of nematode in 0.5 g of soil. H. filipjevi was detected by the method in two of six field samples, and one of those samples contained a mixed population of H. filipjevi and H. avenae. This study is the first to provide a definitive diagnostic assay for H. filipjevi in wheat roots and soil.

The cereal cyst nematode (CCN), Heterodera aveane, has been found in 16 provinces of China, including the Shandong winter-wheat-growing region. This study investigated the population dynamics of H. avenae in the winter wheat ‘Jimai 22’ and ‘Tainong 18’ for two consecutive years in the field. Soil and root samples were collected during the winter-wheat-growing season and H. avenae population densities and life-stages determined. H. avenae population dynamics in roots and soil of the two winter wheat cultivars were similar over the 2-year period. Second-stage juvenile (J2) population densities in wheat roots were greatest during booting stage (April), when mean soil temperature was between 11.8 and 14.4°C. Cysts in rhizosphere soil increased significantly when new cysts were formed after Zadoks (Z) 47 (booting stage). There was a peak in J2 population densities in soil during Z23 and Z30 (tillering and stem elongation, respectively) whereas J2 population densities were the lowest at Z13 (seedling stage). This research provides important information indicating that J2 populations in roots and soil increased after the wheat winter dormancy period. Knowledge of when different life stages of H. avenae occur in winter wheat in Shandong will provide valuable insights to enable the development of an integrated approach to manage this plant-parasitic nematode.

Cereal cyst nematodes (Heterodera avenae and H. filipjevi) and root lesion nematodes (Pratylenchus spp.) have been found to infect cereals in 16 provinces of China. To develop a nematicide that effectively controls nematodes, two novel chemical products, methylene bis thiocyanate (MBT) and MBT + thiamethoxam (MTT); four common pesticides, fipronil + chlorpyrifos (FIC), emamectin benzoate, imidacloprid, and Bacillus thuringiensis; and one fungicide, iprodione, were tested as seed coatings for the control of cereal cysts and root lesion nematodes from 2013 to 2015. Wheat seeds were treated with these seven seed coatings before sowing, and changes in the numbers of Heterodera spp. and Pratylenchus spp. were recorded during three different growth stages. Wheat yields were also compared after harvest. All treatments reduced the numbers of Pratylenchus in wheat and of cysts and eggs of Heterodera in the soil compared with the untreated control. Among the treatments, application of MTT or FIC was more effective than that of the other treatments for nematode control, and the other treatments had similar effects. The results of this study have demonstrated that MTT and FIC applied as seed treatments effectively reduce the number of cysts, inhibit the reproduction of Heterodera and Pratylenchus, and enhance wheat yields. MTT and FIC are thus suitable for controlling nematodes on wheat under natural field conditions.

Three of the cereal cyst nematodes, Heterodera avenae, H. filipjevi and H. latipons are considered to be the most economically important cyst nematodes that affect cultivated cereals around the world. H. filipjevi was first detected in China from Xuchang, Henan Province in 2010 (Peng et al. 2010) and now has been recorded in the Central China of Henan, Shandong and Anhui provinces and the Northwest China of Xinjiang Uygur Autonomous Region (Cui et al. 2020). In June 2019, 42 samples consisting of roots and soil were collected from winter wheat fields in Hebei Province of North China. Cysts were detected in 37 soil samples with a mean of 6.4 ± 1.67 cysts per 100 ml of soil. Cysts and second-stage juveniles (J2s) were extracted from root and soil following Cobb's sieving gravity method. Morphological and molecular studies of J2s and cysts confirmed its identity with H. filipjevi in 5 samples from Handan (N36°10'052" and E114°35'056"; N36°37'054" and E114°22'052"), Xingtai (N36°53'060" and E114°30'011") and Shijiazhuang (N 37°26'048" and E 116°05'039") in Hebei Province, China. Morphologically, the cysts are lemon-shaped, light or dark brown in color. The vulval cone is bifenestrate with horseshoe-shaped semifenestrae, strongly globular bullae, and well-developed underbridge. Measurements (mean +_ sd (range)) of cysts (n=10), body length not including neck is 743.0 ± 36.1 μm (665 - 780 μm), body width is 559.0 ± 50.0 μm (455 - 639 μm), length / width ratio is 1.33 ± 0.07 (1.20 - 1.46); neck length is 99.3 ± 8.8 μm (85 - 122 μm); fenestrae length is 56.8 ± 5.0 μm (49 - 65 μm) and width is 25.5 ± 1.8 μm (21.1 - 27.8 μm); underbridge length is 84.0 ± 8.1 μm (62 - 93 μm); and vulval slit length is 8.6 ± 0.5 μm (7.2 - 9.1 μm). Measurements of J2s (n = 12), body length is 541 ± 11.4 μm (490 - 578 μm); stylet length is 22.3 ± 0.5 μm (22.0 - 25.0 μm) with anchor-shaped basal knobs; tail length is 57.7 ± 3.7 μm (52.7 - 65.2 μm), and hyaline tail terminal length is 36.5 ± 2.8 μm (32 - 39.8 μm). The tail had a sharp terminus. Morphology of the cysts and J2s were consistent with the record of H. filipjevi (Peng et al. 2010; Subbotin et al. 2010). The amplifications of rDNA-internal transcribed spacer (ITS) fragments were generated with a PCR fragment of 1054 bp from single cysts of each population, using primers TW81 and AB28 (Joyce et al. 1994). The PCR tests for each sample were repeated five times. The PCR product was purified and sequenced. All nucleotide sequences of ITS-rDNA were submitted to GenBank under accession numbers MW282843-6. Sequences from the ITS region were more than 99.5% identical to those of H. filipjevi from Egypt (KF225725), Turkey (KR704308, KR704293 and MN848333) and China (KT314234, MT254744 and KY448473). These results from ITS supported its identity as H. filipjevi. The results were also confirmed by species specific sequence characterized amplified region primers of H. filipjevi (Peng et al. 2013). Pathogenicity of the H. filipjevi was confirmed by infection of winter wheat (Triticum aestivum L cv. 'Aikang58') and examination of the nematode development and reproduction. Wheat seeds were germinated in petri dishes and then transplanted into five polyvinyl chloride tubs (3 cm in diameter, 25 cm in length) that contained 150 cm3 of a sterile soil mixture (loamy soil: sand = 1:1), each with 5 cysts (mean of 252.0 eggs/cyst). Plants were grown in an artificial climate box for one week at 14/18°C, two weeks at 16/20°C, five weeks at 18/25°C and two weeks at 22/30°C, under 8 h of darkness/16 h light and normal culturing practices (Cui et al. 2015). The parasitic J2s, third and fourth-stage juveniles, and adult females were observed in roots stained with acid fuchsin at 10, 20, 30, and 50 days after inoculation (DAI), and an average of 32.0 cysts per tubes were extracted 70 DAI. The new cyst' morphological and molecular characteristics were identical to the H. filipjevi cysts from the original soil samples. Three other tubes without cysts were set as control and there were no newly formed cysts. Heterodera avenae and H. filipjevi had been detected in a total of 16 wheat-producing provinces in China, which resulted in losses of 1.9 billion CNY year-1 (Cui et al. 2015). To our knowledge, this is the first report of H. filipjevi in Hebei Province of North China. Cereal cyst nematodes are easily transferred to non-infested areas by many avenues, resulting in increased species and pathotype complexity (Cui et al. 2020). Once H. filipjevi continues to spread in main wheat producing area of China, it could become be a new threat to cereals production. It is time to take effective control methods to prevent H. filipjevi further dispersal, especially through the farming machinery transmission. Hebei Province is one of the most important major grain-producing areas, our findings will be very beneficial for H. filipjevi management and further research on winter wheat in Hebei Province, North China.

The cereal cyst nematode (CCN, Heteroder aavenae) causes serious yield loss on cereal crops, especially wheat, worldwide. Daxing population in Beijing City and Huangyuan population in Qinghai Province, China, are two CCN populations. In this study, the CCN pathotypes of Daxing and Huangyuan populations were characterized by tests on 23 standard “International Test Assortment” with the local species Wenmai 19 as the susceptible control. Tested materials were grouped by three nematode populations' virulence on resistant genes (Rha1, Rha2, Rha3, Cre1) and nonresistant genes, varieties and lines. Both Daxing and Huangyuan populations were avirulent to Ortolan (Ha1). Barley cvs. Ortolan, Siri, Morocco, Bajo Aragon 1-1, and Martin 403-2 were all resistant to both populations. Cultivars Herta, Harlan 43 and wheat Iskamish-K-2-light were all susceptible to Huangyuan population, all of them, however, were resistant to Daxing population. The other five oats were all resistant to the two tested CCN populations. Except Iskamisch K-2-light, all the other wheat cultivars (Capa, Lo-rosxKoga, AUS 10894, and Psathias) were susceptible to Daxing population. Because the pathotypes of the two tested CCN populations in Beijing and Qinghai were not identical to any of the 13 pathotypes previously characterized by the test assortment, we classified Daxing and Huangyuan populations as the new pathotypes, named Ha91.

Soybean cyst nematode Heterodera glycines is one of the most serious soil-borne pathogens in soybean production. However, the researches were limited in China due to lack of an effective pathosystem. In this study, we screened 21 legume Medicago plants in both Medicago truncatula and Medicago sativa to obtain candidate model plants for establishing a new pathosystem for legume-H. glycines interactions. The nematode infection of tested plants was assayed with Race 3 and 4 respectively, which were two dominant H. glycines inbred races in China soybean producing areas. The results showed that the model legume plant M. truncatula A17 failed to allow Race 3 of H. glycines to complete its life cycle, in contrast, it provided the Race 4 population to form several cyst nematodes, however, the female index (FI) value was approximately 1.6. Three M. sativa cultivars, including Xunlu, Aergangjin and Junren, provided either Race 3 or 4 of H. glycines to develop into mature cysts with their FI value below 5 as well. Our results demonstrated that legume plants in both M. truncatula and M. sativa were not likely to be a model plant for H. glycines because of an extreme high resistance.

For sedentary endo-parasitic nematodes, parasitism genes encoding secretory protein expressed in the subventral glands cells always play an important role during the early parasitic process. A new acid phosphatase gene (Ha-acp1) expressed in the subventral glands of the cereal cyst nematode (Heterodera avenae) was cloned and the characteristics of the gene were analyzed. Results showed that the gene had a putative signal peptide for secretion and in situ hybridization showed that the transcripts of Ha-acp1 accumulated specifically in the subventral gland cells of H. avenae. Southern blot analysis suggested that Ha-acp1 belonged to a multigene family. RT-PCR analysis indicated that this transcription was strong at the pre-parasitic juveniles. Knocking down Ha-acp1 using RNA interference technology could reduce nematode infectivity by 50%, and suppress the development of cyst. Results indicated that Ha-acp1 could play an important role in destroying the defense system of host plants.

Glyphosate-resistant (GR) transgenic soybean has never been cultivated commercially in China. It is essential to develop the separation measures required to prevent out-crossing between GR and conventional soybean (Glycine max (L.) Merr.) by characterizing the transgene flow before GR soybean is released. In this study, the transgene flow from a GR transgenic soybean AG5601 to conventional soybeans was characterized. First, natural out-crossing rate was evaluated using 36 conventional soybean varieties interplanted with GR soybean AG5601 transformed with a cp4 EPSPS gene conferring the resistance to herbicide glyphosate in the field in 2007 and 2008 in China. Second, drift distance of cp4 EPSPS gene from GR soybean AG5601 to soybean cv. Zhonghuang13 was evaluated using the progenies harvested from eight directions at different distance. Third, the relationship of gene flow of GR soybean AG5601 with flowering synchronization days or insect pollinators of each variety was analyzed using regression analysis. Thirty-two of 36 tested conventional soybean varieties had surviving progenies after two times of sprays of glyphosate, and 49 of 41,679 progenies were verified to be glyphosate-tolerant heterozygous offspring. The out-crossing rates in positive varieties (having surviving offspring after two times of sprays of glyphosate) ranged from 0.039 to 0.934 %. The farthest distance (drift distance) between soybean AG5601 and cv. Zhonghuang13 at which out-pollinating was still able to be observed was 15 m, with an out-crossing rate of 0.012 %. Regression analysis showed that there was a positive relationship between cross-pollination frequency and flowering synchronization days or pollinator insects. Therefore, when GR soybean is released to the field, it should be critically separated with the conventional soybean in space and cultivation time with efficient insect control during flowering.

Alternative splicing (AS) is common in higher eukaryotes and plays an important role in gene posttranscriptional regulation. It has been suggested that AS varies dramatically among species, tissues, and duplicated gene families of different sizes. However, the genomic forces that govern AS variation remain poorly understood. Here, through genome-wide identification of AS events in the soybean (Glycine max) genome using high-throughput RNA sequencing of 28 samples from different developmental stages, we found that more than 63% of multiexonic genes underwent AS. More AS events occurred in the younger developmental stages than in the older developmental stages for the same type of tissue, and the four main AS types, exon skipping, intron retention, alternative donor sites, and alternative acceptor sites, exhibited different characteristics. Global computational analysis demonstrated that the variations of AS frequency and AS types were significantly correlated with the changes of gene features and gene transcriptional level. Further investigation suggested that the decrease of AS within the genome-wide duplicated genes were due to the diminution of intron length, exon number, and transcriptional level. Altogether, our study revealed that a large number of genes were alternatively spliced in the soybean genome and that variations in gene structure and transcriptional level may play important roles in regulating AS.