Germination Stimulants,Virulence And Genetic Diversity Of Parasitic Plant Orobanche Cumana

Parasitic plants plunder nutrients and water from the host plants to meet their growth and development due to the degradation of roots or leaves and the lack of photosynthetic capacity.Parasitic plants use specialized organs called haustoria to make direct connections with the vascular tissue in the roots or shoots of a host plant.Almost all plants in the Orobanchaceae family are parasitic plants,mainly parasitic on the root of the host plant.Orobanche is one of the most widely studied parasitic plant in the world because of their severe damage to the crop.Orobanche cumana?sunflower broomrape?is a holoparasitic plant which lacks photosynthetic activity,parasitizing roots of sunflower?Helianthus annuus?.O.cumana is a limiting factor for sunflower production in contaminated fields,mainly in Europe and Asia,especially in Spain,France,Turkey,Russia,Ukraine,Israel,Kazakhstan,and China.In recent years,the main producing areas of sunflower in China,Inner Mongolia and Xinjiang,have been heavily invaded by O.cumana.Our study,in cooperation with INRA of France,made a comprehensive exploration mainly on sunflower broomrape in China and Europe in aspects of phenotype,biochemistry,genetics and genomics.Based on the first completion of O.cumana draft genome assembly,the genetic map was constructed and improved by SNP KASP genotyping and validation.Re-sequencing of Chinese O.cumana was implemented for the first time.SSR and SNP molecular markers were used to study the genetic diversity and population structure of O.cumana populations in different regions of the world.To explore the correlation of virulence and genetic diversity of O.cumana populations,the toxicity level or race of Chinese sunflower broomrape was compared with that of French sunflower broomrape in a newly developed system Rhizotron.Our study also applied aeroponic system and improved solid phase extraction?SPE?method to explore the germination stimulants of O.cumana from different host root exudates.The main results were as follows:?1?SSRs markers with high polymorphism information?PIC?that developed and identified by next-generation genome sequencing were used to evaluate the genetic diversity of O.cumana in different regions of the world.The genetic variation levels of 259 materials from 5 different regions in China and 108 materials from 27 different regions in Europe were detected by fluorescence PCR and capillary electrophoresis?CE?.Genetic variation and diversity parameters clearly distinguish Chinese populations from European populations.Principal component analysis and phylogenetic tree divided the O.cumana populations into three groups:?I?Xinjiang?XJ?population of China,where is close to Kazakhstan and Russia,parasitizing on confectionary sunflower;?II?Inner Mongolia population parasitized on confectionary sunflower;and?III?European population parasitized on oil sunflower.The Xinjiang population had the highest number of private alleles and the highest number of alleles,combined with other genetic parameters,suggesting that the Xinjiang population in China may be one of the common ancestors of O.cumana,or it might hybrid with other Orobanche species in local area.?2?O.cumana?IN23?from south of Spain was used as the reference genome to explore the Chinese O.cumana genome for the first time.The next generation sequencing technology?llumina HiSeq 3000?was used to re-sequence the genomes of nine representative Chinese?Inner Mongolia?O.cumana and six Spanish O.cumana.The average depth of re-sequencing was 15.3X,and 276,926 SNPs were detected.Based on the genetic analysis of 276,926 SNPs and 231 SNPs obtained by exome sequencing,it was found that the genetic variation of O.cumana in different regions varied considerably and isolated gene pools were in different geographical environments.It existed five genetic pools of O.cumana in the world:?I?China?Inner Mongolia?;?ii?Cuenca,east-central of Spain;?iii?Guadalquivir Valley,southwest Spain;?iv?France;?v?eastern European region?Hungary,Romania,Turkey,Ukraine?.The genetic divergence between the Chinese populations?Inner Mongolia?and the European populations was significant,while the genetic variations among the European populations had minor variations.The results of genetic diversity revealed by SNP and SSR markers were consistent,suggesting that geographical isolation and host differentiation might lead to adaptive evolution of O.cumana populations.?3?The 91 individuals of F₂ from parents INA and IN12 were genotyped using KASP technology.509 SNPs and 18 SSRS were used to construct linkage map by Carthagene,obtaining 28 genetic groups,with a total map distance of 1479 cM.40,912 SNPs were detected by re-sequencing of INA and IN12,of which 13,511 SNPs were located at 145contigs,accounting for 90%of the genome contigs.The re-sequencing SNPs were strictly screened,and 288 SNPs were selected for SNP genotyping and validation.The newly added and validated 130 SNPs were added to 145 contigs to construct 19 high-density linkage groups.?4?Root exudates of sunflowers were collected and pretreated by solid phase extraction?SPE?under aeroponic system.The components of Orobanche germination stimulants were separated by UHPLC-TQ-MS.Four kinds of germination stimulants were detected,that is,dehydrocostus lactone,costunolide,8-epixanthatin,and heliolactone.?5?The soil-free system rhizotron was used to identify the race/virulence of O.cumana,which was more space-saving,convenient and accurate than the traditional pot experiment.The pot experiment was used for the identification of O.cumana in France,and the rhizotron system was used for the identification of race of O.cumana in China.In our study,a preliminary identification standard for physiological races of O.cumana was established,with six sunflower lines as the identification host,namely,2603,L86,LC1003,LC1093,R96 and P96.The virulence of O.cumana did not distinguish populations in terms of genetic diversity or geographical locations.The number of tubercles of Xinjiang population on sunflower line LD5009 was much higher than that of other populations,suggesting the possibility of adaption of O.cumana in Xinjiang towards the change of host genotype.The results showed that the races of sunflower broomrape from China and France were something between race E and F,and were tentatively named as race X_EF.