Development Of Molecular Markers Tightly Linked To Resistance Genes Of Papaya Ringspot Virus-watermelon Strain(PRSV-W) And Their Application In Breeding

Zucurbita?Cucurbita pepo L.?is an important cultivated vegetable of Cucurbitaceae,which is widely cultivated in the world.The cultivation area and yield of zucchini in China rank first in the world.Zucchini is popular with producers and consumers because of its strong adaptability,high yield,easy management,good taste and high nutritional value.At present,the cultivation area of zucchini has increased year by year,and the annual supply has been realized.The change of production mode also increased the harm of diseases such as viral diseases.The papaya ring spot virus disease watermelon line?PRSV-W?is one of the main diseases that harm the cucurbitaceous crops such as zucchini.After infected with PRSV-W,the zucchini plants showed mottled leaves,vesicles,dwarfism and fruit deformity and so on.It can cause more than 40%of production loss,or even no output in serious cases.At present,there is no effective agent for controlling viral diseases,and breeding resistant varieties is the most economical,safe and effective measure to control PRSV-W virus disease.So far,there have been few studies on the resistance identification techniques and resistance germplasm identification of PRSV-W in China,which leads to the lack of resistant germplasm resources.At the same time,the traditional disease-resistant breeding techniques have low efficiency and long cycle,which seriously hinders the breeding of resistant varieties.As a result,there are few resistant varieties and can not meet the production needs.On the basis of previous work,this study further improved the resistance evaluation and identification technology of zucchini PRSV-W at seedling stage,screened the resistance of zucchini germplasm resources and current main varieties,constructed six generations to reveal the genetic law of PRSV-W resistance,and further conducted the mapping of PRSV-W resistance gene of zucchini.Finally,the molecular marker closely linked to the PRSV-W resistance gene was developed and applied to the molecular marker-assisted breeding of zucchini.The main results are as follows:1.Resistance evaluation and screening of zucchini germplasm resources and main varieties,and disclosure of genetic law of PRSV-W resistanceUsing the improved technical system of resistance evaluation and identification of zucchini PRSV-W at seedling stage,combined with DAS-ELISA and RT-PCR detection techniques,the resistance evaluation and screening of 148 zucchini varieties and germplasm resources were carried out.Thirty-three materials resistant to PRSV-W disease and 115 susceptible materials were obtained.Among them,the inbred line"BV21"showed high disease resistance to PRSV-W,and there was no significant difference between the“BV21”and the control plants after inoculation of PRSV-W disease.On the contrary,the inbred line"BV37"was highly susceptible to PRSV-W.The inbred lines“BV21”and“BV37”were used to prepare the six generations,and the resistance of each individual was identified.The results showed that all of the F₁ plants showed disease resistance.For F₂ population,137 individuals were resistant while individuals were susceptible,with a separation ratio of 3:1??2=1.36,P=0.24?.The BC₁P₁?F₁×BV21?population was resistant to PRSV-W,while in the BC₁P₂?F₁×BV37?population,the separation ratio of disease-resistant and susceptible plants was 1:1.It indicated that the PRSV-W resistance trait of the zucchini material"BV21"was controlled by a pair of dominant resistance genes.2.Mapping of PRSV-W resistance geneUsing 897 pairs of SSR markers,polymorphic molecular markers were screened between resistant and susceptible parents and 149 pairs of polymorphic molecular markers were obtained.Bulked Segregant Analysis?BSA?was used to analyze the plants in the F₂ population by using the inter-parent polymorphism SSR markers obtained by screening.Four molecular markers closely linked to the disease resistance gene CpPRSV-W were obtained,among which SSR47 was the most closely linked marker and the genetic distance from the disease resistance gene was 1.0 cM.The genetic distance between marker SSR113 and the disease resistance gene is 1.5 cM.The genetic distance of other two markers SSR21 and SSR276 was relatively far.In order to narrow the candidate interval of the disease resistance gene,a new InDel marker was developed between the above-mentioned molecular markers SSR47 and SSR113 by resequencing of parental materials,and then the exchanged individual plants were screened.Finally,the disease resistance gene CpPRSV-W was located between the InDel marker ID523 and ID1317,which contains 753 Kb.Based on the zucchini genome annotation information,the candidate region contains 30 candidate genes.3.Development of molecular markers closely linked to PRSV-W resistance gene and its application in molecular marker-assisted selection breedingA new InDel marker was designed in the candidate region of the disease-resistance gene,and the molecular marker ID307 with good polymorphism and stable amplification was obtained.High-efficiency molecular marker-assisted selection breeding technique was established by ID307 to select the resistant zucchini progenies at seedling stage.Since 2018,about 20,000 plants of zucchini materials have been screened for resistance at seedling stage,and 6,707 resistant plants have been obtained.The virus vaccination and identification results showed that the zucchini progenies selected by molecular marker screening obtained PRSV-W resistance.The results of this study provided technical support for the efficient use of PRSV-W disease resistance gene,the rapid creation of disease-resistant zucchini germplasm and breeding of new disease-resistant varieties,and laid a foundation for further isolation and identification of resistance genes and revealing the molecular mechanisms of resistance.