Development Of Molecular Markers Tightly Linked To Resistance Genes Of ZYMV And Their Application In Breeding

Zucchini?Cucurbita pepo L.?is a kind of vegetable widely planted in the world,and its cultivated area and yield in China rank first in the world.Zucchini has become an important economic crop to increase the income of farmers because of its high yield,easy management and good planting benefit.However,the production of zucchini is often endangered by viral diseases,resulting in serious yield and economic losses.Zucchini yellow mosaic virus?ZYMV?is one of the main diseases infecting cucurbitaceae crops such as zucchini.After infected with ZYMV,zucchini plants showed dwarfness,mosaic,leaf deformity,fruit discoloration,bitter and astringent pulp,loss of commodity and edible value,yield reduction,which causes huge economic losses to farmers.At present,the cultivation of disease-resistant varieties is the most effective way to reduce production risks and reduce the loss.However,the domestic disease-resistant breeding techniques are backward and breeding efficiency is low,resulting in fewer varieties resistant to virus diseases.So it is difficult to meet production needs.In this study,a high-resistant germplasm was obtained by establishing the accurate resistance identification technique of zucchini ZYMV at seedling stage and screening the collected germplasm materials.Six generations of populations were prepared by using high resistance and high sensitivity inbred lines to reveal the genetic law of resistance to zucchini ZYMV disease,to develop molecular markers closely linked to resistance genes,and to establish a high efficiency molecular marker assisted selection breeding technology platform,which can be applied to the creation of resistant varieties and the breeding of new resistant varieties of zucchini.The main results are as follows:1.The ZYMV resistance of zucchini material"08-1"is controlled by a pair of dominant resistance genesBased on the established accurate disease resistance evaluation system of ZYMV at seedling stage,207 germplasm materials and main cultivation varieties were identified and 14 high-resistant ZYMV virus disease materials were obtained.Among them,the inbred line"08-1"showed high resistance to ZYMV disease,and there was no significant difference between the"08-1"and the control plants after inoculation of ZYMV disease.The six generations were prepared by using"08-1"and the high-intensity inbred line"Xianyibai 12",and the resistance of each individual of the population was identified.The results showed that all of the F₁ plants were resistant to diseaseand the segregation ratio of resistance and susceptible plants in F₂ population was in accordance with 3:1.BC₁P₁?F₁×08-1?population showed disease resistance,while BC₁P₂?F₁×Xianyibai12?population,the separation ratio of disease-resistant and susceptible plants was in accordance with 1:1.The results showed that the ZYMV resistance trait of inbred line"08-1"was controlled by a pair of dominant resistance genes.2.The ZYMV resistance gene was located in the range of 673 Kb,and the molecular markers closely linked to the ZYMV resistance gene were obtained..A total of 1500 pairs of SSR markers uniformly distributed on 20 chromosomes of zucchini were synthesized,among which 953 pairs could be efficiently amplified.Using the 953 pairs of markers to perform polymorphic marker screening between resistant and susceptible parents,179 pairs of stably amplified polymorphic markers were obtained.Fve SSR markers linked to ZYMV resistance gene CpZYMV were obtained by Bulked Segregant Analysis?BSA?.Among them,SSR103 was the most closely linked to the disease resistance gene CpZYMV,and the genetic distance was only 0.7 cM.The marker on the other side of the disease resistance gene was SSR46,the genetic distance was 1.1 cM.Further,using parental resequencing,a new InDel marker was developed between the above two markers,and the target gene candidate interval was narrowed,and finally the disease resistance gene CpZYMV was positioned between the InDel markers 7716 and 8214.This interval contains 673 Kb.According to the genome annotation information of zucchini,the candidate region contains 40candidate genes.3.High-efficiency molecular marker-assisted selection breeding technology platform construction and disease-resistant breedingA stable and highly amplified molecular marker ID8013 in the candidate region of the disease resistance gene was synthesized.This marker was used to detect and select the zucchini plants with resistance backcross transfer,and the individual plants without the disease resistance gene were eliminated at the seedling stage.Since 2018,the resistance of more than 27,000 zucchini plants have been tested.The results of partial resistance identification of individual plants showed that the detection accuracy of molecular markers can reach more than 99%.The establishment of this technology platform provides technical support for the creation of disease-resistant germplasm and the rapid breeding of disease-resistant new varieties.