Screening Of Rice Varieties Resistance To Brown Leaf Spot Disease And The Development Of Rice Lines With Quantitative Resistance Genes Pi21 And Bsr-d1 Against Rice Blast By CRISPR-Cas9

Rice is one of the most important foods for the world’s rising population.Crops such as rice,wheat,and maize provide approximately half of the calories consumed by humans.Rice is the most important food source for Asian countries,while its yield losses caused by kinds of diseases such as brown leaf spot,blast,and bacterial blight have been one of the major factors leading to the decrease in rice production worldwide annually.Brown leaf spot,a disease caused by Bipolaris oryzae,is a major hindrance to growing rice yield.The disease has spread rapidly in rice fields due to the rapid airborne transmissions and latent infection in the rice seeds.In addition,the most harmful disease that seriously threatens rice production is blast disease caused by Magnaporthe oryzae.Identification and generation of resistant resources to the diseases is one of the critical steps to develop resistant variety to control the diseases in an economical and environmentally friendly manner.In this study,we evaluated the resistance of 25 rice varieties to brown spot leaf and generated some rice lines containing two quantitative resistance genes to blast.The major results are as follows.1)A total of 25 different rice varieties,from different rice planting regions in Pakistan,were evaluated in resistance to brown leaf spot,and some agronomical and yield associated traits,such as chlorophyll contents,plant height,flag leaf length,flag leaf width,panicle length,number of panicles,number of spikelets and 1000-grain weight were also measured.Among them,no immune varieties to brown leaf spot were found,and only 2 varieties(Winner and Diamond 121)reached resistant level,12 varieties reached moderate resistant level,and the remaining varieties were all in the range of moderate susceptible to high susceptible levels.These 25 varieties displayed considerable variations on most of agronomical and yield associated traits tested,indicating a well diversity of the varieties used in the study.Compared with the un-inoculation treatment,all 8 agronomical and yield associated traits were significantly reduced in inoculation treatment,while the two resistant varieties displayed apparently well performance on all agronomical traits in inoculation treatment,indicating the importance of developing resistant variety.2)Seventy-eight simple sequence repeat(SSR)markers were used to genotype these 25 varieties.Among them,53 SSR markers exhibited polymorphism,in which each locus contained 2 to 4 alleles with the average of 2.359.The polymorphism information content(PIC)values for these markers ranged from 0.147 to 0.662 with the average of 0.458.Through clustering analysis using these SSR genotypes,we found that the 25 varieties showed a well genetic diversity.By using association analysis,we found 6 SSR loci significantly associated with the number of panicles(RM1331),the number of spikelets(RM3825,RM231),plant height(RM231),flag leaf length(RM267 and RM424),and 1000-grain weight(RM561),respectively.One marker,RM542,was found significantly associated with the disease incidence,and among the three alleles in this marker locus,the varieties contained allele-III showed significantly higher disease incidence than the varieties contained the other two alleles.The elite alleles in these associated marker loci could be further used in marker assisted breeding programme for improving the trait of interest.3)In the genetic background of a well-known commercial rice variety ’Nanjing 9108(NJ9108)’in Jiangsu province,some rice lines with broad spectrum qualitative resistance genes,Pi21 and Bsr-dl,to blast were generated by CRISPR/Cas9 technology.Total of 25 T0 transgenic plants were obtained,in which 7 plants were confirmed containing mutations through gene sequencing.Five T0 plants were mutated in bsr-dl with 4 types of mutation,and 2 in pi21 with 3 types of mutation.Through continuous detection,we finally obtained 4 types of bsr-dl and 3 types of pi21 homozygous knockout plants(NJ9108bsr-d1KO,NJ9108pi21KO),receptively,in T1 generation.Through crossing,we have also obtained 4 types of hybrids F1 plants with doublegene mutations.Further study will focus on the evaluation of these gene-editing plants against blast.In total,this study provides some valuable rice cultivars with high resistance to brown leaf spot disease as well as some new rice lines with broad-spectrum blast resistance genes in the genetic background of a well-known commercial rice cultivar,which will be very useful for both disease resistance breeding and identification of resistance gene against brown leaf spot in future.