Identification Of Rice Blast Resistance Genes In A New Hybrid Rice Restorer Line Yahui2115

Rice is one of the most major food crops in the word.The stable yield of rice is very important to the safety of world.Because of biologic and abiotic stress, the loss of yield is serious.Rice blast damage is a significant one causing by fungi.Developing resistant cultivars is the most effective, economical, and eco-friendly approach for disease control.But by the rapid genetic evolution of rice blast, the resistant varieties will lost resistance when wildly planted in several years.So, hybrid rice breeding program must take durable effort in order to rich germplasm resources,overcome rice blast,and achieve the high yield aim of rice.So far, more than 101 major blast resistant genes (R genes) have been identified and 23 R genes have been cloned. Most of these cloned blast R genes belong to the nucleotide binding-site leucine-rich repeat (NBS-LRR) family, and some of then are alleles.YaHui 2115(YH2115) is an elit restore, and breeded by agricultural college, Sichuan Agricultural University. Hybrid rice combinations YiXiangYou2115, combined by YaHui2115, have passed new variety certification both in Sichuan and China in 2011 and 2012, respectively.According to our several years and locations identifying,the resistance of YaHui2115 is controlled by dominant gene.We exploited quite a few hybrid rice combinations, that the female parents belong to resistant,middle resistant and susceptible types.The F1 all showed high resistance to rice blast.So we focused on study the reason of blast resistance in YaHui2115.We designed field resistance investigation and molecular biology resaech,finally we verified that the major blast resistant gene is broad spectrum blast gene Pi2. The result is as followed:1.YaHui2115 has a broad broad-spectrum resistance to rice blastThe field blast resistanceYaHui2115 and monogenic lines of blast resistance genes are investgated and it showed:The resistance frequency, average leaf blast, average disease index of YaHui2115 is 100%,0.22%,0.05, respectively; The monogenic lines of Pi2, Pi9, Pizt, Pi1, Pi5, Pikm, the resistance frequency, average leaf blast, average disease index are 96.63%,90.57%,68.97%,28.74%,23.17%, 93.87%; 0.58%,0.60%,56.48%,4.66%,65.41%,0.50%; 0.10,0.07,23.60, 0.59,36.03,0.08, respectively. Pi2, Pi9, Pikm, Pil already have strong resistance in Sichuan Province and should enhance hybrid breeding. Other R genes now have partial resistance or even lose resistance. YaHui2115 has the most powerful resistance and widest resistant spectrum. We exploited quite a few hybrid rice combinations, that the female parents belong to resistant,middle resistant and susceptible types. The leaf blast and neck blast level is between 0-3, resistance frequency is between 88.15%-100%, disease index is between 0.01-0.25, The F1 all showed high resistance to rice blast.2. RT-PCR analysis infers Pi2 is the major R gene of YaHui2115 to rice blastTo identify the functional blast resistance genes in YH2115, RT-PCR was carried out monitoring the expression levels of reported blast R genes in YH2115 infected with M. oryzae. it showed:Pi2,Pib and Pia-RGA4 had much higher expression in YH2115. But Pib and Pia-RGA4 lost blast resistance in the field. So,Pi2 should be a major contributor of resistance in YH2115.3. The allele clone of R genes in YaHui2115 Confirm full length of Pi2, Pi25 existWe amplified the full length of cloned R genes from YH2115. By sequencing the amplified fragments,we got the full length of Pi2, Pit, Pid2, Pi25, Pib, Pita, Pia.We found that the sequence of Pi2,Pi25 in YH2115 were identical to the resistance allele reported4. LTH/YaHui2115 F2 group is developed, Pi2 linked marker can effective distinguish resistant and susceptive plants, It verifies Pi2 existing inYaHui2115.We built up a F2 population of YH2115 crossed with LTH,based on natural infection and artificial inoculation in the field, we employedthe previously reported Pi2linkage marker AP22 to genotype these F2 plants.These data indicated that the Pi2 linkage marker AP22 could effectively distinguish susceptible and resistant plants from F2 population generated from YH2115 and LTH; and that YH2115 has a functional blast resistance gene Pi2.5. RNAi experiment was performed for interfere Pi2, the positive plants show lower expression of Pi2, more susceptible. It confirms Pi2 is the major R gene in YaHui2115.To confirm the role of Pi2 in YH2115’s blast resistance, RNAi experiment was performed. We constructed a RNAi expression vector "pANDA-Pi2-RNAi"for Pi2.Fourteen YH2115 transgenic lines were obtained, with Pi2 expression level reduced by 55%-95%. The transgenic plants were challenged with M. oryzae using punch inoculation method. The results showed that the Pi2 knocking-down plants were more susceptible to blast disease, and the lesion size was significantly larger than that in YH2115 in response to different M. oryzae strains. And transgenic plants showed up to about 140% relative mycelial growth biomass, more number of spores than in YaHui2115.These results further confirmed that Pi2 contributed to YH2115’s blast resistance.