Tao CHEN,Chong HE,Yadong ZHANG,Zhen ZHU,Qingyong ZHAO,Lihui ZHOU,Xin YU,Cailin WANG

Institute of Food Crops,Jiangsu Academy of Agricultural Sciences,Jiangsu High Quality Rice R&C Center,Nanjing Branch of China National Center for Rice Improvement,Nanjing 210014,China

Rice blast,caused byMagnaporthe grisea(Hebert)Barr.,is one of the most destructive diseases to all the rice-growing areas across the world[1-4].According to statistics,the global yield loss caused by rice blast reaches 11%-30%every year,which is equivalent to rations of 6.00×107people a year[5].The damages of rice blast in China are also very serious.Since the 1990s,the annual incidence area of rice blast has reached 3.80×106hm2,and the annual grain loss has also reached hundreds of millions of kilograms[6].Rice blast,especially spike neck blast,is a major disease in rice-growing areas in Jiangsu Province.In the early 1990s,the incidence area of rice blast was usually 1.67×106hm2.In recent two years,although the incidence area has been reduced,rice blast still probably occurs.At present,chemical control and promotion of resistant varieties are usually used to control rice blast.Since chemical agents may pollute the environment,the breeding of resistant varieties has become the most economical and effective way to control rice blast.For the breeding of resistant varieties,the identification of genotype and distribution of resistance genes in rice-growing areas is the premise.

Japan first carried out studies on rice blast resistance genes in the middle 1960s.So far,a total of 68 rice blast resistance loci and 83 major genes have been reported.Among them,Pb-1,Pi-a,Pi-b,Pi-d2,Pi-d3,Pik,Pik-h/Pi54,Pik-m,Pik-p,Pi-sh,Pi-t,Pi-ta,Piz-t,Pi-1,Pi-2,Pi-5,Pi-9,Pi-21,Pi-25,Pi-36,Pi-37,Pi-50,Pi-56,Pi-CO39 have been successfully cloned(National Rice Data Center).Pi-tais a rice blast resistance gene that has been studied most extensively.It originated from indica rice,and is located near the centromere of the 12thchromosome in rice[7-11].There is a direct interaction betweenPi-taand nontoxic geneAVR-Pita.Moreover,they follow the gene-for-gene hypothesis[12].Pi-bcomes from the rice variety BL1,and is located near the end of the long arm of the 2ndchromosome in rice.It is linked to RFLP-marked RZ123,C379 and C2782B[13-14].Pi-bis resistant to most of the blast physiological races in Japan and is induced and regulated by environmental factors,such as temperature and illumination[15].It has been reported thatPi-taandPi-bhave lasting and stable resistance to rice blast injaponicarice,so they are widely used in the rice blastresistance breeding.Pi-taandPi-bare also two rice blast resistance genes that have been first cloned.

In this study,the rice blast resistance of 40japonicarice varieties and 665 newjaponicarice lines in Jiangsu Province was identified by using the functional markers of dominant genesPi-taandPi-band their sensitive allelespi-taandpi-b.Thus the distribution ofPi-taandPi-bin rice materials would be defined.In addition,the source of resistance genes in some new lines was also traced so as to explore excellent blast resistant rice materials,thereby providing a basis for rice blast resistance breeding in Jiangsu Province.

Material and Methods

Tested materials

The tested materials included 40japonicarice varieties (including Wujing 13,Wuxiangjing 14,Wujing 15 and Nanjing 44,which were the parents of some newjaponicarice lines)and 665 newjaponicarice lines(including 256 lines from 2011 and 409 lines from 2012)bred by the Institute of Food Crops of Jiangsu Academy of Agricultural Sciences.The materials above were planted in two consecutive years.In each of the two years,the materials were all sowed on May 10thand transplanted on June 10th.For each material,there were total 8 lines with 40 plants per line.The plant and row spacing was 13.2 cm×26.4 cm.

DNA extraction and primer synthesis

At the tillering stage,the fresh leaves were collected from each plot.Then the DNA in rice leaves was extracted[16].The primers used for the amplification of targeted genes were shown in Table 1.All the 4 pairs of primers were synthesized by the ShanghaiInvitrogen Biotechnology Co.,Ltd.

PCR amplification and agarose gel electrophoresis

The PCR reaction system (20 μl)was as follows:2.0 μl 10× Buffer(25 mmol/L),0.4 μl dNTP (2.5 mmol/L),1 μl upstream primer (4 pmol/μl),1 μl downstream primer(4 pmol/μl),2 μl DNA template (approximately20 ng/μl),0.4 μlTaqDNA polymerase(2 U/μl),13.2 μl sterile ddH2O.The PCR reaction conditions were as follows:pre-denaturation at 95℃for 5 min;denaturation at 94℃for 30 s,annealing at 58℃for 30 s,extension at 72℃for 90 s,35 cycles;extension at 72℃for 10 min.The amplified products were examined by 1%agarose gel electrophoresis.The gel was stained with nuclear dye Dured and then analyzed with the UV gel imaging analysis system.

Results and Analysis

Molecular detection of resistance genes Pi-ta and Pi-b and their sensitive alleles

The functional primer pairs,YL155/YL87,YL183/YL87,Pi-bdomF/PibdomR and Lys145F/Lys145R were used to detect the resistance genesPi-taandPi-band theirsensitive genespi-taandpi-bin the 40japonicarice varieties and 665 newjaponicarice lines in Jiangsu Province.For thePi-ta-carried rice,a 1 042 bp band was amplified by YL155/YL87 (Fig.1 A);for the pi-ta-carried rice,a 1 042 bpband was amplified by YL183/YL87(Fig.1 B);for thePi-b-carried rice,a 365 bp band was obtained byPibdomF/Pi-bdomR(Fig.1 C);for thepib-carried rice,a 803 bp band was obtained by Lys145F/Lys145R(Fig.1 D).

Genotypic detection of Pi-ta and Pib in japonica rice varieties in Jiangsu

Among the 40japonicarice varieties in Jiangsu,16 varieties carried resistance genePi-ta,accounting for 40.0%of the total.There were 24 rice varieties carrying resistance genePi-b,accounting for 60.0%.There were 11 rice varieties carrying bothPi-taandPi-b,including Nanjing 44,Nanjing 45,Wuyunjing No.7,Wuxiangjing 14,Wujing 15,Wuyunjing 21,Changnongjing No.5,Yandao No.9,Wuyunjing No.8,Wuyunjing 20 and Yangjing 805,accounting for 27.5%.Changnongjing No.3,Wuyujing No.3,Huaidao No.10,Yangjing 806 and Ningjing No.1 carried only resistance genePi-ta,representing 12.5%of the total varieties.Nanjing 46,Nanjing 5055,Nanjing 9108 and other 10 rice varieties carried only resistance genePi-b,representing 32.5%.However,there were 11 rice varieties carrying none ofPi-taorPi-b,such as Huaidao No.9,Lianjing No.5,Lianjing No.6,Yangfujing No.8,Zhendao 88 and Xudao No.6,accounting for27.5% ofthe totaljaponicarice varieties(Table 2).

Genotypic detection of Pi-ta and Pib in new japonica rice lines in Jiangsu

Among the 665 newjaponicarice lines,240 lines carried the resistance genePi-ta,accounting for 36.1%of the total lines;while sensitive allelePitawas detected in all the remaining lines,accounting for 63.9%.There were 622 rice lines carrying resistance genePi-b,representing 93.5%of the total lines;while the remaining lines all carried sensitive allelepi-b,representing 6.5%.Both of the two resistance genesPi-taandPi-bwere detected in 223 rice lines,accounting for 33.5%of the total rice lines.There were 17 and 339 rice lines carried onlyPi-taorPi-b,accounting for 2.6%and 60.0%of the total.However,none ofPi-taandPi-bwas detected in 26 rice lines,accounting for 3.9%of the total tested rice lines (Table 3).The detection results also showed that the distribution frequency ofPi-bwas significantly higher than that ofPi-ta;in terms of genotype,the number of lines carrying onlyPi-b(pi-ta/Pi-b)was highest,followed by number of lines carrying bothPi-taandPi-b(Pi-ta/Pi-b),number of lines carrying none ofPi-taandPi-b(pi-ta/pi-b)and number of lines carrying onlyPi-ta(Pi-ta/pi-b).

Source analysis of resistance genes Pi-ta and Pi-b in japonica rice lines

There were 368 out of the 665 rice lines originated from the 4 combinations,including Wujing 13/Kanto 194,Wuxiangjing 14/Kanto 194,Wujing15//Wujing 13/Kanto 194 and Nanjing 44//Wujing 13/Kanto 194.In this study,the sources of resistance genesPi-taandPi-bin the parents and offspring of the 4 combinations were analyzed.The results showed that since Kanto 194 carried none ofPi-taandPi-b,the resistance genePibin the offspring of Wujing 13/Kanto194 was from Wujing 13;the resistance genesPi-taandPi-bin the offspring of Wuxiangjing 14/Kanto 194 came from Wuxiangjing 14;the resistance genePi-tain the offspring of Wujing 15//Wujing 13/Kanto 194 and Nanjing 44//Wujing 13/Kanto 194 was from Wujing 15 or Nanjing 44,whilePibmight come from Wujing 13,Wujing 15 or Nanjing 44.

Table 1 Primer sequences and expected fragment sizes for detection of Pi-ta and Pi-b

Table 2 Distribution of Pi-ta and Pi-b genes in the 40 japonica rice varieties in Jiangsu

Table 3 Genotypic distribution of Pi-ta and Pi-b in new rice lines

Table 4 Genotypic distribution of Pi-ta and Pi-b in the offspring of the four combinations

The further analysis on the distribution frequencies of the 4 genotypes in the offspring of each of the combinations showed,among the offspring of Wujing 13/Kanto 194,there were none rice lines carrying bothPi-taandPi-b,which was caused byPi-ta-free parents;among the offspring of Wuxiangjing 14/Kanto 194,there were 1 rice line carrying bothPi-taandPi-band 19 rice lines carrying only resistance genePi-b,accounting for 5.0%and 95.0%of the total lines originated from the combination of Wuxiangjing 14/Kanto 194,but the rice lines withPi-ta/pi-bandpi-ta/pi-bwere all zero;among the offspring of Wujing 15//Wujing 13/Kanto 194,there were 16 rice lines withPi-ta/Pi-b,60 lines withpi-ta/Pi-band 2 lines withpi-ta/pi-b,accounting for 20.5%,76.9%and 2.6%of the total lines,respectively,but there were no rice lines carrying only resistance genePi-ta;among the offspring of Nanjing 44//Wujing 13/Kanto 194,there were 80 rice lines withPi-ta/Pi-b,6 lines withpi-ta/Pi-band 2 lines withpi-ta/pi-b,accounting for 54.0%,44.6%and 1.4%,respectively,but there were also no rice lines carrying only resistance genePi-ta(Table 4).

Discussion

Production practice has proved that the breeding and utilization of resistant rice varieties is the most economical and effective control measure for rice blast.However,due to the complexity and variability of rice blast fungus,the disease resistant varieties usually lose their resistance in a few years after their promotion,resulting in the occurrence and spreading of rice blast.So the rice blast resistance breeding has attracted a lot of attention from breeders.At present,a lot of rice blast resistance genes have been reported.The effective combination of few blast resistance genes is the key to determine the success of breeding.Pi-taandPi-bare broad-spectrum rice blast resistance genes[21-23].Liuet al.[24]detected the genotypes ofPi-taandPi-bin the 36 major rice varieties in Heilongjiang and identified the dominant mixed stains for inoculation.The results showed the rice blast resistance genesPi-taandPi-bhad very high utilization value.Liet al.[25]detected the genotypes ofPi-taandPi-bin the 176 local rice varieties in Yunnan.They found that the rice varieties carryingPi-taorPi-bshowed relatively good resistance to rice blast.Yanget al.[26]found that a certain amount of cultivated rice varieties in Zhejiang and Jiangsu carried the blast resistance genesPi-taandPi-b,which was an important reason for the resistance in rice varieties.Wanget al.[27]detected the genotypes of some experimentaljaponicarice varieties and lines in Jiangsu,and the results showed thatPi-taandPi-bwere positively related to the spike neck blast resistance injaponicarice in Jiangsu.Therefore,the further definition of distribution ofPi-taandPi-binjaponicarice varieties and newjaponicarice lines in Jiangsu Province will be conducive to the screening of excellent blast resistant rice germplasms and the breeding of excellent rice varieties and lines.

However,the traditional identification of blast resistance genes requires a lot of vaccination and genetic analysis.This will undoubtedly require a long time and heavy workload.Even worse,it is easily affected by environmental conditions,and has low accuracy and low operability.So the traditional identification of blast resistance genes cannot meet the needs of modern breeding for disease resistance.With the development of molecular biology,more and more blast resistance genes are mapped and cloned.The detection of marker bands of target genes, under assistance of molecular markers closely linked to resistance genes,especially functional markers produced by difference in gene sequences,can detectthe genotype as early as at the seedling stage,thereby improving the efficiencies of screening of resistance gene sources,identification of resistance genes and breeding selection[20-28].The results of this study proved that the detection of functional markers can identify the genotypes ofPi-taandPi-binjaponicarice,thereby laying a good foundation for the further development of rice blast resistance breeding.

The results of this study also showed that the distribution frequency ofPi-binjaponicarice varieties in Jiangsu was 60.0%,and ofPi-tawas 40.0%;the distribution frequencies ofPi-bandPi-tain newjaponicarice lines were 93.5%and 36.1%,respectively.Among thejaponicarice varieties and lines in Jiangsu,the distribution frequency ofPi-tawas relatively stable,but the distribution frequency ofPi-bin new lines was higher than that in commercial varieties.It was indicated that artificial selection is conducive to the improvement of distribution frequency ofPi-bin rice.Meanwhile,the genotypic analysis also showed that the Lianjing serial varieties bred by the Lianyungang Academy of Agricultural Sciences,such as Lianjing No.4,Lianjing No.5,Lianjing No.6 and Lianjing No.7,all didn’t carry the resistance genesPi-taorPi-b.However,the rice varieties bred by the Jiangsu(Wujin)Rice Research Institute,such as Wuyunjing No.7,Wuyunjing No.8,Wuyunjing 20,Wuyunjing 21,Wujing 15 and Wuxiangjing 14,all carried the two resistance genes.This may be caused byPi-taandPi-b-carried parent materials from Jiangsu or Zhejiang.

Considering the sources ofPi-taandPi-bin some newjaponicarice lines,the resistance genePi-tamay come from Wuxiangjing 14,Wujing 15 or Nanjing 44,andPi-bmay come from Wujing 13,Wuxiangjing 14,Wujing 15 or Nanjing 44.The analysis on the distribution frequencies of genotypes in the offspring the four combinations(Wujing 13/Kanto 194,Wuxiangjing 14/Kanto 194,Wujing 15//Wujing 13/Kanto 194,Nanjing 44//Wujing 13/Kanto 194)showed,the genotypic distribution frequencies ofpi-ta/Pi-bandPi-ta/Pi-bwere much higher than those ofPi-ta/pi-bandpi-ta/pi-b.Although the parent genotypes were same between the two combinations,Wujing 15//Wujing 13/Kanto 194 and Nanjing 44//Wujing 13/Kanto 194,the distribution frequency ofPi-ta/Pi-bin the offspring of Nanjing 44//Wujing 13/Kanto 194 was significantly higher than that of Wujing 15//Wujing 13/Kanto 194.Since the rice varieties and lines mentioned above are all bred by artificial selection,it is speculated that the differences in genotype frequenciesofresistance genesmaybe caused by the linkage among some genes encoding the important agronomic traits in rice.

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