水稻主栽品种空育131抗稻瘟病位点的扫描及其基因组重构建

doi:10.11983/CBB16107

摘要/Abstract

摘要： 空育131粳稻(Oryza sativa ssp. japonica)品种因具有早熟质优、丰产稳产及耐低温冷害等优点成为黑龙江省的第一大主栽品种。为了挽救其近年来由于感染稻瘟病而从生产上退出的局面, 通过对主栽品种空育131基因组的重测序和扫描, 明确其遗缺多个优良抗稻瘟病Pi基因(Pi2、Pi9、Pi36、Pi5-1、Pb1、Pid3、Pi25、Pikh、Pi1、Pik-m、Pik-p和Pi56t等), 并通过回交育种的方法, 将MP水稻材料中的Pb1广谱抗瘟基因片段导入空育131染色体组中。该基因组的再构建过程尽可能不改变原品种的其它优良性状, 并利用控制目标导入片段长短的策略来缩短Pb1位点附近的连锁累赘。在目前得到的导入系中, 目标导入片段长约700 kb, 背景回复率为99.38%。表型鉴定结果显示, 该导入系可能和亲本MP水稻材料发挥同等的抗瘟能力。

Abstract: The rice variety Kongyu 131 (Oryza sativa ssp. japonica) is the most widely grown elite cultivar in Heilongjiang province because of its high quality, early maturity, high yield and cold resistance. However, because it has been cultivated in the same areas for many years, it is now highly susceptible to rice blast. By re-sequencing and scanning the whole genome of Kongyu 131, we found that it lacks more than 12 cloned blast resistance genes, including Pi2, Pi9, Pi36, Pi5-1, Pb1, Pid3, Pi25, Pikh, Pi1, Pik-m, Pik-p and Pi56t. To improve the blast resistance, we successfully introgressed Pb1 (Panicle blast 1) into Kongyu131. Without changing the other agronomic characters, we used 5 single nucleotide polymorphism markers to control the length of the introduced target fragment derived from the donor cultivar MP. Inoculating test results indicated that the improved line, containing a 700 kb target fragment and sharing 99.38% genetic background with Kongyu 131, showed the same blast resistance as MP.

张晓慧, 冯晓敏, 林少扬. 水稻主栽品种空育131抗稻瘟病位点的扫描及其基因组重构建. 植物学报, 2017, 52(1): 30-42.

Xiaohui Zhang, Xiaomin Feng, Shaoyang Lin. Scanning for Pi Loci and Rebuilding an Improved Genome of Elite Rice Variety Kongyu 131. Chinese Bulletin of Botany, 2017, 52(1): 30-42.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB16107

https://www.chinbullbotany.com/CN/Y2017/V52/I1/30

图/表 8

表1 水稻品种空育131稻瘟病感病性鉴定结果(按国际水稻所稻瘟病抗性评价分级标准)

Table 1 The results of rice cultivar Kongyu 131’s susceptibility to rice blast with score 1-9 (Scale by IRRI Standard Evaluation System for rice)

Year	Artifical inoculation			Natural infection
Year	Seedling blast	Leaf blast	Panicle blast	Seedling blast	Leaf blast	Panicle blast
1995	8	8	9	6	5	9
1996	9	9	9	9	9	9
1998	unknown	9	9	unknown	9	9
1999	unknown	8	9	unknown	8	9
2002	unknown	9	9	unknown	8	9
2003	unknown	8	9	unknown	7	9
2004	unknown	7	9	unknown	7	7
2005	9	9	9	9	9	9

表2 分子标记SNP1-SNP5

Table 2 Molecular markers SNP1-SNP5

Markers	Chr.		Position (IRGSP1.0)	Forward primer (5′-3′)	Reverse primer (5′-3′)
SNP1	11		24107429	CTTACAACCGAATTGCTTATCAC	TATAGGATAAGAGTCGAAAGCATC
SNP2	11		24552815	CCTCAACAGGATCCAGATTCAATAC	CAAGATCAATTGAGTTAGACAAC
SNP3		11	24717067	CCAGACATAATGCTTAAAGTAG	CATAGTCGCAGTTCATCTTATG
SNP4		11	24747927	GACATATTGTATGCAATCTACTCG	TTCACAGAAGCTAGTATAAGAG
SNP5	11		25104609	AAGTAATATACGCTACTATCTTG	CGACGTGATGGGTAAGAACAG

表3 Pi基因在水稻品种空育131上的扫描比对结果

Table 3 The results of the whole genome scanning of rice cultivar Kongyu 131 for Pi genes

Pi gene	Chr.	GenBank ID	BLAST results (with CDS)	Be contained or lacked in Kongyu 131
Pib	2	AB013448.1	High sequence homology	Uncertain
Pita/Pi4a	12	AF207842.1	3 SNP sites	Uncertain
Pi2/Piz5	6	DQ352453.1	53 SNP sites	Lacked
Pi9	6	DQ285630	Sequence deletion	Lacked
Pizt	6	DQ352040	High sequence homology	Uncertain
Pi36	8	DQ900896.1	Multi-SNP sites	Lacked
Pi37	1	DQ923494.1	4 SNP sites	Uncertain
Pi5-1/Pi3/Pii	9	EU869185.1	29 SNP sites	Lacked
Pit	1	AB379815.1	6 SNP sites	Uncertain
Pish	1	Os01g0782100	No difference	Contained
Pb1	11	AB570371.1	Structural variation	Lacked
Pia	11	AB604622 and AB604627	High sequence homology	Uncertain
pi-21	4	AB430853.1	1 SNP site	Uncertain
Pi-d2	6	FJ915121.1	6 SNP sites	Uncertain
Pi-d3	6	FJ773285	Terminator codon mutation	Lacked
Pi25	6	HM448480	18 SNP sites	Lacked
Pik	11	HM048900.1	2 SNP sites	Uncertain
Pikh/Pi-54	11	AY914077(Pikh)/HE589445 (Pi54)	Structural variation	Lacked
Pi1	11	HQ606329cds	11 SNP sites	Lacked
Pik-m	11	AB510262.1	Structural variation	Lacked
Pik-p	11	HM035360	Multi-SNP sites	Lacked
Pi56t	9	None	Multi-SNP sites	Lacked

图1 水稻Pi25 CDS (coding sequence)和空育131 (BKY)的等位序列比对结果

Figure 1 Blast results using rice Pi25 CDS (coding sequence) in the genome of Kongyu 131 (BKY)

图2 水稻Pik1基因第3外显子和空育131 (BKY)的等位序列比对结果

Figure 2 Blast results using rice Pik1-exon3 in the genome of Kongyu 131 (BKY)

图3 水稻选拔个体的染色体组基因型^(A) BC3F1-02个体染色体组成; (B) BC3F2-0050个体染色体组成。表示来源于空育131的染色体片段; 表示来源于MP的染色体片段; 表示片段来源未鉴定。

Figure 3 Graphical genotype of the selected individuals of rice^(A) Graphical genotype of BC3F1-02; (B) Graphical genotype of BC3F2-0050. stands for the fragment derived from Kongyu 131, for the fragment derived from MP and for the unidentified loci.

图4 水稻选拔个体的染色体组基因型^(A) BC3F3-0032个体染色体组成; (B) BC3F4-22个体染色体组成。表示来源于空育131的染色体片段; 表示来源于MP的染色体片段; 表示片段来源未鉴定。

Figure 4 Graphical genotype of the selected individuals of rice^(A) Graphical genotype of BC3F3-0032; (B) Graphical genotype of BC3F4-22. stands for the fragment derived from Kongyu 131, for the fragment derived from MP and for the unidentified loci.

图5 水稻导入系接菌实验结果^(A) 水稻幼苗期(5叶)接菌实验结果及其相对病斑面积; (B) 水稻成株期(11叶) ±Pb1近等基因系接菌实验结果及其相对病斑面积。

Figure 5 The results of inoculating the improved line of rice^(A) The phenotypic results after inoculation on rice seedlings (5 leaves) and the bar chart of relative lesion area; (B) The phenotypic results after inoculation on ±Pb1 near-isogenic lines in rice adult stage (11 leaves) and the bar chart of relative lesion area.

参考文献 24

[1]	董志峰, 马荣才, 彭于发, 管华诗 (2001). 转基因植物中外源非目的基因片段的生物安全研究进展. 植物学报 43, 661-672.
[2]	姜玉英, 曾娟, 陆明红, 刘杰 (2013). 2013年全国主要粮食作物重大病虫害发生趋势预报. 植物保护 39, 1-4.
[3]	姜玉英, 曾娟, 陆明红, 刘杰 (2014). 2014年全国主要粮食作物重大病虫害发生趋势预报. 植物保护 40, 1-4.
[4]	姜玉英, 曾娟, 陆明红, 刘杰 (2015). 2015年全国三大谷类作物重大病虫害发生趋势预报. 植物保护 41, 1-4.
[5]	宋成艳, 王桂玲, 辛爱华, 丛万彪 (2007). 黑龙江省水稻品种空育131稻瘟病菌生理小种种类及发病原因分析. 黑龙江农业科学 (1), 41-42.
[6]	孙向东 (2005). 黑龙江省粮食主产区主要作物品种种植情况分析. 黑龙江农业科学 (2), 12-14.
[7]	Bryan GT, Wu KS, Farrall L, Jia Y, Hershey HP, Mc- Adams SA, Faulk KN, Donaldson GK, Tarchini R, Valent B (2000). A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.Plant Cell 12, 2033-2045.
[8]	Chen J, Shi Y, Liu W, Chai R, Fu Y, Zhuang J, Wu J (2011). A Pid3 allele from rice cultivar Gumei2 confers resistance to Magnaporthe oryzae.J Genetics Genomics 38, 209-216.
[9]	Chen X, Shang J, Chen D, Lei C, Zou Y, Zhai W, Liu G, Xu J, Ling Z, Cao G, Ma B, Wang Y, Zhao X, Li S, Zhu L (2006). A B-lectin receptor kinase gene conferring rice blast resistance.Plant J 46, 794-804.
[10]	Fukuoka S, Okuno K (2001). QTL analysis and mapping of pi21, a recessive gene for field resistance to rice blast in Japanese upland rice.Theor Appl Genet 103, 185-190.
[11]	Hayashi N, Inoue H, Kato T, Funao T, Shirota M, Shimizu T, Kanamori H, Yamane H, Hayano-Saito Y, Matsumoto T, Yano M, Takatsuji H (2010). Durable panicle blast-resistance gene Pb1 encodes an atypical CC-NBS- LRR protein and was generated by acquiring a promoter through local genome duplication.Plant J 64, 498-510.
[12]	Hua L, Wu J, Chen C, Wu W, He X, Lin F, Wang L, Ashikawa I, Matsumoto T, Wang L, Pan Q (2012). The isolation of Pi1, an allele at the Pik locus which confers broad spectrum resistance to rice blast.Theor Appl Genet 125, 1047-1055.
[13]	Inoue H, Hayashi N, Matsushita A, Liu X, Nakayama A, Sugano S, Jiang CJ, Takatsuji H (2013). Blast resistance of CC-NB-LRR protein Pb1 is mediated by WRKY- 45 through protein-protein interaction.Proc Natl Acad Sci USA 110, 9577-9582.
[14]	Lin F, Chen S, Que Z, Wang L, Liu X, Pan Q (2007). The blast resistance gene Pi37 encodes a nucleotide binding site-leucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome 1.Genetics 177, 1871-1880.
[15]	Okuyama Y, Kanzaki H, Abe A, Yoshida K, Tamiru M, Saitoh H, Fujibe T, Matsumura H, Shenton M, Galam DC, Undan J, Ito A, Sone T, Terauchi R (2011). A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes.Plant J 66, 467-479.
[16]	Peleman JD, van der Voort JR (2003). Breeding by design.Trends Plant Sci 8, 330-334.
[17]	Qu S, Liu G, Zhou B, Bellizzi M, Zeng L, Dai L, Han B, Wang GL (2006). The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of a multigene family in rice. Genetics 172, 1901-1914.
[18]	Shang J, Tao Y, Chen X, Zou Y, Lei C, Wang J, Li X, Zhao X, Zhang M, Lu Z, Xu J, Cheng Z, Wan J, Zhu L (2009). Identification of a new rice blast resistance gene, Pid3, by genomewide comparison of paired nucleotide-binding site-leucine-rich repeat genes and their pseudogene alleles between the two sequenced rice genomes.Genetics 182, 1303-1311.
[19]	Takahashi A, Hayashi N, Miyao A, Hirochika H (2010). Unique features of the rice blast resistance Pish locus revealed by large scale retrotransposon-tagging. BMC Plant Biol 10,175.
[20]	Tanaka T, Antonio BA, Kikuchi S, Matsumoto T, Nagamura Y, Numa H, Sakai H, Wu J, Itoh T, Sasaki T, Aono R, Fujii Y, Habara T, Harada E, Kanno M, Kawahara Y, Kawashima H, Kubooka H, Matsuya A, Nakaoka H, Saichi N, Sanbonmatsu R, Sato Y, Shinso Y, Suzuki M, Takeda J, Tanino M, Todokoro F, Yamaguchi K, Yamamoto N, Yamasaki C, Imanishi T, Okido T, Tada M, Ikeo K, Tateno Y, Gojobori T, Lin YC, Wei FJ, Hsing Y, Zhao Q, Han B, Kramer MR, McCombie RW, Lonsdale D, O'Donovan CC, Whitfield EJ, Apweiler R, Koyanagi KO, Khurana JP, Raghuvanshi S, Singh NK, Tyagi AK, Haberer G, Fujisawa M, Hosokawa S, Ito Y, Ikawa H, Shibata M, Yamamoto M, Bruskiewich RM, Hoen DR, Bureau TE, Namiki N, Ohyanagi H, Sakai Y, Nobushima S, Sakata K, Barrero RA, Sato Y, Souvorov A, Smith-White B, Tatusova T, An S, An G, OOta S, Fuks G, Messing J, Christie KR, Lieberherr D, Kim H, Zuccolo A, Wing RA, Nobuta K, Green PJ, Lu C, Meyers BC, Chaparro C, Piegu B, Panaud O, Echeverria M (2008). The rice annotation project database (RAP-DB): 2008 update.Nucleic Acids Res 36, 1028-1033.
[21]	von Bubnoff A (2008). Next-generation sequencing: the race is on.Cell 132, 721-723.
[22]	Wang ZX, Yano M, Yamanouchi U, Iwamoto M, Monna L, Hayasaka H, Katayose Y, Sasaki T (1999). The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes.Plant J 19, 55-64.
[23]	Zhai C, Lin F, Dong Z, He X, Yuan B, Zeng X, Wang L, Pan Q (2011). The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication.New Phytol 189, 321-334.
[24]	Zhou B, Qu S, Liu G, Dolan M, Sakai H, Lu G, Bellizzi M, Wang GL (2006). The eight amino-acid differences within three Leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea.Mol Plant-Microbe Interact 19, 1216-1228.