1、QTL Ana lysis of Cold Tolerance a t the Booting Stage in Y unnan R ice Var ietyChongtuiYE Chang2rong1, KA TO A k ira2, SA ITO Ko ji2, ISE Kazuo3, DA IL u2yuan1, YAN G Q in2zhong1(1C rop Germp lasm R esou rce Institu te, Y unnan A cad emy of A g ricu ltu ral S ciences, K unm ing 650205, Ch ina; E 2m
2、ail: y echang rp ublic. km. y ; 2 H okkaid o N ational A g ricu ltu ral E xp erim ent S tation, S app oro 062, J ap an; 3 J ap an International R esearch Center f or A g ricu ltu ral S ciences,T sukuba 305, J ap an)云南稻种冲腿的孕穗期耐冷性 Q TL 分析叶昌荣 1 加藤 明 2 齐藤 浩二 2 伊势 一男 3 戴陆园 1 杨勤忠 1(1 云南省农业科学院 农作物品种资源站 , 云
3、南 昆明 650205; E2m ail: yechangrpublic. km. yn. cn; 2 北海道农业试验场 , 日本 札幌 062; 3 日本国际农林水产业研究中心 , 日本 筑波 305)摘 要 : 利用 352 个探针分析了日本品种农林 20 和云南品种冲腿之间的限制性片段长度多态性 (RFL P ) , 从中筛选出157 个在农林 20 和冲腿间显示多态性的探针 , 其中 55 个被用于其杂交后代 (农林 20 冲腿 ) 的 70 个 F 3 系统的孕穗期耐冷性数量性状位点 (Q TL )分析。结果初步显示出一些与水稻孕穗期耐冷性有关的 Q TL , 它们主要分布在第 1、
4、 3、 4、 5、 6、 7、 8、 10 和第 12 染色体上。可能在第 3 和第 7 染色体上具有对孕穗期耐冷性作用较大的 Q TL。关键词 : 限制性片段长度多态性 ; 数量性状位点 ; 耐冷性 ; 孕穗期 ; 水稻中图分类号 : Q 943; S511. 032 文献标识码 : A 文章编号 : 100127216 (2001) 0120013204Abstract: The restriction fragm ent length po lymo rphism (RFL P) betw een Japanese variety (No rin 20) and Yunnan vari2
5、ety (Chongtui) w ere analyzed w ith 352 m arkers in this study. O ne hundred and fifty2seven m arkers had po lymo rphous bandsbetw een the two varieties w ere detected. F ifty2five selected RFL P m arkers w ere used fo r the quantitative trait loci (Q TL )analysis of the co ld to lerance at boo ting
6、 stage of 70 F 3 lines of the cro ss of No rin 20 Chongtui. The results show ed that som eloci are related to the co ld to lerance. The Q TL s are m ainly distributed on chromo som e 1, 3, 4, 5, 6, 7, 8, 10 and 12 w ith m ajo rones on chromo som e 3 and chromo som e 7.Key words: restriction fragm en
7、t length po lymo rphism ; quantitative trait loci; co ld to lerance; boo ting stage; riceYunnan P rovince of Ch ina is one of the centers of geneticdiversity of rice along w ith east N epal, Bhutan, A ssam in In2dia, M yanm ar, and no rth Thailand 1 . Fo r the special terrainand clim ate, rice is cu
8、ltivated from an elevation of 76 m to2670 m. There are p lenty of rice germp lasm resources inYunnan P rovince, especially in southern Yunnan. To m akeuse of these resources better, a sino2Japan cooperative re2search p ro ject betw een Yunnan A cadem y of A gricultural Sci2ences and Japan Internatio
9、nal Research Center fo r A gricultur2al Sciences (J IRCA S) w as initiated to imp rove rice varietiesw ith h igh yield, co ld to lerance and disease resistance in Yun2nan P rovince in 1982. N o table results have been ach ieved bym eans of the researchers hard wo rk fo r mo re than 15years 2 . “ H e
10、xi” serial varieties bred from th is p ro ject havebecom e the m ain cultivars in Yunnan P rovince. Fo r m any ricevarieties are cultivated over the elevation of 2000 m in Yun2nan, the coo l temperature causes decrease of rice yield everyyear. Research ing the co ld to lerance of rice is therefo re
11、veryimpo rtant in Yunnan P rovince.Japanese researchers have m ade good p rogress in thearea of rice co ld to lerance recently. P lenty of RFL P m arkersw ere developed by Japan rice genom e research p rogram. Ah igh2density RFL P linkage m ap of rice w ith 2275 m arkersw as constructed using the cr
12、o ss of N ipponbare Kasalath 3 .Som e Q TL s related to the co ld to lerance at boo ting stage ofrice w ere m apped on the ch romo som es 4, 5 . To use the ad2vanced techniques to study the co ld to lerance of Yunnan ricevarieties, w e detected the RFL P betw een Yunnan varietyRece ived date: 2000-0
13、1-10; Revised date: 2000-08-29.基金项目 : 云南省攻关项目 ; 国家自然科学基金资助项目。第一作者简介 : 叶昌荣 (1972- ) , 男 , 硕士 , 助理研究员。31中国水稻科学 (Ch inese J R ice S ci) , 2001, 15 (1) : 13 16 “ Chongtui” and Japanese variety“ N o rin20” and analyzed theQ TL s of co ld to lerance in Yunnan rice variety.1 M aterials and M ethods1. 1 M a
14、ter ialsTwo rice varieties, Chongtui, N o rin 20 and their F3 pop2ulations (70 lines) w ere used fo r RFL P analysis. Chongtui isan up land rice variety cultivated in southern YunnanP rovince, Ch ina. Its resistant to coo l temperature during itsgrow ing period. N o rin 20 is a Japanese variety susc
15、ep tible tocoo l temperature.1. 2 RFL P detectionGenom ic DNA fo r analysis w as iso lated from theseedlings of the varieties (o r lines) acco rding to the CTABm ethod (M urray and Thomp son, 1980) and digested w ithfour restriction endonucleases, B am H , B g l , H ind andE coR , respectively. The
16、digested DNA w ere separated by0. 8% agaro se gel electropho resis in 10 TA E, denatured in0. 1 mo l L N aOH (1. 5 mo l L N aC l) and transferred to H y2bond2N + nylon m em branes (Am ersham ). ECL direct nucleicacid labeling and detection system s (Am ersham ) w ere usedfo r southern hybridization.
17、 ECL is a good non2radioactive la2beling m ethod 6 . Th ree hundred and fifty2two RFL P m ark2ers p reviously m apped by Saito et al. 7 (designated as XN pbnum ber) and Kurata et al. 8 (designated as C, G, R and Lnum ber) w ere used as DNA p robes to detect the RFL P be2tw een the two varieties. The
18、 p robe DNA w ere labeled by us2ing the ECL labeling system and hybridized w ith the DNAfragm ents on the m em branes at 42 . The m em branes w erew ashed in 0. 5 SSC (0. 4% SD S) containing urea, and ex2po sed to X2ray film s to detect the hybridized signals.1. 3 Cold tolerance testThe seeds from 7
19、0 F2 p lants derived from the cro ss N o rin20 Chongtui w ere used fo r the Q TL analysis. A ll the F3 linesw ere tested the co ld to lerance. To test the co ld to lerance ofthese m aterials, five p lants of each line w ere cultivated inp lastic trays and grow n in the green house till the earliestp
20、 lants fo rm young panicles. Then they w ere grow n in a deepcoo l2w ater condition at temperature of 19 till the flow eringof the latest p lants. The num ber of filled grains and emp tygrains on each panicle w ere counted after the seeds m atured.The co ld to lerance of each line w as estim ated by
21、 the averagepercentage rate of fertile grains.1. 4 QTL analysisThe seeds from tested F3 lines w ere used fo r analysis ofthe geno type of each line. DNA iso lation and RFL P geno typew ere investigated using the selected RFL P m arkers as them ethods described above. The association betw een RFL Pm
22、arkers and Q TL s fo r co ld to lerance w as analyzed by MA PLsoftw are 9, 10 .2 R esults2. 1 RFL P between Chongtui and Nor in 20Th ree hundred and fifty2two m arkers w ere used to detectthe RFL P betw een N o rin 20 and Chongtui. O ne hundred andfifty2seven m arkers of them show ed po lymo rphous
23、bands be2tw een the two varieties. The num ber of used m arkers isshow ed in Table 1. The po lymo rphous m arkers have coveredalmo st the w ho le rice genom e. Because N o rin 20 is w idelydifferent from Chongtui, the percentage of RFL P m arkers oneach ch romo som e is quite h igh. The frequency of
24、 the RFL Pm arkers is 44. 6%. Its very useful fo r studying the genes inthe two varieties. Its po ssible no t only to analyze the co ldto lerance, but also to m ap o ther genes w ith the populationsderived from the cro ss of N o rin 20 Chongtui.2. 2 The cold tolerance of F3 linesTable 1. Number of m
25、arkers used to detect RFL P on each chromosome.Chromo som e no. N um ber of used m arkers N um ber of R FL P m arkers Percentage of R FL P m akers %1 46 21 45. 652 41 12 29. 273 48 19 39. 584 30 13 43. 335 26 10 38. 466 31 13 41. 947 22 15 68. 188 20 11 55. 009 23 9 39. 1310 16 10 62. 5011 29 13 48.
26、 2812 20 10 50. 00To tal 352 157 44. 6041 Chinese Journal of R ice Science,Vo l. 15, No. 1, 2001F ig. 1. D istribution of the fertility of F 3 lines.No rin 20= 55. 83% ; Chongtui= 93. 15%.The fertile grains and the emp ty grains on each panicle ofthe co ld w ater treated m aterials w ere counted. A
27、verage per2centages of the fertile grains of each line w ere counted to eval2uate the co ld to lerance of the line. N o rin 20 and Chongtui are55. 8% and 93. 2% , respectively. The fertile rates of 70 F3lines are distributed from 45. 0% to 94. 8% w ith an averageof 74. 8% (F ig. 1). There is a no ta
28、ble difference betw eenN o rin 20 and Chongtui. It w as confirm ed that Chongtui is re2sistant to coo l tempera ture and N o rin 20 issuscep tible. Itspo ssible to use these m aterials fo r Q TL analysis of co ld to ler2ance.2. 3 QTL related to cold toleranceThe association betw een 55 m arkers and
29、the co ld to ler2ance (fertile rate) w as calculated by using MA PL softw are.The m axim um logarithm of odds (LOD ) sco re near XN pb98(Ch r. 1) , C112 (Ch r. 1) , XN pb373 (Ch r. 3) , C63 (Ch r.3) , C944 (Ch r. 3) , XN pb48 (Ch r. 3) , XN pb282 (Ch r. 4) ,C624 (Ch r. 5 ) , C498 (Ch r. 6 ) , G1091
30、(Ch r. 6 ) , andXN pb152 (Ch r. 7) , XN pb41 (Ch r. 8) , XN pb291 (Ch r. 10)and G124A (Ch r. 12) are greater. W e infer that there areQ TL s related to co ld to lerance in these locations (F ig. 2).M o st of these loci are the sam e as the repo rted Q TL s 5, 11 .Its likely that there are m ajo r Q
31、TL s on ch romo som e 3 andch romo som e 7 fo r the h igher LOD sco res.3 D iscussionsFo r the coo l temperature often causes rice dam age inno rthern Japan, lo ts of studies about the co ld to lerance of ricehave been carried out in Hokkaido (Japan). Saito et al. ana2lyzed the Q TL related to the c
32、o ld to lerance at boo ting stageF ig. 2. The Q TL s related to the co ld to lerance of rice at boo ting stage.51Ye Changrong et al. : Q TL A nalysis of Co ld To lerancein rice variety“ N o rin2PL 8” and indicated that the loci nearXN pb345 on ch romo som e 3 and XN pb235 on ch romo som e 4are respo
33、nsible fo r the co ld to lerance 11 . The fine location ofthese loci have been done up to now. Kato et al. studied theco ld to lerance of Javanica varieties, Silew ah and Padi L abouA lum bis (P. L. A. ) indicated that sm all po rtions of ch romo2som e 1, 4 and o r 7 of Silew ah and ch romo som e 8,
34、 11 and o r12 of P. L. A. contain the genes fo r co ld to lerance 5 . O therresearches by Kato et al. indicated that there are som e loci onch romo som e 1, 3, 4, 5, 6, 7, 8, 10, 11 and 12 related to co ldto lerance of rice 12 . W e suggested that the Q TL s on ch romo2som e 1, 3, 4, 5, 6, 7, 8, 10
35、and 12 are related to the co ld to ler2ance of rice. M o st of them are the sam e location as the re2po rted results.Q TL s such as the co ld to lerance of rice at the boo tingstage are very comp lex. A lthough our results show ed consis2tency to o ther studies, the details couldnt be know n yet be2
36、cause the num ber of m arkers used fo r the Q TL analysis inth is study w as no t enough. There are som e interesting ques2tions aro se from the p resent study. To know these details,its very necessary to continue to study on these m aterials us2ing mo re RFL P m arkers, especially the RFL P m arker
37、s nearthe locations that show ed som e association to the co ld to ler2ance.Acknowledgemen ts: Th is study w as suppo rted by JapanInternational Research Center fo r A gricultural Sciences (J IR2CA S) and all the experim ents w ere done in the P lant B io tech2no logy L abo rato ry of Hokkaido N ati
38、onal A gricultural Experi2m ent Station. W e are also grateful to M r. Sugaw ara A k ira,Kom atsu To sh im itsu, Yo sh ida H ito sh i andM iss Sato M arem ifo r their helpful co2operation.L iterature C ited:1 N agam ine T , X iong J H , X iao Q. Genetic variation in severalisozym es of indigenous ri
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40、 A , et al. A high density rice genet2ic linkage m ap w ith 2275 m arkers using a single F2 populationJ . Genetics, 1998, 148: 479 4944 Saito K, M iura K, N agano K, et al. Genetic analysis of the co ldto lerance genes on chromo som e 3 and 4 in rice“ No rin2PL 8” J .B reed ing S cience, 1997, 47 (s
41、upp l 2) : 1725 Kato A , Kiuchi H , Saito K. A nalysis of the genetic loci invo lvedin coo l to lerance of a coo l2to lerant rice strain Hokkai PL 5.B reed ing S cience, 1997, 47 (supp l 2) : 1146 Ye Changrong (叶昌荣 ) , Dai L uyuan (戴陆园 ). The p rincip lesand app lication of three non2radioactive lab
42、eling m ethods inmo lecular bio logy J . S ou thw est Ch ina J ou rnal of A g ricultu ralS cience (西南农业学报 ) , 1998, 11 (3) : 100 1057 Saito A , Yano M , Kishimo to N , et al. L inkage m ap of restric2tion fragm ent length po lymo rphism loci in rice J . J ap an JB reed , 1991, 41: 665 6678 Kurata N
43、, N agam ura Y, Yam amo to K, et al. A 300 kilobase in2terval genetic m ap of rice including 883 exp ressed sequencesJ .N atu re Genetics, 1994, 8: 365 3729 U kai Y, O hsaw a R , Saito A , et al. MA PL : A package of com 2puter p rogram s fo r construction of DNA po lymo rphism linkagem ap s and ana
44、lysis of Q TL J . B reed ing S cience, 1995, 45: 139 14210 U kai Y. Theo ry of Q TL analysisJ . B reed ing R esearch, 1999,1: 25 3111 Saito K, M iura K, N agano K, et al. Chromo som al location ofquantitative trait loci fo r coo l to lerance at the boo ting stage inrice variety“ No rin2PL 8” J . B r
45、eed ing S cience, 1995, 45: 33734012 Kato A , Saito K, Yo shida H. M o lecular app roaches to coo l to l2erance study in m ajo r Japanese crop sA . In: P roceedings of theJapan2R ussia W o rkshop Sappo ro on Low Temperature Physio lo2gy and B reeding of No rthern C rop s C . Sappo ro, Japan, Jan2uary 25- 28, 1994. 25 2861 Chinese Journal of R ice Science,Vo l. 15, No. 1, 2001
