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基因差异表达导致油菜温敏雄性不育系的育性转换

2022-05-26 来源:步旅网
Agricultu ral Science&Technology,2017,18(1O)1795—1799,1804 Copyright c 201 7,Information Institute of HAAS.All rights rese rved Molecular Biology and Tissue Culture Differentially Expressed Genes Related to Fertility Conversion i n Thermo—sensitive Gen ic Male Steri le(TG MS)Li nes of Brassica juncea Libo SUI ,Haiyang ZHAO ,Lijun AI ,Liping LI ,Chuanli ZHANG。,Chao SUN’,Tianxiang TANG’,Liangbin LIN 1 College of Agronomy and Biotechnology.Yunnan Agricultural University,Kunming 650201,China; 2 The Center for Basic Agricultural Experiments and Feaching, Yunnan Agricultural University,Kunming 650201 China 3 College of Tropical Crops,Yunnan Agricultu ral University,Pu er 665000.China Abstract I()b .iective Thil s study was performed o scrt een unctif ona genes rell ated o t基因差异表达导致油菜温敏雄 the fertility conversion of thermo-sensitive genic male sterile(TGMS)lines of Brassi・ 性不育系的育性转换 ca funcea L. fMethodl A B funcea TGMS Iine K1 21 S was selected as the experi- mentaI material The lotaI RNAs were isolated frOm fertile and sterile pollens at dif- 隋丽波 ,赵海洋 ,艾丽君’,李丽萍 ,张传 ferent development stages.incIuding mother celI stage,tetrad stage,tricellular pollen }1 .孙超 ,唐天向 .林良斌 (1.厶南农、I stage and maturity stage DDRT—PCR was carried out to identify diferentially ex— . ’ 农 J 物投术。’; :院, J妈比叫65020l; pressed genes 【Result1 A total of 44 differentially expressed cDNA fragments were ! 南农、l 夫 、 :农科々、J 伙验墩 、 ,2 南 identified with Dot blot And seven candidate genes related to fertility conversion of K121 S were screened out bv BLASTN.including callose synthase gene.aldehyde } _lJ】65020l;3 南农、l 人 ‘; :热带作物 :院. dehVdrOaenase gene and RNA potymerase I transcription factor RF『』\/3 gene which ^i订} r r 665()00) we re differentiaIly expressed at the lranscripti0nal IeveI,H ATPase gene,fructose 摘 要 lI 1的1获 0汕浆 敏雄一I1-不育系 diphosphate aldolase-class I gene.Ieucine-rich repeat receptor—like se ne/threOnine— l I s肯 r 转换f}f跫的J 能 lq 『力法1以 I 『protein kinase gene and a}kafine/neufraI irivertase gene.wl1}cf l were diferentially ex— 装濉敏雄 rf:/f 育糸KI!IS乃 料,分圳H ・叮 pressed at the post—transcriptional leve1. 『Conclusionl The results of this study wil l.I J譬F翻I 育 J笙Ff10化籽}f: Ⅲ胞期、pL1分f水 help to explain the molecular mechanism of thermo-sensitive genic male sterility of B juncea i 、 梭期阳啦 I{ f内饱’皆挝IIV.RNA.fIIJI J Key words Brassica funcea L:The1‘mo.sensitive male genic sterility;DDRT—PCR: …) r f)I:I{,J’ 上进f r J l 达雎异 示.jt获 Fertility conversion related genes r 44条差 表达n勺‘・I) A"段, i宋l经测 }{l At4 I1序列比刈分析,筛选 7个候选 n0 异表达 j K1 21s的行 #e换l仃关,抑洲 胝匝合J戊脯堪 、乙n≯脱 脯壤I 、R A聚 O iilmsepeodrt aranpt e siso uornce so f tohfe  mveogs—t  aAndd iutitoilnizaaltyio,ns ofm hee tgeeronseis/ [QaTnLds sroe laotn,\嗍T转 J}; F fff{^ 墉 :转采水平莠片丧 i .i打 H 一^Tl】 趣鼻 l 、l炎 电 磷 安 筝匀 缚 etable oi lworldwide and ex・- ed to thermos—。sensitive genic mal—e  . Jt 甫禽 馘腋 的 馘麟,.":氨酸类受体 澈胁旗 、娥I1 1/)ti 转化牌麟f :转 建后水 hibits heterosis(hybrid vigor).which Is sterility of rapeseed have been I{ 、剪接时 &达 沧l}嵌研究结粜刈揭 used to improve crop yield and quality mapped.For exarnple,Xu et a1.㈣ fIII策濉缴/4i i4 的分rⅣ【珥!挺 I J 敞 in breeding.Compared with widely mapped lhe sterility related gene of used cytoplasmic male sterile(CMS)the reverse thermo—sensitive genic 关键词 浆;濉敏雄 f『:I)I)RT-P(:lt分 lines. 1hermo—sensitive genic male male—sterile Iine Huiyou 50S to linkage :岢性转换 sterile(TGMS1 Iines have many ad— group A1 0,and identified three SSR vantages,such as simple seed pro— markers BrGM¥536,BrGMS569 and duction procedu res and short breeding BrGMS287.which were closely Iinked cyclei Al presen1.the studies about to the sterility gene In the study of 基金项目 国家自然平}学基金I3I160289k云 南 目农 1L厅“油莱产业休系连设”项目:云南 (TGMS)Iines of oilseed rape are Dong et a1.I8】.RAPD markers were 抖枝厅”官春云陀士工作站”项目 mainIY focused on lhe selection of used to Iocate fhe gene related to fer— 作者简介 隋丽波(1993一), ,黑龙江林I] sterile I Jnes1 0一 conditiot is for fertility tility conversion in a Brassica napus 人,硕士研究生.研究方向:基闰资源挖掘与 conversion .genetic mechanisms l6 1 TGMS Iine 41 7S,and finalty screened 利用.E—mail:4l 24635ll0、I}IIll・…¨:赵海洋 《l992一}.男,黑龙江鸡西人,硕士研究生.研究 方向:农业生物技术与种质创审 .E—mail: Supported by National NaturaI Science Foundation of China (31 1 60289);Rapeseed 596297I】:@ ff lJl¨III 】H等贡献堆音 Industry Construction P rogram of Department of Agriculture of Yunnan Province;Fund 通讯作静.林良斌,教扳.博士,博士生导师, for Workstation of Academician Guan Chunyun fr0m Department of Science and Technology of Yunnan Province 主蚤从事: b幕分子生物学与疗刊 研究.} 111|Iij: liI1IIangbii1-63(aI63 I)111:,l炙l勺.讲师.硕士.主 These authors contributed equally to this work : :}Corresponding authors.Liangbin LIN.E—mail:linliangbin-63@1 63.com;Tianxiang 皂 事油暮栽培与f『种研究.E II1.1iI: TANG,E・mail:512673189@qq.com 5 l 2673 1 89@(Iq OI11 Received:August 1 2,201 6 Accepted:September 1 6,201 7 收稿日期2Ol 7一(】8一l! 修回日期2()l 7—09一l6 1796 2017 Out a marker BA392 Iinked to the gene,with a genetic distance of 6.0 cM between them.Zhou et a1.【11]mapped the gene related to the fertility conver- Trizol reagent.purified using DNase 1. The concentration and purity of the RNA samples were measured using A naturation at 94 oC for 3 min,followed bv 3O cycles of denaturation at 94℃ for 45 s.annealing at 35℃for 60 s and extension at 72℃for 2 min.and BIO-RADDNA/Protein Analyzer.and their integrity was detected by gel Synthesis of the first strand cDNA The first strand cDNA was syn- thesized using TaKaRa reverse tran. sion of a TGMS line K121S fBrassica markers OI1 1一G1 1a.Nal4一G10 and Na1 O—F06.which were closely related to this gene,and their genetic distance to this gene js 1.6.11.0 and 19.7 cM. terminated with finaI extension at 72℃ for8 min. Recovery of diferentially ex- ectrophoresis. funcea L.),and identified three SSR elpressed DNA fragments The DDRT—PCR products were separated on a 6%polyac ̄lamide ge1. scription kit(Code No.:62 1 0A).Fo1. 1owing the instruction of PrimeScript II Reverse TranSCriDtaSe.The reverse transcription reaction was carried out respectively.Then,two SCAR markers SS1 23—250 and SBA485—482.which were closely related to the fertility con. and the target DNA bands were cut out of the ge1.placed in a 0.5 ml centrifuge tube each.After 20 Ul of ddH2O was added.the tubes were incubated in a version gene in K1 21 S were mapped. in a mixture containing 1 uf Of 50 pmoVL and their genetic distance to the gene was 3.94 and 7.O9 cM㈦.However.the molecular mechanism of fertility con— version of rapeseed TGMS Iines has been rarely reported.In this study.by using the B.iuncea TGMS line K1 21 S developed by Yunnan Academy of Agricultural Sciences as the experi. mentaI material{ .the genes differen- tially expressed at low and high tern— peratures were screened with DDRT- PCR.and the genes related to fertility conversion were identified by se・ quencing and functionaI prediction。to provide a theoreticaI basis to reveaI the molecular mechanism for fertility conversion of K121 S. Materials and Methods Materials The B.iuncea TGMS Iine K1 21 S. which was developed by Yunnan Academy of Agricultural Sciences,is completely sterile at a temperature be— Iow 1 0.8 oC and completely fertile at a temperature above 1 8-2℃. Methods Plant cultivation K1 21 S was planted in pots.one seedling was retained in each pot and the others were removed at 5 leaf stage.Before bolting stage,the pots were equally divided into two groups: one was placed in a climatic chamber at 10℃.and the other in a climatic chamber at 24℃.In both chambers. the seedlings were exposed to a light intensity of 2 200 Ix.and 14 h of Iight perday. RNA extraction and purification The pollens of 1.2 fpollen mother cell stage),2.6(tetrad stage),3.5(tri— cellular pollen stage)and 4.5(mature pollen stage1 mm in diameter were collected.and RNA was extracted with 0ligo dT,1 ul Of1 0 mmol/L dNTPs and boiling water bath for 1 5 min.Subse— 8 uI of template RNA.The mixture was quently,2 I of the solution from each incubated at 65℃for 5 min.and im- tube was amplified again in the DDRT. mediately cooled on ice,and then,an— PCR system described above,and the other 10 Ul of above mixture,4 LlI of 5x products was run on 1.5%agarose gel PrimeScript ll Buffer,0.5 uI of 40 U/pI electrophoresis.The target DNA RNase Inhibitor,1 ul of 200 U/pl bands were cut out of the gel,recov— PrimeScript ll RTase,and 4.5 uI of ered and purified following the instruc— RNase—free ddH20 were added.The tions of Agarose GeI DNA Fragment reaction mixture was jncubated at Recovery Kit of Takara. 42℃for 30 min and then at 95℃for Dotblot 5min. The RNA samples extracted frOm DDRT・PCR fertile and sterile pollens at diferent A totaI of 24 random primers stages were used as the template to (Table 1)and three anchor primers 5’- produce cDNA probes,which were A 3’(T1 ), 5’一 used in Dot blot to identify diferentially C一3’(T14C),5’。 expressed cDNA fragments㈣. G-3’(T14G)were Cloning and sequence analysis of alI synthesized by TaKaRa Biotech- cDNA fragments nologv Co.,Ltd.The DDRT—PCR re— The positive DNA fragments were action system contained 4.0 uI of cD— Iigated into PMD1 8.T vector.trans— NA,1 uI Of 1 0 pmol/L anchor primer, formed into Escherich『a coil DH5a 1 ul 0f 10 pmol/L random primer,1 u1 competent cells,and inoculated onto 0f 10 mmol/L dNTPs,5 ul Of 10 x Ex agar plates.The white single colonies Taq Buffer(Mg Plus),0.25 uI of 5 U/pl were inoculated into 1 0 uI sterilized Ex/a口DNA polymerase,and ddH20 ddH20 to carry out colony PCR.The was added to a totaI volume of 5O uI. reaction system containing 2 uI of sin- The reaction was started with prede- gle colony—containing ddH20,1 uI Of Table 1 Random primers No. Sequence(5’-3’) No. Sequence(5’-3’) DD01 GATCATAGCC DD1 3 CTGCTTGATG DD02 GATCCAGTAC DD1 4 GATCGCATTG DD03 AAACTCCGTC DD1 5 TGGTAAAGGG DD04 GATCATGGTC DD1 6 TTTTGGCTCC DD05 GTTTTCGCAG DD1 7 TACCTAAGCG DD06 GATCTGACAC DD1 8 GATCTAACCG DDO7 TGGAT1_GGTC DD1 9 TCGGTCATAG DD08 GGAACCAATC DD20 GATCTGACTG DD09 GATCAATCGC DD21 TCGATACAGG DD1 0 TACAACGAGG DD22 GATCAAGTCC DD1 1 GATCTCAGAC DD23 GGTACTAAGG DD1 2 GATCACGTAC DD24 CTTTCTACCC 2O17 1797 10 pmol/L universa J primer 1,1 UI Of 1 O um0I,L of universaf primer 2,1 Ul 0f 10 mmOI/L dNTPs.5 uI of 10x Ex Taq fragment were sequenced by Sangon to previous studies,the diferential ex— pression of seven candidate genes was considered to be related to fertility Biotech(Shanghai)Co.,Ltd. Sequence analysis of diferentially Functional prediction revealed that most of the 44 diferentially ex— pressed cDNA fragments were in— fragments Buffer(Mg Plus)and 0-25 ul of 5 U/pl expressed cDNA Ex丁aq DNA polymerase.The positive clones were then extracted with alka- Iine Iysis method.and sequenced by conversion the TGMS Iine K1 21 S (Table 4).The deposition and degra- dation of callose js a dynamic process during pollen development.Callose accumulates in the tapetaI cells at the Sangon Biotech(Shanghai)Co.,Ltd The resulting sequences were sear- volved in energy metabolism,and a few were involved in celI division.re- sponse to heat shock,transcription early stage of m[crospore develop。 ment. and then around the mi— crospores until the pollen matures I . ched jn BLASTN database of NCBI. and signaI transduction.etc.According Results and Analysis selected primer combinations for DDRT.PCR Table 2 Selected primer combinations for DDRT—PCR The first strand cDNA samples of fertile and sterile tricellular pollens were equally bulked,and the mixture was used to select the primers that could produce no less than three poly— morphic DNA bands in DDRT—PCR. As a result.a totaI of 26 pairs of primers were screened out,and used in later DDRT—PCR assay fTable 2). Diferentially expressed cDNA frag— ments in ferl:ile and sterile pollens The selected primer combinations were used to identify the diferentially expressed genes between fertile and UU + ++ ++ UL,Zi 一 + + DD10 DD11 DD12 “一++ 一 —十++一 4-4-4- 4- 一 一 DD22 DD23 DD24 -4+4- 一 + --i一 一 -44--4 一 + sterile pollens at diferent stages bv DDRT.PCR assay.As a result a total Of 1 37 polymorphic cDNA fragments ”means that no potymorphic DNA band is produced by the Drimer combination;“+” were obtained,most of which were 300 to 900 bp long fTable 3 and Fig.1).Among them,the diferentially expressed fragments in fertile and sterile pollens at tricellular pollen stage were more than that at other stages. means that one to three polymorphic DNA bands are produced by the primer combination; “++”means that four to eight polymorphic DNA bands are produced by the primer combi— nation;‘ 4--44-”means that more than eight polymorphic DNA bands are produced by the primer combination. 1) The reason may be that the primer combinations were selected by using the cDNA of tricellular pollens as the template in DDRT—PCR.or that tricel- Jular pollen stage was a critica J period for fertility conversion of K1 21 S.The polymorphic cDNA fragments pro— duced by DDRT—PCR were validated by Dot blot,and 44 were proven posi— tive(Table 3).The false positive rate of DDRT—PCR was about 7O%. Cloning of diferentially expressed cDNA fragments The 44 positive cDNA fragments were Iigated into PMD1 8一T vector. transformed into E.coil DH5c(compe— tent cells.and inoculated onto LB agar plates containing 1 0O mg/L ampicillin and X—galll PTG for blue—white screen— ing.The white single colonies were (A)PCR products amplified from the pollens at mother cell stage by the primers HT2/DD20—4:(B)PCR products amplified from the pollens at tetrad stage by the primers HT2/DD1 9-2;(C)PCR products amplified from the pollens at tricellular pollen stage by the primers HT3/DD1 4—3;(D)PCR products amplified from the pollens at mature pollen stage by the primers HT3/DD22—1. further identified by colony PCR(Fig.2). Three positive clones for each cDNA shows the diferentially expressed fragments. Fig.1 Products of DDRT—PCR by PAGE 1798 2017 study,we found that the protein was 2 000 bp 1 200 bp 1 000 bp 750 bp expressed in fertile pollens at tetrad stage,and not expressed in sterile pollens at this stage,which affected rRNA transcription.ribosome forma. tion and protein synthesis,Ieading to male sterility eventually. 300 bp 1 50 bp Previous studies have shown that Lanes 1 and 2,PCR products amplified fr0m the pollens at mother cel stage bv the Iprimers HT3/DD22・1 and HT2/DD1 9—2 respectively;lanes 3 and 4.PCR products ampli. led frf0m the pollens at tetrad stage bv the primers HT1/DD09-3 and HT2/DD20—4 re— spectively;Ianes 5 and 6.PCR products anaplified frOm the pollens at tricellular pollen stage by the primers HT3/DD13—2 and HT2/DD18—3 respectively;Ianes 7 and 8。PCR products amplified frOm the pollens at mature pollen stage bv the primers HT2/DD 1 4 1 and HT3/DD14—3 respectively. normally expressed Ieucine-rich repeat receptor・・like serine/threonine-.protein kinase, aIkaIine/neutraI invertase. fructose diphosphate aldolase—class I and H 一ATPase is necessary for pollen development.Leucine-rich repeat receptor-like se rine,lhreOnine-protein kinase is involved in floral organ de— velopment—related signaI transduction. Fig.2 Recombinant plasmids characterized bv colony PCR Table 3 Diferentially exp ressed cDNA fragments by DDRT—PCR Ken—Ichi ef a1. showed that in msD 7 fwhich encodes Ieucine—rich repeat receptor-like se rine/lhreOnine—protein kinase)mutants,the formation of an— ther walI Iayers was disordered.and the development of pollen mother cells was arrested at various stages of mei- otic prophase I.which resulted in com— plete male sterility.AIkaline/neutral in- The results of the present study showed that the cDNA fragment of codes a putative mitOchOnd riaI aide— vertases,key enzymes involved in su- crose metabolism,catalyze the hy— drolysis of sucrose into glucose and fructose.Welham et a1.㈣isolated a hyde dehvdroqenase (TobAIdh2A)in ~『c0fiana tabacum.and shows 76% identity with maize rf2.evaluated its function using specific inhibitors.And callose synthase gene was expressed in fertile tricellular pollens.But it was not expressed in sterile tricellular pol— Lotus iaponicus mutant of LffNV1 (which encodes a nodule—enhanced isoform),and found that the anthers of the mutant contained no pollen,and lens.then callose could not be synthe— sized,and thus the pollens could not develop normally.resulting in male the results revealed that aldehyde de— hydrogenase plays an important role in tobacco pollen development.Our re— sterility eventually.Cui et a1.115]cloned nuclear restorer gene of male—ster— suits also showed that the cDNA flag— ment of aldehyde dehVdrOqenase flowers opened later,suggesting that this gene is cruciaI to whole plant de— velopment.I n the study of Tang ef aL【1引. the function of eight FBA family genes ile T—cytoplasm maize by transposon gene was specifically expressed in fertile pollens at tetrad stage。and not expressed in sterile pollens.suggest— tagging,and proved that rf2 encodes mitOchOndrial aldehyde dehydroge— (AtFBA1—8)was jdentified and aria— Iyzed in Arabidopsis thaliana.and the results showed that the mutations of nase,indicating that the male sterility Of T—cytoplasm maize is caused by the Iack of mitochondrial aldehyde dehy— ing that the Iack of aldehyde dehydro— genase wilI Iead to male sterility of K1 21 S.RNA polymerase I transcrip— tion factor RRN3 is involved in the initi— ation of rRNA transcription.1n this fba8-7 and fba8—2 inhibited pollen ger— drogenase.Opden Camp et a1.[16j cloned a cDNA fragment which en— Table 4 BLASTN analysis of candidate genes uirnation.indicating that AfFBA8 plays an important role in pollen germination. 2017 Kasamo et a1. reported that the free functional genes related to its fertility conversion. 1799 fication and genetic analysis of the fertil— ity restoring gene for dominant male sterility accession “Shaan—GM S”in subunit of H 4-ATPase was separated fr0m its membrane subunit under chill— In this study.seven candidate ing stress,so that the activity of the enzyme was completely lost,resulting pollen abortion.Our results showed the cDNA fragments of alI Ieucine-rich genes related to K1 21 S fertility con- version were screened out by se— quence alignment and functional anal— ysis of the 44 diferentially expressed cDNA fragments.Among l}1em.cal- Brassica napus L.『J1.JournaI of North— west A&F University (Nat.Sci.Ed.), 2004,(04):9—12,18. 『71 TAO FF,YANG X L_PENG Y,et a1. Analysis of the inherited character of a thermo-sensitive genic male sterile Iine repeat receptor-like se rine,lhreOnine— protein kinase.alkaline/neutraI Inver- tase,fructose diphosphate aldolase— class I and H ,ATPase were diferen- tially expressed in fertile and sterile pollens at mother celJ  stage,tricellular pollen stage and mature pollen stage. lose synthase gene,aldehyde dehy- drogenase gene and RNA polymerase I transcription factor RRN3 gene were expressed in fertile pollens。and not 1 00S in日rassica napus L.fJ].Journal of Hunan Agricultural University(Natural Sciences),201 6,42(2):1 25-1 28. f81 DONG JG,LI HB,LlU CS,ef a1.Inheri- tance and RAPD analysis on thermo・ expressed jn sterile pollens of K1 21 S. Leucine-rich repeat receptor—like ser- sensitive CMS 417S in Brassica napus L_『J1.Acta Botanica Boreali Occidentalia So,we speculated that these genes were expressed in both fertile and sterile pollens,but then diferentially spliced,which causes the diferences in their cDNA fragments between fer- tile and sterile pollens.Namely,the sites that are complementary to the random primers were cut off as introns and thus could not be amplified in sterile pollens,but were recognized as exons and amplified in fertile pollens. Therefore,the genes encoding the four enzymes are normally expressed in fertile pollens.and abnormally ex. pressed in sterile pollens.resulting in TGMS.which was consistent with pre vious studies[21]. UISCUSSlon ln the late 1 990s.DDRT-RCR was a commonly used and effective technique for studying gene differential expression and functional genes【 . However。this technique has high false positive rate,and many diferentially expressed cDNA fragments obtained by this method are false—positive.and need to be verified by Northern blot and other techniques.In our study.to decrease the faIse positive rate of DDRT—PCR.the annealing lempera. ture was increased,and the number of thermaI cycles was decreased.How— ever.about 70%of the differentially expressed cDNA fragments were proven false・positive.In recent years, gene chip and particularly transcrip— tome sequencing have attracted in— creasing attention due to their high throughput.However,compared with gene chip and transcriptome sequenc— ing.DDRT.RCR is simple to operate and low—cost.Therefore.DDRT・RCR technique was adopted to identify the diferentially expressed genes in fertile and sterile pollens of K1 21 S.to screen ine/threonine-protein kinase gene.al— kaline/neutraIinvertase gene.fructose diphosphate aldolase—class I gene and H -ATPase gene were expressed in both fertile and sterile pollens,but dif— ferentially spliced to controI fertility conversion of K1 2 1 S.These results proved that the thermo—sensitive genic male sterility in oilseed rape is a very complex biological phenomenon.We have proposed that the molecular mechanism for fertility conversion of thermo-sensitive genic male sterile Iines in oilseed rape is controlled by many genetic factors.and jt is a com. plex regulatory system involving multi— pie pathways and factors .Whether the seven candidate genes found in this study are actually related to K1 21 S fertility conversion still needs to be verified. 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The,f2 nUCIear rest0rer aene Of maIe. ste rile T-cvtoplasm maize fJ1.Science, 1 996,272:1 334—1 336. 『1 61 OPDEN CAMP R G L.KUHL毒 ElER C.Aldehyde dehydrDqenase in tObacc0 poflenfJ1.Plant MoI BiOl,1 997,35:355— 365. 『171 KEN—ICHI N,KAZUMARU M,MlT— SUGU E.efa The Ms尸1 aene is nec- essarV tO rest—ct the number 0f cells ente ring intO maIe and female Sp0rO一 0enesjs and tO jnjtjate an廿1er waI JfOr— ma1i0n in rjce『J1.The PIant Cell,2003, 15:1728—1739. 『181 WELHAM T。PlKE J,HORST l,efa^A CytOSOlIC InvertaSe lS reqUlrea tOr n0r— maI grOwth and ceI ldeveIODment in the (Continued on page 1 804) 2017 tech Seed Science Research Institute Co..Ltd.is now completely indepen- dent frOm the derivatives of Peiai 64S proved lines of Huazhan(bred current- ly1 are independent frOm the closely- related lines of 931 1.and have already diferentiated into 2 subgroups.The 2015,29(1):28-34. 【31 YANG YR(杨玉蓉),SUN JC(孙建昌), WANG XS(王兴盛),et a1.Comparative analysis of genetic diversity for diferent and Guangzhan 63S,and have the excellent characteristics of high com- average gene distance(0.403 3)in the improved Iines and the closely.related lines of Huazhan bred by Lonqping High-tech Seed Science Research ln. stitute Co..Ltd.was remarkably lower period rice varieties jn Ningxia f宁夏不 bining ability,and distant germplasm with strong stress resistance could be introduced in the further improvement of this subgroup.such as Aus rice germplasm with strong stress resis— tance and high nutrition efficiency.Aus rice group as an independent Indica rice subgroup mainly includes early— maturing drought-・resistant waterlog-・ ging・resistant varieties planted in Aus and lndia s West Bengal state[6-a.This 同年代水稻品种的遗传多佯性比较)IJ]. JournaI of Plant Genetic Resources f}j§ 物遗传资源学报),2014,15(3):457— 464. [4】MURRAY MG,Thompson WF.Rapid jsolation of high molecular weight plant than that(0。531 9)in the closely-relat- ed Iines of 931 1.indicating that the homogenization problem exists to a DNA【J】.Nucleic Acids Research,1 980, 8f19):4321—4326. [5】LIU K,MUSE SV.Power Marker:an in— tegrated analysis environment for ge- certain degree.Previous combining experiments indicated that Longke 638S and Jing 41 55S have higher rials,and in the improvement process Of Huazhan derivative group。germ- plasms with high quality,high resis— tance and wide adaptability in Huanan lines should be further introduced to continuously expand the genetic dis- tance between the sterile Iine groups netic marker analysis【J】.Bioinformatics, 2005,21(9):2 128-2 129. season(March—July)in Bangladesh combining ability to Huanan Iine mate— group contains a large quantiW of ex- cellent genes.for instance.80%-90% of varieties In it contains Pup1 gene[ ̄. and there are also germplasms with high tolerance to high temperature恻. 【6】GLASZMANN JC.Isozymes and classi— fication of Asian rice varieties『J].Theo- reticaI and Applied Genetics,1 987,74 (1):21-30. 『71 PARSONS BJ,NEWBURY HJ,JACK- SON MT.The genetic structure and conservation of Aus,aman and boro rices frOm Bangladesh『J1.Genetic Re- waterlogging[删and wide affinity[11】_The introduction of Aus germplasm could further expand the genetic distance between medium Indica type sterile sources&Crop Evolution,1 999,46(6): 587-598. of Longping High.tech Seed Science Research Institute Co..Ltd..so as to create more abundant genetic diversi- 『81 CHIN JH,LU X,HAEFELE SM,et a1. Erratum to:Development and applica- tion of gene.based markers for the ma— lines of Longping High-tech Seed Sci— ence Research Institute Co..Ltd.and Indica or inter Indica—Japonica type restoring line group,and further lays a genetic basis for the formation of strong—heterosis combination. Because of the characteristics of high combining ability,high disease resistance,high quality and wide adaptability of the combinations fOrm- ed fr0m Huazhan with Longke 638S ty basis for the formation of strong— heterosis combinations. jor rice QTL Phosphorus uptake 1[J】. Theoretical and Applied Genetics,201 0, 2Of6):1 073-1 086. 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