高抗白粉病和条锈病小麦-卵穗山羊草衍生后代1003的分子细胞遗传学鉴定
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摘要
卵穗山羊草中蕴含着许多小麦改良所需的优良基因,是小麦重要的三级基因库。为了解其更多遗传特性,本研究利用细胞学、原位杂交、分子标记、形态学和抗病性鉴定等技术对小麦-卵穗山羊草SY159的衍生后代1003进行鉴定。细胞学鉴定结果表明,1003含有44条染色体,减数第一次分裂中期含有22个二价体且配对良好,减数第一次分裂后期含有44条染色体且均等分离;基因组原位杂交(Genomic in situ hybridization,GISH)分析显示,1003含有42条小麦染色体和2条卵穗山羊草染色体;EST和PLUG分子标记分析表明,导入的染色体属于7M染色体;荧光原位杂交(Fluorescence in situ hybridization,FISH)分析表明,1003中含有38条与中国春标准核型相一致的染色体,4A、5A和7M的FISH信号有变异;苗期抗病性鉴定结果表明,1003对白粉病生理小种E09免疫,对条锈病生理小种条中23(CYR23)高抗;形态学调查表明,1003的农艺性状介于双亲之间,千粒重高于双亲。因此,1003是一个具有白粉病和条锈病抗性的小麦-卵穗山羊草二体异附加系,可为小麦品种改良和抗病育种提供新的种质资源。
The Ae.geniculata Roth contains many excellent genes for wheat improvement and it is an important tertiary gene bank for wheat. Here,we used cytological,in situ hybridization(Genomic in situ hybridization,GISH and Fluorescence in situ hybridization,FISH) and molecular marker techniques to identify the progeny 1003 of wheat-Ae.geniculataRoth SY159. Cytological observation showed that 1003 contained 44 chromosomes,and the pollen mother cells possessed 22 bivalents with good pairing at metaphase I(PMCs MI),and the pollen mother cells held 44 chromosomes that were equally separated at anaphase I(PMCs AI). GISH analysis showed that 1003 contained 42 wheat chromosomes and 2 Ae.geniculata Roth chromosomes.Molecular marker analysis of expressed sequence tag(EST) and PCR based landmark unique gene(PLUG) indicated that the additions belonged to the 7 M chromosome of Ae.geniculata Roth. FISH analysis showed that there were 38 chromosomes in 1003 that were consistent with the standard karyotype of wheat Chinese Spring,while the FISH signals of 4 A,5 A and 7 M were altered. Identification of resistance to powdery mildew showed that 1003 was immune to the powdery mildew physiological race E09. Stripe rust screen indicated that 1003 was high resistant to CYR23 of stripe rust physiological races. Morphological survey showed that the agronomic traits of 1003 were between parents.Therefore,1003 is a wheat-Ae.geniculataRoth disomic addition line with powdery mildew and stripe rust resistance,which will provide new germplasm resources for wheat variety improvement and disease resistance breeding.
The Ae.geniculata Roth contains many excellent genes for wheat improvement and it is an important tertiary gene bank for wheat. Here,we used cytological,in situ hybridization(Genomic in situ hybridization,GISH and Fluorescence in situ hybridization,FISH) and molecular marker techniques to identify the progeny 1003 of wheat-Ae.geniculataRoth SY159. Cytological observation showed that 1003 contained 44 chromosomes,and the pollen mother cells possessed 22 bivalents with good pairing at metaphase I(PMCs MI),and the pollen mother cells held 44 chromosomes that were equally separated at anaphase I(PMCs AI). GISH analysis showed that 1003 contained 42 wheat chromosomes and 2 Ae.geniculata Roth chromosomes.Molecular marker analysis of expressed sequence tag(EST) and PCR based landmark unique gene(PLUG) indicated that the additions belonged to the 7 M chromosome of Ae.geniculata Roth. FISH analysis showed that there were 38 chromosomes in 1003 that were consistent with the standard karyotype of wheat Chinese Spring,while the FISH signals of 4 A,5 A and 7 M were altered. Identification of resistance to powdery mildew showed that 1003 was immune to the powdery mildew physiological race E09. Stripe rust screen indicated that 1003 was high resistant to CYR23 of stripe rust physiological races. Morphological survey showed that the agronomic traits of 1003 were between parents.Therefore,1003 is a wheat-Ae.geniculataRoth disomic addition line with powdery mildew and stripe rust resistance,which will provide new germplasm resources for wheat variety improvement and disease resistance breeding.
引文
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[16]ISHIKAWAG,NAKAMURA T,ASHIDAT,et al.Localization of anchor loci representing five hundred annotated rice genes to wheat chromosomes using PLUG markers [J].Theoretical and Applied Genetics,2009,118:499.
[17]盛宝钦.用反应型记载小麦苗期白粉病 [J].植物保护,1988,14(1):49.SHENG B Q.Score wheat seedlings resistance to powdery mildew by infection type [J].Plant Protection,1988,14(1):49.
[18] 韩德俊,王琪琳,张立,等.“西北-华北-长江中下游”条锈病流行区系当前小麦品种(系)抗条锈病性评价 [J].中国农业科学,2010,43(14):2889.HAN D J,WANG Q L,ZHANG L,et al.Evaluation of resistance of current wheat cultivars to stripe rust in northwest China,north China and the middle and lower reaches of Changjiang river epidemic area [J].Scientia Agricultura Sinica,2010,43(14):2889.
[19] 李北,徐琪,杨宇衡,等.重庆麦区小麦品种(系)抗条锈性评价与基因分析 [J].中国农业科学,2017,50(3):413.LI B,XU Q,YANG Y H,et al.Stripe rust resistance and genes in Chongqing wheat cultivars and lines [J].Scientia Agricultura Sinica,2017,50(3):413.
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[21]李立会,李秀全,杨欣明.小麦野生近缘植物种质资源描述规范和数据标准 [M].北京:中国农业出版社,2006:11-20.LI L H,LI X Q,YANG X M.Descriptors and data standard for wild relatives of wheat [M].Beijing:China Agriculture Press,2006:11-20.
[22]BADAEVA E D,DEDKOVA O S,GAY G,et al.Chromosomal rearrangements in wheat:their types and distribution [J].Genome,2007,50(10):907-26.
[23]HUANG X Y,ZHU M Q,ZHUANG L F,et al.Structural chromosome rearrangements and polymorphisms identified in Chinese wheat cultivars by high resolution multiplexoligonucleotide FISH [J].Theoretical and Applied Genetics,2018,131:1967.
[24] 张志明,汤才国,杨三维,等.小麦(Triticumaestivum L.)野生资源的发掘、利用研究进展 [J].生物技术进展,2016,6(5):305.ZHANG Z M,TANG C G,YANG S W,et al.Advances on gene discovery and utilization of wild relatives of Triticum aestivum L [J].Current Biotechnology,2016,6(5):305.
[25]CIFUENTESA M,GARCIA-AGüEROB V,BENAVENTE E,et al.A comparative analysis of chromosome pairingat metaphase I in interspecific hybrids between durum wheat(Triticum turgidum L.) and the most widespread Aegilops Species [J].Cytogenetic and Genome Research,2010,129 (1-3):124.
[26]王玉海,何方,鲍印广,等.高抗白粉病小麦-山羊草新种质 TA002 的创制和遗传研究 [J].中国农业科学,2016,49(3):418.WANG Y H,HE F,BAO Y G,et al.Development and genetic analysis of a novel wheat-Aegilops germplasm TA002 resistant to powdery mildew [J].Scientia Agricultura Sinica,2016,49(3):418.
[2]WULFF B B H,DHUGGA K S.Wheat—the cereal abandoned by GM [J].Science,2018,361(6401):451.
[3]张爱民,阳文龙,李欣,等.小麦抗赤霉病研究现状与展望 [J].遗传,2018,40(10):858.ZHANG A M,YANG W L,LI X,et al.Current status and perspective on research against Fusarium head blight in wheat [J].Hereditas,2018,40(10):858.
[4]ISTVANMOLNA R,HANA S,MICHELLE L W,et al.Syntenic relationships between the U and M genomes of Aegilops,wheat and the model species Brachypodium and rice as revealed by COS markers [J].PLoS ONE,2013,8(8):e70844.
[5]WANG Y J,QUAN W,PENG N N,et al.Molecular cytogenetic identification of a wheat-Aegilops geniculata Roth 7M disomic addition line with powdery mildew resistance [J].Molecular Breeding,2016,36(40):1.
[6]KLYMIUKV,YANIV E,HUANGL,et al.Cloning of the wheat Yr15 resistance gene sheds light on the plant tandem kinase-pseudokinase family [J].Nature Communications,2018,9(1):3735.
[7]张羽,张先平,李小鹏,等.分子标记在小麦抗条锈病遗传育种中的应用研究进展 [J].分子植物育种,2018,16(18):6032.ZHANG Y,ZHANG X P,LI X P,et al.Research progress of the application of molecular marker in genetic breeding of resistance to stripe rust in wheat [J].Molecular Plant Breeding,2018,16(18):6032.
[8]韩德俊,康振生.中国小麦品种抗条锈病现状及存在问题与对策 [J].植物保护,2018,44(5):1.HAN D J,KANG Z S.Current status and future strategy in breeding wheat for resistance to stripe rust in China [J].Plant Protection,2018,44(5):1.
[9]杨立军,曾凡松,龚双军,等.68个主推小麦品种的白粉病抗性分析及基因推导 [J].中国农业科学,2013,46(16):3354.YANG L J,ZENG F S,GONG S J,et al.Evaluation of resistance to powdery mildew in 68 Chinese major wheat cultivars and postulation of their resistance genes [J].Scientia Agricultura Sinica,2013,46(16):3354.
[10]何中虎,兰彩霞,陈新民,等.小麦条锈病和白粉病成株抗性研究进展与展望 [J].中国农业科学,2011,44(11):2193.HE Z H,LAN C X,CHEN X M,et al.Progress and perspective in research of adult-plant resistance to stripe rust and powdery mildew in wheat [J].Scientia Agricultura Sinica,2011,44(11):2193.
[11]朱晨,王艳珍,陈春环,等.抗条锈病、耐盐小麦-十倍体长穗偃麦草5J(E)二体异附加系的分子细胞学鉴定 [J].农业生物技术学报,2017,25(5):689.ZHU C,WANG Y Z,CHEN C H,et al.Molecular cytogenetic identification of Triticumaestivum-Thinopyrumponticum 5J(E) disomic addition line with stripe rust resistance andsalt tolerance [J].Journal of Agricultural Biotechnology,2017,25(5):689.
[12]HAN F P,LAMB J C,BIRCHLER J A.High frequency of centromere inactivation resulting in stable dicentric chromosomes of maize [J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(9):3238.
[13] ALLENGC,FLORES-VERGARA M A,KRASNYANSKI S,et al.A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide [J].Nature Protocols,2006,1(5):2320.
[14]TANG Z X,YANG Z J,FU S L.Oligonucleotides replacing the roles of repetitive sequences pAs1,pSc119.2,pTa-535,pTa71,CCS1,and pAWRC.1 for FISH analysis [J].Journal of Applied Genetics,2014,55(3):313.
[15]TANG S Y,TANG Z X,QIU L,et al.Developing new oligo probes to distinguish specific chromosomal segments and the A,B,D genomes of wheat(Triticum aestivum L.) using ND-FISH [J].Frontier in Plant Science,2018,9:1104.
[16]ISHIKAWAG,NAKAMURA T,ASHIDAT,et al.Localization of anchor loci representing five hundred annotated rice genes to wheat chromosomes using PLUG markers [J].Theoretical and Applied Genetics,2009,118:499.
[17]盛宝钦.用反应型记载小麦苗期白粉病 [J].植物保护,1988,14(1):49.SHENG B Q.Score wheat seedlings resistance to powdery mildew by infection type [J].Plant Protection,1988,14(1):49.
[18] 韩德俊,王琪琳,张立,等.“西北-华北-长江中下游”条锈病流行区系当前小麦品种(系)抗条锈病性评价 [J].中国农业科学,2010,43(14):2889.HAN D J,WANG Q L,ZHANG L,et al.Evaluation of resistance of current wheat cultivars to stripe rust in northwest China,north China and the middle and lower reaches of Changjiang river epidemic area [J].Scientia Agricultura Sinica,2010,43(14):2889.
[19] 李北,徐琪,杨宇衡,等.重庆麦区小麦品种(系)抗条锈性评价与基因分析 [J].中国农业科学,2017,50(3):413.LI B,XU Q,YANG Y H,et al.Stripe rust resistance and genes in Chongqing wheat cultivars and lines [J].Scientia Agricultura Sinica,2017,50(3):413.
[20]李振岐,曾士迈.中国小麦锈病 [M].北京:中国农业出版社,2002:370-373.LI Z Q,ZENG S M.Wheat rust in China [M].Beijing:China Agriculture Press,2002:370-373.
[21]李立会,李秀全,杨欣明.小麦野生近缘植物种质资源描述规范和数据标准 [M].北京:中国农业出版社,2006:11-20.LI L H,LI X Q,YANG X M.Descriptors and data standard for wild relatives of wheat [M].Beijing:China Agriculture Press,2006:11-20.
[22]BADAEVA E D,DEDKOVA O S,GAY G,et al.Chromosomal rearrangements in wheat:their types and distribution [J].Genome,2007,50(10):907-26.
[23]HUANG X Y,ZHU M Q,ZHUANG L F,et al.Structural chromosome rearrangements and polymorphisms identified in Chinese wheat cultivars by high resolution multiplexoligonucleotide FISH [J].Theoretical and Applied Genetics,2018,131:1967.
[24] 张志明,汤才国,杨三维,等.小麦(Triticumaestivum L.)野生资源的发掘、利用研究进展 [J].生物技术进展,2016,6(5):305.ZHANG Z M,TANG C G,YANG S W,et al.Advances on gene discovery and utilization of wild relatives of Triticum aestivum L [J].Current Biotechnology,2016,6(5):305.
[25]CIFUENTESA M,GARCIA-AGüEROB V,BENAVENTE E,et al.A comparative analysis of chromosome pairingat metaphase I in interspecific hybrids between durum wheat(Triticum turgidum L.) and the most widespread Aegilops Species [J].Cytogenetic and Genome Research,2010,129 (1-3):124.
[26]王玉海,何方,鲍印广,等.高抗白粉病小麦-山羊草新种质 TA002 的创制和遗传研究 [J].中国农业科学,2016,49(3):418.WANG Y H,HE F,BAO Y G,et al.Development and genetic analysis of a novel wheat-Aegilops germplasm TA002 resistant to powdery mildew [J].Scientia Agricultura Sinica,2016,49(3):418.