拟斯卑尔脱山羊草的FISH核型分析

doi:10.3864/j.issn.0578-1752.2017.08.003

摘要/Abstract

摘要： 【目的】建立拟斯卑尔脱山羊草的FISH核型，分析明确不同来源拟斯卑尔脱山羊草的FISH核型特点，比较不同拟斯卑尔脱山羊草及其与普通小麦的FISH核型差异。【方法】以荧光标记的寡核苷酸Oligo-pTa535和Oligo-pSc119.2为探针，利用荧光原位杂交（fluorescence in situ hybridization，FISH）技术分析pTa535和pSc119.2在不同拟斯卑尔脱山羊草、四倍体小麦和普通小麦染色体上的杂交信号分布特点；以禾本科植物着丝粒专化寡核苷酸Oligo-CCS1为探针，明确拟斯卑尔脱山羊草的着丝粒位置，测量拟斯卑尔脱山羊草染色体相关参数；通过FISH核型比较明确不同拟斯卑尔脱山羊草及其与小麦核型的多态性差异。【结果】Oligo-pTa535主要分布在小麦的D和A组染色体上，在小麦的B组染色体上仅有零星分布，在5份拟斯卑尔脱山羊草的染色体中未显示Oligo-pTa535杂交信号。Oligo-pSc119.2杂交信号主要分布在小麦的B组染色体上，在小麦的A、D组染色体中分布较少，但在5份拟斯卑尔脱山羊草染色体上均有广泛分布。根据Oligo-pTa535和Oligo-pSc119.2杂交信号在小麦染色体上的分布特点，可以将小麦的不同染色体相互区分开来。Oligo-pSc119.2杂交信号在不同倍性、不同品种的小麦B组染色体上的分布特点基本相似，而不同来源拟斯卑尔脱山羊草的Oligo-pSc119.2的FISH核型差异较大，甚至在同一细胞内的2条同源染色体上Oligo-pSc119.2杂交信号的分布也具有明显差异。不同来源的拟斯卑尔脱山羊草与小麦B染色体组的FISH核型存在明显差异。PI542238的7对染色体均为中间着丝粒染色体，核型公式为2n=14=14m。其余4份拟斯卑尔脱山羊草的4S染色体均为近中着丝粒染色体，其余染色体均为中间着丝粒染色体，核型公式皆为2n=14=12m+2sm。【结论】拟斯卑尔脱山羊草染色体上含有丰富的与pSc119.2高度同源的重复序列，不含有与pTa535高度同源的重复序列。不同来源的拟斯卑尔脱山羊草之间以及同一来源的拟斯卑尔脱山羊草的个体间甚至同一个体内的同源染色体间在pSc119.2的分布上均具有遗传多样性。以Oligo-pSc119.2为探针建立的拟斯卑尔脱山羊草染色体FISH核型与小麦B组染色体的核型具有显著差异。利用荧光标记的Oligo-pTa535和Oligo-pSc119.2为探针进行FISH分析，可以准确区分拟斯卑尔脱山羊草的不同染色体，并能将拟斯卑尔脱山羊草与小麦的染色体区分开来。

关键词: 普通小麦, 拟斯卑尔脱山羊草, 核型分析, FISH, 寡核苷酸

Abstract: 【Objective】The objective of this study is to reveal the karyotypic polymorphism of Aegilops speltoides (Aegilops short for Ae. hereafter) and the karyotypic difference between common wheat and Ae. speltoides via the establishment of FISH karyotype of Ae. speltoides.【Method】Multicolor fluorescence in situ hybridization (mc-FISH) was employed to detect the distribution of Oligo-pSc119.2 and Oligo-pTa535 in chromosomes of Ae. speltoides; centromere-specific oligonucleotide CCS1 was used to identify the location of centromeres on chromosomes; FISH karyotype comparison was conducted to show the karyotypic differences between Ae. speltoides and wheat.【Result】In wheat, oligo-pTa535 signals were observed mainly on chromosomes in A and D genomes, and only very sporadic signals were found in B genome. However, oligo-pTa535 signals were absent in Ae. speltoides of five accessions. Oligo-pSc119.2, compared to a little distribution in A and D genomes, shined plentiful fluorescence in the whole genome B in wheat, and especially in S genomes in Ae. speltoides of five accessions used in this study. Different pairs of chromosomes in wheat could be distinguished from each other according to the distribution of Oligo-pSc119.2 and Oligo-pTa535 on chromosomes of wheat. FISH patterns produced by Oligo-pSc119.2 in wheat showed similarity among wheat materials whether of different ploidy or of different varieties of same ploidy, and that in Ae. speltoides varied depending on accessions. Even homologous chromosomes in one cell in Ae. speltoides exhibited differences in FISH pattern. Oligo-pSc119.2 FISH patterns of five accessions each showed obvious differences from that of B genomes in wheat. Four of five Ae. speltoides accessions possess six pairs of metacentric chromosomes except for homologous pairs 4S of submetacentric chromosomes, which were involved in the karyotype formula 2n = 14 = 12m + 2sm, and the rest one, PI542238, however, houses seven pairs of metacentric chromosomes which resulted in the karyotype formula 2n = 14 = 14m. 【Conclusion】 Chromosomes of Ae. speltoides house rich repetitive DNA sequences highly homologous to pSc119.2 and lack that highly homologous to pTa53. The distribution of pSc119.2 on chromosomes of Ae. speltoides showed differences between accessions, between plants of one accession and even between homologous chromosomes in one plant. FISH patterns produced by Oligo-pSc119.2 on Ae. speltoides chromosomes exhibited significant difference from that on chromosomes of B genomes in wheat. FISH analysis, using Oligo-pSc119.2 and Oligo-pTa535 as probes, not only could differentiate the chromosomes in wheat from that in Ae. speltoides, but also could dishtinguish the chromosomes from each other whether in wheat or in Ae. speltoides.

Key words: Triticum aestivum, Aegilops speltoides, karyotypic analysis, FISH, oligonucleotides

董磊，董晴，张文利，胡晓龙，王洪刚，王玉海. 拟斯卑尔脱山羊草的FISH核型分析[J]. 中国农业科学, 2017, 50(8): 1378-1387.

DONG Lei, DONG Qing, ZHANG WenLi, HU XiaoLong, WANG HongGang, WANG YuHai. Karyotypic Analysis of Aegilops speltoides Revealed by FISH[J]. Scientia Agricultura Sinica, 2017, 50(8): 1378-1387.

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参考文献

[1] 周阳, 何中虎, 张改生, 夏兰琴, 陈新民, 高永超, 井赵斌, 于广军. 1BL/1RS易位系在我国小麦育种中的应用. 作物学报, 2004, 30(6): 531-535.

ZHOU Y, HE Z H, ZHANG G S, XIA L Q, CHEN X M, GAO Y C, JING Z B, YU G J. Utilization of 1BL/1RS translocation in wheat breeding in China. Acta Agrinomica Sinica, 2004, 30(6): 531-535. (in Chinese)

[2] 张丽, 张怀渝, 任正隆, 罗培高. 小麦-黑麦1BL/1RS易位系在小麦遗传改良中的应用现状及前景分析. 分子植物育种, 2010, 8(14): 1-8.

ZHANG L, ZHANG H Y, REN Z L, LUO P G. The progress and prospect of 1BL/RS translocation line in wheat genetic improvement. Molecular Plant Breeding, 2010, 8(14): 1-8. (in Chinese)

[3] 钟冠昌, 穆素梅, 张正斌. 麦类远缘杂交. 北京: 科学出版社, 2002: 92-97.

ZHONG G C, MU S M, ZHANG Z B. Wide hybridization in Triticeae. Beijing: China Science Press, 2002: 92-97. (in Chinese)

[4] MAESTRA B, NARANJO T. Homoeologous relationships of Aegilops speltoides chromosomes to bread wheat. Theoretical and Applied Genetics, 1998, 97: 181-186.

[5] SALSE J, CHAGUÉ V, BOLOT S, MAGDELENAT G, HUNEAU C, PONT C, BELCRAM H, COULOUX A, GARDAIS S, EVRARD A, SEGURENS B, CHARLES M, RAVEL C, SAMAIN S, CHARMET G, BOUDET N, CHALHOUB B. New insights into the origin of the B genome of hexaploid wheat: Evolutionary relationships at the SPA genomic region with the S genome of the diploid relative Aegilops speltoides. BMC Genomics, 2008, 9: 555.

[6] PETERSEN G, SEBERG O, YDE M, BERTHELSEN K. Phylogenetic relationships of Triticum and Aegilops and evidence for the origin of the A, B, and D genomes of common wheat (Triticum aestivum). Molecular Phylogenetics and Evolution, 2006, 39(1): 70-82.

[7] MAGO R, VERLIN D, ZHANG P, BANSAL U, BARIANA H, JIN Y, ELLIS J, HOXHA S, DUNDAS I. Development of wheat-Aegilops speltoides recombinants and simple PCR-based markers for Sr32 and a new stem rust resistance gene on the 2S#1 chromosome. Theoretical and Applied Genetics, 2013, 126: 2943-2955.

[8] JIA J, DEVOS K M, CHAO S, MILLER T E, READER S M, GALE M D. RFLP-based maps of the homoeologous group-6 chromosomes of wheat and application in the tagging of Pm12, a powdery mildew resistance gene transferred from Aegilops speltoides to wheat. Theoretical and Applied Genetics, 1996, 92: 559-565.

[9] NAIK S, GILL K S, PRAKASA RAO V S, GUPTA V S, TAMHANKAR S A, PUJAR S, GILL B S, RANJEKAR P K. Identification of a STS marker linked to the Aegilops speltoides- derived leaf rust resistance gene Lr28 in wheat. Theoretical and Applied Genetics, 1998, 97: 535-540.

[10] PETERSEN S, LYERLY J H, WORTHINGTON M L, PARKS W R, COWGER C, MARSHALL D S, BROWN-GUEDIRA G, MURPHY J P. Mapping of powdery mildew resistance gene Pm53 introgressed from Aegilops speltoides into soft red winter wheat. Theoretical and Applied Genetics, 2015, 128: 303-312.

[11] YU G T, KLINDWORTH D L, FRIESEN T L, FARIS J D, ZHONG S B, RASMUSSEN J B, XU S S. Development of a diagnostic co-dominant marker for stem rust resistance gene Sr47 introgressed from Aegilops speltoides into durum wheat. Theoretical and Applied Genetics, 2015, 128: 2367-2374.

[12] DUBCOVSKY J, LUKASZEWSKI A J, ECHAIDE M, ANTONELLI E F, PORTER D R. Molecular characterization of two Triticum speltoides interstitial translocations carrying leaf rust and greenbug resistance genes. Crop Science, 1998, 38: 1655-1660.

[13] NOORI S A S. Assessment for salinity tolerance through intergeneric hybridisation: Triticum durum × Aegilops speltoides. Euphytica, 2005, 146: 149-155.

[14] YUDINA R S, LEONOVA I N, SALINA E A, KHLESTKINA E K. Change in salt tolerance of bread wheat as a result of the introgression of the genetic material of Aegilops speltoides and Triticum timopheevii. Russian Journal of Genetics: Applied Research, 2016, 6(3): 244-248.

[15] PSHENICHNIKOVA T A, SIMONOV A V, ERMAKOVA M F, CHISTYAKOVA A K, SHCHUKINA L V, MOROZOVA E V. The effects on grain endosperm structure of an introgression from Aegilops speltoides Tausch. into chromosome 5A of bread wheat. Euphytica, 2010, 175: 315-322.

[16] AWLACHEW Z T, SINGH R, KAUR S, BAINS N S, CHHUNEJA P. Transfer and mapping of the heat tolerance component traits of Aegilops speltoides in tetraploid wheat Triticum durum. Molecular Breeding, 2016, 36: 78.

[17] FRIEBE B, BADAEVA E D, KAMMER K, GILL B S. Standard karyotypes of Aegilops uniaristata, Ae. mutica, Ae. comosa subspecies comosa and heldreichii (Poaceae). P1ant Systematics Evolution, 1996, 202: 199-210.

[18] FERNÁNDEZ-CALVIN B, ORELLANA J. Metaphase-I bound-arm frequency and genome analysis in wheat-Aegilops hybrids. 2. Cytogenetical evidence for excluding Ae. sharonensis as the donor of the B genome of polyploid wheats. Theoretical and Applied Genetics, 1993, 85(5): 587-592.

[19] FRIEBE B, QI L L, NASUDA S, ZHANG P, TULEEN N A, GILL B S. Development of a complete set of Triticum aestivum-Aegilops speltoides chromosome addition lines. Theoretical and Applied Genetics, 2000, 101: 51-58.

[20] TEOH S B, MILLER T E, READER S M. Intraspecific variation in C-banded chromosomes of Aegilops comosa and Ae. Speltoides. Theoretical and Applied Genetics, 1983, 65: 343-348.

[21] FRIEBE B, SCHUBERT V, BLIITHNER W D, HAMMER K. C-banding pattern and polymorphism of Aegilops caudata and chromosomal constitutions of the amphiploid T. aestivum-Ae. caudata and six derived chromosome addition lines. Theoretical and Applied Genetics, 1992, 83: 589-596.

[22] FRIEBE B, JIANG J, TULEEN N, GILL B S. Standard karyotype of Triticum umbellulatum and the characterization of derived chromosome addition and translocation lines in common wheat. Theoretical and Applied Genetics, 1995, 90: 150-156.

[23] FRIEBE B, TULEEN N A, GILL B S. Standard karyotype of Triticum searsii and its relationship with other S-genome species and common wheat. Theoretical and Applied Genetics, 1995, 91: 248-254.

[24] BADAEVA E D, DEDKOVA O S, ZOSHCHUK S A, AMOSOVA A V, READER S M, BERNARD M, ZELENIN A V. Comparative analysis of the N-genome in diploid and polyploid Aegilops species. Chromosome Research, 2011, 19: 541-548.

[25] MIRZAGHADERI G, HOUBEN A, BADAEVA E D. Molecular- cytogenetic analysis of Aegilops triuncialis and dentification of its chromosomes in the background of wheat. Molecular Cytogenetics, 2014, 7: 91.

[26] 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. Journal of Applied Genetics, 2014, 55: 313-318.

[27] FERNFINDEZ-CALVFN B, ORELLANA J. Metaphase I-bound arms frequency and genome analysis in wheat-Aegilops hybrids. 3. Similar relationships between the B genome of wheat and S or S^Lgenomes of Ae. speltoides, Ae. longissima and Ae. sharonensis. Theoretical and Applied Genetics, 1994, 88: 1043-1049.

[28] 王玉海, 何方, 鲍印广, 明东风, 董磊, 韩庆典, 李莹莹, 王洪刚. 高抗白粉病小麦-山羊草新种质TA002的创制和遗传研究. 中国农业科学, 2016, 49(3): 418-428.

WANG Y H, HE F, BAO Y G, MING D F, DONG L, HAN Q D, LI Y Y, WANG H G. Development and genetic analysis of a novel wheat-Aegilops germplasm TA002 resistant to powdery mildew. Scientia Agricultura Sinica, 2016, 49(3): 418-428. (in Chinese)

[29] WANG Y H, WANG H G. Characterization of three novel wheat-Thinopyrum intermedium addition lines with novel storage protein subunits and resistance to both powdery mildew and stripe rust. Journal of Genetics and Genomics, 2016, 43: 45-48.

[30] KRUPPA K, TÜRKÖSI E, MAYER M, TÓTH V, VIDA G, SZAKÁCS É, MOLNÁR-LÁNG M. McGISH identification and phenotypic description of leaf rust and yellow rust resistant partial amphiploids originating from a wheat × Thinopyrum synthetic hybrid cross. Journal of Applied Genetics, 2016, 57: 427-437.

[31] LI G R, WANG H J, LANG T, LI J B, LA S X, YANG E N, YANG Z J. New molecular markers and cytogenetic probes enable chromosome identification of wheat-Thinopyrum intermedium introgression lines for improving protein and gluten contents. Planta, 2016, 244: 865-876.

[32] 陈雷, 李萌, 王洋洋, 邱玲, 汤述尧, 唐宗祥, 符书兰. 小麦-黑麦1BL/1RS 易位系中的染色体结构变异. 麦类作物学报, 2015, 35(8): 1038-1043.

CHEN L, LI M, WANG Y Y, QIU L, TANG S Y, TANG Z X, FU S L. Structural variation of chromosomes in wheat-rye 1BL/1RS translocation lines. Journal of Triticeae Crops, 2015, 35(8): 1038-1043. (in Chinese)

[33] SCHNEIDER A, RAKSZEGI M, MOLNÁR-LÁNG M, SZAKÁCS É. Production and cytomolecular identification of new wheat-perennial rye (Secale cereanum) disomic addition lines with yellow rust resistance (6R) and increased arabinoxylan and protein content (1R, 4R, 6R). Theoretical and Applied Genetics, 2016, 129: 1045-1059.

[34] ZHUANG L F, LIU P,LIU Z Q, CHEN T T, WU N, SUN L, QI Z J. Multiple structural aberrations and physical mapping of rye chromosome 2R introgressed into wheat. Molecular Breeding, 2015, 35: 133.

[35] FU S L, REN Z L, CHEN X M, YAN B J, TAN F Q, FU T H, TANG Z X. New wheat-rye 5DS-4RS·4RL and 4RS-5DS·5DL translocation lines with powdery mildew resistance. Journal of Plant Research, 2014, 127: 743-753.

[36] DELGADO A, CARVALHO A, MARTÍN A C, MARTÍN A, LIMA-BRITO J. Use of the synthetic Oligo-pTa535 and Oligo-pAs1 probes for identification of Hordeum chilense-origin chromosomes in hexaploid Tritordeum. Genetic Resources and Crop Evolution, 2016, 63: 945-951.

[37] CUADRADO A, JOUVE N. Evolutionary trends of different repetitive DNA sequences during speciation in the genus secale. The Journal of Heredity, 2002, 93(5): 339-345.

[38] KATO A, JONATHAN C L, BIRCHLER J A. Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize. Proceedings of the National Academy of Sciences of the USA, 2004, 101(37): 13554-13559.

[39] MOLNÁR I, KUBALÁKOVÁ M, ŠIMKOVÁ H, FARKAS A, CSEH A, MEGYERI M, VRÁNA J, MOLNÁR-LÁNG M, DOLE?EL J. Flow cytometric chromosome sorting from diploid progenitors of bread wheat, T. urartu, Ae. speltoides and Ae. Tauschii. Theoretical and Applied Genetics, 2014, 127: 1091-1104.

[40] BADAEVA E D, AMOSOVA A V, MURAVENKO O V, SAMATADZE T E, CHIKIDA N N, ZELENIN A V, FRIEBE B, GILL B S. Genome differentiation in Aegilops. 3. Evolution of the D-genome cluster. Plant Systematics and Evolution, 2002, 231: 163-190.

[41] BADAEVA E D, AMOSOVA A V, SAMATADZE T E, ZOSHCHUK S A, SOSHTAK N G, CHIKIDA N N, ZELENIN A V, RAUPP W J, FRIEBE B, GILL B S. Genome differentiation in Aegilops. 4. Evolution of the U-genome cluster. Plant Systematics Evolution, 2004, 246: 45-76.

[42] LINC G, SEPSI A, MOLNÁR-LÁNG M. A FISH karyotype to study chromosome polymorphisms for the Elytrigia elongata E genome. Cytogenetic and Genome Research, 2012, 136: 138-144.

[43] 颜济, 杨俊良. 小麦族生物系统学（第一卷第二版）, 小麦-山羊草复合群. 北京: 中国农业出版社, 2013: 43-53.

YAN J, YANG J L. Triticeae Systematics (volume 1 2^nd edition), Triticum- Aegilops complx. Beijing: China Agriculture press, 2013: 43-53. (in Chinese)

[44] 董玉琛, 郑殿升. 中国小麦遗传资源. 北京: 中国农业出版社,2000: 152.

DONG Y C, ZHENG D S. The wheat genetic resource in China. Beijing: China Agriculture Press, 2000: 152. (in Chinese)