抗白粉病和条锈病的小麦-十倍体长穗偃麦草5E (5D)二体异代换系的细胞遗传学鉴定及分子标记开发

doi:10.1016/j.jia.2024.04.012

摘要/Abstract

摘要： 十倍体长穗偃麦草（2n=10x=70）是普通小麦的野生近缘种之一，因其含有大量抗生物、非生物胁迫的基因，被认为是改良小麦的宝贵遗传资源。本研究以普通小麦7182和十倍体长穗偃麦草杂交的BC₁F₇衍生系CH97为研究对象。细胞学证据显示，CH97有42条染色体，在减数第一次分裂中期形成21个二价体，随后在减数第一次分裂后期二价体分离并移向两极。通过FISH（荧光原位杂交）、GISH（基因组原位杂交）、mc-GISH（多色基因组原位杂交）和液相芯片综合分析明确了CH97由40条小麦染色体和2条十倍体长穗偃麦草E^e基因组染色体组成，并且缺失了一对小麦的5D染色体，以及1B、6B和7B染色体发生变异。这些结果证实CH97是一个稳定的小麦-十倍体长穗偃麦草5E(5D)二体异代换系。接种试验表明，CH97在整个生育期对小麦白粉病和条锈病表现出高抗，而普通小麦亲本7182呈现高感。与7182相比，CH97的穗粒数、千粒重和粒长均有增加。此外，基于简化基因组测序技术，开发并验证了5E染色体的特异分子标记，开发效率达33.3%，这有助于通过标记辅助选择提高小麦的抗病育种效率。总之，CH97代换系具有的抗病性和改良的农艺性状，为小麦染色体工程育种提供了宝贵的新种质。

Abstract: Thinopyrum ponticum (2n=10x=70), a wild relative of common wheat (Triticum aestivum L.), is considered an invaluable genetic resource for wheat improvement due to its abundance of genes that confer resistance to biotic and abiotic stresses. This study focused on the CH97 line, derived from the BC₁F₇progeny of a cross between wheat cv. 7182 and Th. ponticum. Cytological evidence showed that CH97 has 42 chromosomes, forming 21 bivalents at meiotic metaphase I, with the bivalents subsequently separating and moving to opposite poles during meiotic anaphase I. Through a combination of FISH (fluorescence in situ hybridization), GISH (genomic in situ hybridization), mc-GISH (multicolor genomic in situ hybridization), and liquid array analysis, it was determined that CH97 comprises 40 wheat chromosomes and two alien chromosomes from the E^e genome of Th. ponticum, featuring the absence of a pair of 5D chromosomes and variations in 1B, 6B, and 7B chromosomes. These findings confirm that CH97 is a stable wheat-Th. ponticum 5E (5D) alien disomic substitution line. Inoculation experiments revealed that CH97 exhibits high resistance to wheat powdery mildew and stripe rust throughout the growth period, in contrast to the highly susceptible common wheat parent 7182. Compared to 7182, CH97 displayed improvements in spikelets per spike, thousand-kernel weight, and kernel length. Additionally, utilizing SLAF-seq technology, chromosome 5E-specific molecular markers were developed and validated, achieving a 33.3% success rate, which facilitates marker-assisted selection to enhance disease resistance in wheat. Overall, the CH97 substitution line, with its resistance to diseases and improved agronomic traits represents valuable new germplasm for wheat chromosome engineering and breeding.

Key words: Thinopyrum ponticum , disomic substitution line , powdery mildew , stripe rust SLAF-seq , liquid array

. 抗白粉病和条锈病的小麦-十倍体长穗偃麦草5E (5D)二体异代换系的细胞遗传学鉴定及分子标记开发[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.04.012.

Xiaofang Cheng, Yi Xiao, Luhui Wang, Xiaoying Yang, Pingchuan Deng, Jixin Zhao, Changyou Wang, Chunhuan Chen, Tingdong Li, Wanquan Ji. Cytogenetic characterization and molecular marker development of a novel wheat-Thinopyrum ponticum 5E (5D) disomic substitution line with resistance to powdery mildew and stripe rust [J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.04.012.

参考文献

Allen G C, Flores-Vergara M A, Krasnyanski S, Kumar S, Thompson W F. 2006. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols, 1, 2320-2325.

Bashir T, Mishra R C, Hasan M M, Mohanta T K, Bae H. 2018. Effect of hybridization on somatic mutations and genomic rearrangements in plants. International Journal of Molecular Sciences, 19, 3758.

Blanco A, Gadaleta A, Cenci A, Carluccio A V, Abdelbacki A M, Simeone R. 2008. Molecular mapping of the novel powdery mildew resistance gene Pm36 introgressed from Triticum turgidum var. dicoccoides in durum wheat. Theoretical and Applied Genetics, 117, 135-142.

Chen P D, Qi L L, Zhou B, Zhang S Z, Liu D J. 1995. Development and molecular cytogenetic analysis of wheat-haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew. Theoretical and Applied Genetics, 91, 1125-1128.

Chen S Q, Huang Z F, Dai Y, Qin S W, Gao Y Y, Zhang L L, Gao Y, Chen J M. 2013. The development of 7E chromosome-specific molecular markers for Thinopyrum elongatum based on SLAF-seq technology. PLoS ONE, 8, e65122.

Conner R L, Kuzyk A D, Su H. 2003. Impact of powdery mildew on the yield of soft white spring wheat cultivars. Canadian Journal of Plant Science, 83, 725-728.

Deng P C, Du X, Wang Y Z, Yang X Y, Cheng X F, Huang C X, Li T T, Li T T, Chen C H, Zhao J X, Wang C Y, Liu X L, Tian Z R, Ji W Q. 2024. GenoBaits^®WheatplusEE: A targeted capture sequencing panel for quick and accurate identification of wheat-Thinopyrum derivatives. Theoretical and Applied Genetics, 137, 36.

Du X, Feng X B, Li R X, Jin Y L, Shang L H, Zhao J X, Wang C Y, Li T D, Chen C H, Tian Z R, Deng P C, Ji W Q. 2022. Cytogenetic identification and molecular marker development of a novel wheat-Leymus mollis 4Ns (4D) alien disomic substitution line with resistance to stripe rust and Fusarium head blight. Frontiers in Plant Science, 13, 1012939.

Dvořák J, Chen K C. 1984. Phylogenetic relationships between chromosomes of wheat and chromosome 2E of Elytrigia elongata. Canadian Journal of Genetics and Cytology, 26, 128-132.

Everts K L, Leath S. 1992. Effect of early season powdery mildew on development, survival, and yield contribution of tillers of winter wheat. Phytopathology, 82, 1273.

Feng X B, Du X, Wang S W, Deng P C, Wang Y F, Shang L H, Tian Z R, Wang C Y, Chen C H, Zhao J X, Ji W Q. 2022. Identification and DNA marker development for a wheat-Leymus mollis 2Ns (2D) disomic chromosome substitution. International Journal of Molecular Sciences, 23, 2676.

Fouche S, Plissonneau C, McDonald B A, Croll D. 2018. Meiosis leads to pervasive copy-number variation and distorted inheritance of accessory chromosomes of the wheat pathogen Zymoseptoria tritici. Genome Biology and Evolution, 10, 1416-1429.

Fu S L, Lv Z L, Qi B, Guo X, Li J, Liu B, Han F P. 2012. Molecular cytogenetic characterization of wheat-Thinopyrum elongatum addition, substitution and translocation lines with a novel source of resistance to wheat Fusarium Head Blight. Journal of Genetics and Genomics, 39, 103-110.

Gong B R, Zhang H, Yang Y L, Zhang J W, Zhu W, Xu L L, Wang Y, Zeng J, Fan X, Sha L N, Zhang H Q, Wu D D, Chen G Y, Zhou Y H, Kang H Y. 2022. Development and identification of a novel wheat-Thinopyrum scirpeum 4E (4D) chromosomal substitution line with stripe rust and powdery mildew resistance. Plant Disease, 106, 975-983.

Gong B R, Zhao L, Zeng C Y, Zhu W, Xu L L, Wu D D, Cheng Y R, Wang Y, Zeng J, Fan X, Sha LN, Zhang H Q, Chen G Y, Zhou Y H, Kang H Y. 2023. Development and characterization of a novel wheat-tetraploid Thinopyrum elongatum 6E (6D) disomic substitution line with stripe rust resistance at the adult stage. Plants-Basel, 12, 2311.

Guo Y W, Bai F T, Wang J T, Fu S Y, Zhang Y, Liu X Y, Zhang Z B, Shao J J, Li R, Wang F, Zhang L, Zheng H L, Wang X H, Liu Y B, Jiang Y. 2023. Design and characterization of a high-resolution multiple-SNP capture array by target sequencing for sheep. Journal of Animal Science, 101, skac383.

Guo Z F, Wang H W, Tao J J, Ren Y H, Xu C, Wu K S, Zou C, Zhang J N, Xu Y B. 2019. Development of multiple SNP marker panels affordable to breeders through genotyping by target sequencing (GBTS) in maize. Molecular Breeding, 39, 37.

Guo Z F, Yang Q, Huang F F, Zheng H J, Sang Z Q, Xu Y F, Zhang C, Wu K S, Tao J J, Prasanna B M, Olsen M S, Wang Y B, Zhang J A, Xu Y B. 2021. Development of high-resolution multiple-SNP arrays for genetic analyses and molecular breeding through genotyping by target sequencing and liquid chip. Plant Communications, 2, 100230.

Gyawali Y, Zhang W, Chao S M, Xu S, Cai X W. 2019. Delimitation of wheat ph1b deletion and development of ph1b-specific DNA markers. Theoretical and Applied Genetics, 132, 195-204.

Ha M, Lu J, Tian L, Ramachandran V, Kasschau K D, Chapman E J, Carrington J C, Chen X M, Wang X J, Chen Z J. 2009. Small RNAs serve as a genetic buffer against genomic shock in Arabidopsis interspecific hybrids and allopolyploids. Proceedings of the National Academy of Sciences of the United States of America, 106, 17835-17840.

Han F P, Lamb J C, Birchler J A. 2006. High frequency of centromere inactivation resulting in stable dicentric chromosomes of maize. Proceedings of the National Academy of Sciences of the United States of America, 103, 3238-3243.

He F, Bao Y G, Qi X L, Ma Y X, Li X F, Wang H G. 2017. Molecular cytogenetic identification of a wheat-Thinopyrum ponticum translocation line resistant to powdery mildew. Journal of Genetics, 96, 165-169.

Hou L Y, Jia J Q, Zhang X J, Li X, Yang Z J, Ma J, Guo H J, Zhan H X, Qiao L Y, Chang Z J. 2016. Molecular mapping of the stripe rust resistance gene Yr69 on wheat chromosome 2AS. Plant Disease, 100, 1717-1724.

Huang Z P, Liu J Q, Lu X Q, Guo Y F, Li Y Y, Liu Y Q, Zhang R Q, Xing L P, Cao A Z. 2023. Identification and transfer of a new Pm21 haplotype with high genetic diversity and a special molecular resistance mechanism. Theoretical and Applied Genetics, 136, 10.

Ishikawa G, Nakamura T, Ashida T, Saito M, Nasuda S, Endo T R, Wu J, Matsumoto T. 2009. Localization of anchor loci representing five hundred annotated rice genes to wheat chromosomes using PLUG markers. Theoretical and Applied Genetics, 118, 499-514.

Jia H W, Feng H, Yang G T, Li H W, Fu S L, Li B, Li Z S, Zheng Q. 2022. Establishment and identification of six wheat-Thinopyrum ponticum disomic addition lines derived from partial amphiploid Xiaoyan 7430. Theoretical and Applied Genetics, 135, 3277-3291.

Kruppa K, Turkosi E, Mayer M, Toth V, Vida G, Szakacs E, Molnar-Lang M. 2016. 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, 57, 427-437.

Li D Y, Li T H, Wu Y L, Zhang X H, Zhu W, Wang Y, Zeng J, Xu L L, Fan X, Sha L N, Zhang H Q, Zhou Y H, Kang H Y. 2018. FISH-based markers enable identification of chromosomes derived from tetraploid Thinopyrum elongatum in hybrid lines. Frontiers in Plant Science, 9, 526.

Li D Y, Zhang J W, Liu H J, Tan B W, Zhu W, Xu L L, Wang Y, Zeng J, Fan X, Sha L N, Zhang H Q, Ma J, Chen G Y, Zhou Y H, Kang H Y. 2019. Characterization of a wheat-tetraploid Thinopyrum elongatum 1E (1D) substitution line K17-841-1 by cytological and phenotypic analysis and developed molecular markers. BMC Genomics, 20, 963.

Li H. 2012. Exploring single-sample SNP and INDEL calling with whole-genome de novo assembly. Bioinformatics, 28, 1838-1844.

Li J C, Li J J, Zhao L, Zhao J X, Wu J, Chen X H, Zhang L Y, Dong P H, Wang L M, Zhao D H, Wang C P, Pang Y H. 2023. Rapid identification of Psathyrostachys huashanica Keng chromosomes in wheat background based on ND-FISH and SNP array methods. Journal of Integrative Agriculture, 22, 2934-2948.

Li J J, Zhao L, Lü B Y, Fu Y, Zhang S F, Liu S H, Yang Q H, Wu J, Li J C, Chen X H. 2023. Development and characterization of a novel common wheat-Mexico Rye T1DL·1RS translocation line with stripe rust and powdery mildew resistance. Journal of Integrative Agriculture, 22, 1291-1307.

Li M Z, Wang Y Z, Liu X J, Li X F, Wang H G, Bao Y G. 2021. Molecular cytogenetic identification of a novel wheat-Thinopyrum ponticum 1J^S (1B) substitution line resistant to powdery mildew and leaf rust. Frontiers in Plant Science, 12, 727734.

Li M Z, Yuan Y Y, Ni F, Li X F, Wang H G, Bao Y G. 2022. Characterization of two wheat-Thinopyrum ponticum introgression lines with pyramiding resistance to powdery mildew. Frontiers in Plant Science, 13, 943669.

Linc G, Sepsi A, Molnar-Lang M. 2012. A FISH karyotype to study chromosome polymorphisms for the Elytrigia elongata E genome. Cytogenetic and Genome Research, 136, 138-144.

Liu B, Liu T G, Zhang Z Y, Jia Q Z, Wang B T, Gao L, Peng Y L, Jin S L, Chen W Q. 2017. Discovery and pathogenicity of CYR34, a new race of Puccinia striiformis f sp. Tritici in China. Acta Phytopathologica Sin, 47, 681-687.

Liu L Q, Luo Q L, Li H W, Li B, Li Z S, Zheng Q. 2018. Physical mapping of the blue-grained gene from Thinopyrum ponticum chromosome 4Ag and development of blue-grain-related molecular markers and a FISH probe based on SLAF-seq technology. Theoretical and Applied Genetics, 131, 2359-2370.

Liu S B, Wang H G. 2005. Characterization of a wheat-Thinopyrom intermedium substitution line with resistance to powdery mildew. Euphytica, 143, 229-233.

Lopes M S, El-Basyoni I, Baenziger P S, Singh S, Royo C, Ozbek K, Aktas H, Ozer E, Ozdemir F, Manickavelu A, Ban T, Vikram P. 2015. Exploiting genetic diversity from landraces in wheat breeding for adaptation to climate change. Journal of Experimental Botany, 66, 3477-3486.

Ma H, Singh R P, Mujeebkazi A. 1995. Suppression/expression of resistance to stripe rust in synthetic hexaploid wheat (Triticum-turgidum × T. tauschii). Euphytica, 83, 87-93.

Mago R, Miah H, Lawrence G J, Wellings C R, Spielmeyer W, Bariana H S, McIntosh R A, Pryor A J, Ellis J G. 2005. High-resolution mapping and mutation analysis separate the rust resistance genes Sr31, Lr26 and Yr9 on the short arm of rye chromosome 1. Theoretical and Applied Genetics, 112, 41-50.

Qu L J, Foote T N, Roberts M A, Money T A, Aragon-Alcaide L, Snape J W, Moore G. 1998. A simple PCR-based method for scoring the ph1b deletion in wheat. Theoretical and Applied Genetics, 96, 371-375.

Sheng B Q, Duan X Y. 1991. Improvement of scale 0-9 method for scoring adult plant resistance to powdery mildew of wheat. Beijing Agricultural Sciences, 1, 38-39.

Sun X W, Liu D Y, Zhang X F, Li W B, Liu H, Hong W G, Jiang C B, Guan N, Ma C X, Zeng H P, Xu C H, Song J, Huang L, Wang C M, Shi J J, Wang R, Zheng X H, Lu C Y, Wang X W, Zheng H K. 2013. SLAF-seq: An efficient method of large-scale de novo SNP discovery and genotyping using high-throughput sequencing. PLoS ONE, 8, e58700.

Tang S Y, Tang Z X, Qiu L, Yang Z J, Li G R, Lang T, Zhu W Q, Zhang J H, Fu S L. 2018. Developing new oligo probes to distinguish specific chromosomal segments and the A, B, D genomes of wheat (Triticum aestivum L.) using ND-FISH. Frontiers in Plant Science, 9, 1104.

Tang Z X, Yang Z J, Fu S L. 2014. Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis. Journal of Applied Genetics, 55, 313-318.

Tong S F, Mo Z, Rui X, Li D F, Zhang L F, Yang L. 2022. Genome-wide detection for runs of homozygosity analysis in three pig breeds from Chinese Taihu Basin and Landrace pigs by SLAF-seq data. Journal of Integrative Agriculture, 21, 3293-3301.

Wang Y F, Cheng X F, Yang X Y, Wang C Y, Zhang H, Deng P C, Liu X L, Chen C H, Ji W Q, Wang Y J. 2021. Molecular cytogenetics for a wheat-Aegilops geniculata 3M^g alien addition line with resistance to stripe rust and powdery mildew. BMC Plant Biology, 21, 575.

Wang Y Z, Cao Q, Zhang J J, Wang S W, Chen C H, Wang C Y, Zhang H, Wang Y J, Ji W Q. 2020. Cytogenetic analysis and molecular marker development for a new wheat-Thinopyrum ponticum 1J^S (1D) disomic substitution line with resistance to stripe rust and powdery mildew. Frontiers in Plant Science, 11, 1282.

Xing L P, Hu P, Liu J Q, Witek K, Zhou S, Xu J F, Zhou W H, Gao L, Huang Z P, Zhang R Q, Wang X, Chen P D, Wang H Y, Jones J D G, Karafiatova M, Vrana J, Bartos J, Dolezel J, Tian Y C, Wu Y F, et al. 2018. Pm21 from Haynaldia villosa encodes a CC-NBS-LRR protein conferring powdery mildew resistance in wheat. Molecular Plant, 11, 874-878.

Yang G T, Tong C Y, Li H W, Li B, Li Z S, Zheng Q. 2022. Cytogenetic identification and molecular marker development of a novel wheat-Thinopyrum ponticum translocation line with powdery mildew resistance. Theoretical and Applied Genetics, 135, 2041-2057.

Yang G T, Deng P C, Ji W Q, Fu S L, Li H W, Li B, Li Z S, Zheng Q. 2023. Physical mapping of a new powdery mildew resistance locus from Thinopyrum ponticum chromosome 4AgS. Frontiers in Plant Science, 14, 1131205.

Yang X F, Wang C Y, Chen C H, Zhang H, Tian Z R, Li X, Wang Y J, Ji W Q. 2014. Chromosome constitution and origin analysis in three derivatives of Triticum aestivum-Leymus mollis by molecular cytogenetic identification. Genome, 57, 583-591.

Yu Z H, Wang H J, Xu Y F, Li Y S, Lang T, Yang Z J, Li G R. 2019. Characterization of chromosomal Rearrangement in new wheat-Thinopyrum intermedium addition lines carrying Thinopyrum-specific grain hardness genes. Agronomy-Basel, 9, 18.

Zhan H X, Li G R, Zhang X J, Li X, Guo H J, Gong W P, Jia J Q, Qiao L Y, Ren Y K, Yang Z J, Chang Z J. 2014. Chromosomal location and comparative genomics analysis of powdery mildew resistance gene Pm51 in a putative wheat-Thinopyrum ponticum introgression line. PLoS ONE, 9, e113455.