基于ITS Sanger测序的香蕉杂交和自交后代的鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2024-0501

摘要/Abstract

摘要：

【目的】以核糖体内部转录间隔区（internal transcribed spacer, ITS）为分子标记，鉴定新创制的杂交和自交香蕉子代，为香蕉育种和遗传研究奠定基础。【方法】提取亲本及其疑似后代的DNA，PCR扩增和Sanger测序，利用软件“DNAMAN”和“SnapGene”进行序列比对，分析ITS的多态性，从而确定杂交和自交后代。当疑似子代植株的ITS序列与亲本相比，在一个或多个碱基位点上显示出差异时，该植株被归类为阳性植株；反之，若未发现此类差异，则判定其为假阳性植株。【结果】以‘广粉1号’栽培蕉和‘天野BB1号’野生蕉、‘粉杂1号’栽培蕉自交以及‘尖苞片蕉1号’和‘尖苞片蕉2号’共3个组合的疑似后代为材料，通过性状观察，发现只有部分子代植株与亲本存在某些差异，无法准确判断杂交和自交是否成功；而利用前期建立的香蕉ITS Sanger测序法，发现其与亲本的ITS存在显著差异并有一定联系，说明这些材料都是阳性植株。【结论】‘广粉1号’和‘天野BB1号’‘粉杂1号’自交以及‘尖苞片蕉1号’和‘尖苞片蕉2号’的疑似后代均为阳性植株。ITS Sanger测序法可用于香蕉杂交和自交后代的鉴定。ITS Sanger测序法对相同杂交或自交组合产生的不同子代也有一定的基因分型效果。

关键词: 香蕉, 种质资源, 杂交, 自交, 分子鉴定, ITS

Abstract:

【Objective】The aim of this study is to utilize the Internal Transcribed Spacer（ITS）region as a molecular marker in identifying newly developed hybrid and selfing offspring banana progeny, thereby laying the foundation for advancements in banana breeding and genetics research.【Method】DNA of parents and their suspected offspring was extracted, then PCR amplifification and Sanger sequencing were conducted, and sequence alignment was performed using software “DNAMAN” and “SnapGene” to analyze the polymorphism of ITS, thereby determining the hybrid and selfing offspring. The plant was classified as a positive plant when the ITS sequence of the suspected offspring plant showed differences at one or more base sites compared with the parent; conversely, if no such differences were found, the plant was determined to be a false positive plant.【Result】The study encompasses three sets of putative progeny derived from cultivated banana crossed with wild banana（‘Guang Fen No. 1’ × ‘Tian Ye BB No. 1’）, selfed offspring of a cultivated variety（‘Fen Za No.1’）, and a cross between two wild bananas（‘Musa acuminata No. 1’ × ‘Musa acuminata No. 2’）. Applying the established banana ITS Sanger sequencing protocol, the notable disparities linked to the ITS sequences of these candidate offspring compared to their parents were identified. Phenotypic observations further confirmed variances in the offspring plants, suggesting they were positive plants. In the hybrid offspring of ‘Guang Fen No.1’ and ‘Tian Ye BB No.1’, polymorphisms in the “410 bp region” were observed in the ITS Sanger sequencing profiles, accompanied by distinct changes in bunch characteristics. Among four selfed offspring of ‘Fen Za No.1’, polymorphic peaks in the “50 bp” and “410 bp regions” were detected, with two lines exhibiting distinctive leaf traits. For the ten seedlings resulting from the ‘Musa acuminata No. 1’ × ‘Musa acuminata No. 2’ cross, significant differences in the sequencing profiles were noted at “115 bp” “431 bp” and “452 bp” relative to their parents.【Conclusion】The ITS Sanger sequencing approach emerges as a complementary tool to phenotypic assessments and SSR-based identification methods, facilitating rapid, flexible, and convenient material authenticity confirmation in distinguishing banana hybrid or selfed offspring. This methodology aids breeders and researchers in efficiently confirming their materials accuracy in breeding and genetic studies.

Key words: banana, germplasm resources, hybrid, selfing, molecular identification, ITS

曾鸿运, 许林兵, 吴元立, 黄秉智. 基于ITS Sanger测序的香蕉杂交和自交后代的鉴定[J]. 生物技术通报, 2024, 40(12): 53-60.

ZENG Hong-yun, XU Lin-bing, WU Yuan-li, HUANG Bing-zhi. Identification of Banana Hybrid and Selfing Offspring Based on Internal Transcribed Spacer（ITS）Sanger Sequencing[J]. Biotechnology Bulletin, 2024, 40(12): 53-60.

图/表 3

图1 ‘广粉1号’与‘天野BB1号’的杂交后代的鉴定 A：田间性状；B：ITS Sanger测序峰图“420 bp区域”

Fig. 1 Identification of hybrids from the cross between ‘Guang Fen No. 1’ and ‘Tian Ye BB1 No. 1’ A: Field traits. B: Sanger sequencing chromatogram of the “420 bp region” of ITS

图2 ‘粉杂1号’自交后代的鉴定 A：田间性状；B：ITS Sanger测序峰图“50 bp区域”；C：ITS Sanger测序峰图“420 bp区域”

Fig. 2 Identification of selfing offspring of ‘Fen Za No.1’ A: Field characteristics. B: Sanger sequencing chromatogram of the “50 bp region” of ITS. C: Sanger sequencing chromatogram of the “420 bp region” of ITS

图3 ‘尖苞片蕉1号’和‘尖苞片蕉2号’的杂交后代的鉴定 A：ITS序列比对；B：ITS Sanger测序峰图“110 bp区域”；C：ITS Sanger测序峰图“440 bp区域”

Fig. 3 Identification of hybrids derived from the cross between ‘Musa acuminata No.1’ and ‘Musa acuminata No.2’ A: Alignment of ITS sequences. B: Sanger sequencing chromatogram of the “110 bp region” of ITS. C: Sanger sequencing chromatogram of the “440 bp region” of ITS

参考文献 44

[1]	Perrier X, De Langhe E, Donohue M, et al. Multidisciplinary perspectives on banana(Musa spp.)domestication[J]. Proc Natl Acad Sci USA, 2011, 108(28): 11311-11318. doi: 10.1073/pnas.1102001108 pmid: 21730145
[2]	Fu N, Ji MY, Rouard M, et al. Comparative plastome analysis of Musaceae and new insights into phylogenetic relationships[J]. BMC Genomics, 2022, 23(1): 223.
[3]	Dewi S, Ahmad F, Oktorida Khastini R, et al. Bananas and their wild relatives in pandeglang, Indonesia[J]. HAYATI J Biosci, 2023, 30(6): 1071-1091.
[4]	Tripathi JN, Ntui VO, Malarvizhi M, et al. Improvement of nutraceutical traits of banana: new breeding techniques[M]// Compendium of Crop Genome Designing for Nutraceuticals. Singapore: Springer, 2023: 1-33.
[5]	Rocha AJ, Soares JMDS, Nascimento FDS, et al. Improvements in the resistance of the banana species to Fusarium wilt: a systematic review of methods and perspectives[J]. J Fungi, 2021, 7(4): 249.
[6]	Olufemi OS. Exploring banana production in Africa for food security and economic growth—a short review[J]. Food Nutr Chem, 2024, 2(1): 125.
[7]	Navy T, Mardy S. A review on industrial banana production in Cambodia[J]. Int J Sustain Appl Sci, 2023, 1(6): 783-788.
[8]	Platonovskiy NG, Ibrasheva LR, Obukhova NI, et al. International banana trade: volumes, countries, and trends[M]//Popkova EG, Bogoviz AV, Sergi BS, et al. Sustainable Development of the Agrarian Economy Based on Digital Technologies and Smart Innovations. Cham: Springer, 2024: 25-30.
[9]	Martin G, Cottin A, Baurens FC, et al. Interspecific introgression patterns reveal the origins of worldwide cultivated bananas in New Guinea[J]. Plant J, 2023, 113(4): 802-818.
[10]	Martin G, Cardi C, Sarah G, et al. Genome ancestry mosaics reveal multiple and cryptic contributors to cultivated banana[J]. Plant J, 2020, 102(5): 1008-1025.
[11]	吴元立, 黄秉智, 杨护, 等. 抗枯萎病优质特色香蕉新品种粉杂1号的选育[J]. 果树学报, 2022, 39(12): 2450-2454.
	Wu YL, Huang BZ, Yang H, et al. reeding of Fenza No. 1, a new high-quality and special banana variety with high resistance against Fusarium oxysporum f. sp. cubense[J]. J Fruit Sci, 2022, 39(12): 2450-2454.
[12]	Penna S, Ghag SB, Ganapathi TR, et al. Induced genetic diversity in banana[M]// Genetic Diversity in Horticultural Plants. Cham: Springer, 2019: 273-297.
[13]	Aguilar Morán JF. Improvement of Cavendish banana cultivars through conventional breeding[J]. Acta Hortic, 2013(986): 205-208.
[14]	曾鸿运, 吴元立, 黄秉智. 中国香蕉育种研究进展[J]. 果树学报, 2023, 40(11): 2446-2465.
	Zeng HY, Wu YL, Huang BZ. Research and utilization progress in banana germplasm resources in China[J]. J Fruit Sci, 2023, 40(11): 2446-2465.
[15]	Chabi M, Dassou AG, Adoukonou-Sagbadja H, et al. Variation in symptom development and infectivity of banana bunchy top disease among four cultivars of Musa sp[J]. Crops, 2023, 3(2): 158-169.
[16]	Bolfarini ACB, Souza JMA, Putti FF, et al. Physicochemical characteristics of unripe and ripe banana ‘FHIA 18’ submitted to phosphorus fertilizer over three production cycles[J]. Semina Ciências Agrárias, 2020, 41(1): 33.
[17]	Annor GA, Asamoah-Bonti P, Sakyi-Dawson E. Fruit physical characteristics, proximate, mineral and starch characterization of FHIA 19 and FHIA 20 plantain and FHIA 03 cooking banana hybrids[J]. SpringerPlus, 2016, 5(1): 796.
[18]	Meghwal M, Jayachandran A. Advances in production technology of banana[M]// Amit K, Om P, Rahul D, et al. A Textbook on Advances in Production Technology of Tropical and Subtropical Fruits. New Delhi: New Vishal Publication, 2023:60.
[19]	Bugaud C, Alter P, Daribo MO, et al. Comparison of the physico-chemical characteristics of a new triploid banana hybrid, FLHORBAN 920, and the Cavendish variety[J]. J Sci Food Agric, 2009, 89(3): 407-413.
[20]	Hu WF, Yang BM, He ZH, et al. Magnesium may be a key nutrient mechanism related to Fusarium wilt resistance: a new banana cultivar(Zhongjiao No. 9)[J]. PeerJ, 2021, 9: e11141.
[21]	Ssali RT, Kiggundu A, Lorenzen J, et al. Inheritance of resistance to Fusarium oxysporum f. sp. cubense race 1 in bananas[J]. Euphytica, 2013, 194(3): 425-430.
[22]	Arinaitwe IK, Teo CH, Kayat F, et al. Evaluation of banana germplasm and genetic analysis of an F1 population for resistance to Fusarium oxysporum f. sp. cubense race 1[J]. Euphytica, 2019, 215(10): 175.
[23]	Uwimana B, Nakato GV, Kanaabi R, et al. Identification of the loci associated with resistance to banana Xanthomonas wilt(Xanthomonas vasicola pv. musacearum)using DArTSeq markers and continuous mapping[J]. Horticulturae, 2024, 10(1): 87.
[24]	Ahmad F, Martawi NM, Poerba YS, et al. Genetic mapping of Fusarium wilt resistance in a wild banana Musa acuminata ssp. malaccensis accession[J]. Theor Appl Genet, 2020, 133(12): 3409-3418.
[25]	Chen A, Sun JM, Martin G, et al. Identification of a major QTL-controlling resistance to the subtropical race 4 of Fusarium oxysporum f. sp. cubense in Musa acuminata ssp. malaccensis[J]. Pathogens, 2023, 12(2): 289.
[26]	Anuradha C, Ramajayam D, Mayilvaganan M, et al. Molecular characterization of Red banana and its somaclonal variant: a comprehensive study[J]. 3 Biotech, 2024, 14(1): 19.
[27]	Biswas MK, Yi GJ. Genes and markers: application in banana crop improvement[M]// Banana: Genomics and Transgenic Approaches for Genetic Improvement. Singapore: Springer, 2016: 35-50.
[28]	Inta W, Traiperm P, Ruchisansakun S, et al. Evolution and classification of Musaceae based on male floral morphology[J]. Plants, 2023, 12(8): 1602.
[29]	Brisibe EA, Ubi GM, Ekanem NG. Descriptive and multivariate analyses of morphotaxonomic and yield-related traits in inflorescence dichotomous cultivars of Musa species(AAB genome)[J]. Genet Resour Crop Evol, 2021, 68(8): 3357-3372.
[30]	Sawant GB, Dalvi VV, Ambavane AR, et al. Evaluation of qualitative trraits in banana(Musa spp.)genomes[J]. International Journal of Genetics, 2018, 10(11):548-551.
[31]	Pillay M. The genetic homogeneity of Uganda's East African highland bananas(Mutika/Lujugira)does not match the extensive morphological variation identified in this subgroup[J]. Int J Plant Biol, 2024, 15(2): 267-280.
[32]	Christelová P, Valárik M, Hřibová E, et al. A platform for efficient genotyping in Musa using microsatellite markers[J]. AoB Plants, 2011, 2011: plr024.
[33]	Hinge VR, Shaikh IM, Chavhan RL, et al. Assessment of genetic diversity and volatile content of commercially grown banana(Musa spp.)cultivars[J]. Sci Rep, 2022, 12: 7979.
[34]	Taheri S, Lee Abdullah T, Yusop M, et al. Mining and development of novel SSR markers using next generation sequencing(NGS)data in plants[J]. Molecules, 2018, 23(2): 399.
[35]	Harnelly E, Kusuma HI, Thomy Z, et al. Internal Transcribed Spacer(ITS)gene as an accurate DNA barcode for identification of macroscopic fungus in Aceh[J]. Biodiversitas, 2022, 23(5): 2369-2378.
[36]	Nehal N, Choudhary B, Nagpure A, et al. DNA barcoding: a modern age tool for detection of adulteration in food[J]. Crit Rev Biotechnol, 2021, 41(5): 767-791.
[37]	Li LF, Häkkinen M, Yuan YM, et al. Molecular phylogeny and systematics of the banana family(Musaceae)inferred from multiple nuclear and chloroplast DNA fragments, with a special reference to the genus Musa[J]. Mol Phylogenet Evol, 2010, 57(1): 1-10.
[38]	Liu AZ, Kress WJ, Li DZ. Phylogenetic analyses of the banana family(Musaceae)based on nuclear ribosomal(ITS)and chloroplast(trnL-F)evidence[J]. Taxon, 2010, 59(1): 20-28.
[39]	Hřibová E, Čížková J, Christelová P, et al. The ITS1-5.8S-ITS2 sequence region in the Musaceae: structure, diversity and use in molecular phylogeny[J]. PLoS One, 2011, 6(3): e17863.
[40]	Nwakanma DC, Pillay M, Okoli BE, et al. PCR-RFLP of the ribosomal DNA internal transcribed spacers(ITS)provides markers for the A and B genomes in Musa L[J]. Theor Appl Genet, 2003, 108(1): 154-159. pmid: 12955208
[41]	Zeng H, Huang B, Xu L, et al. Banana classification using sanger sequencing of the ribosomal DNA internal transcribed spacers(ITS)[J/OL]. Preprints, 2024. DOI: 10.20944/preprints202402.1260.v1.
[42]	Batte M, Nyine M, Uwimana B, et al. Significant progressive heterobeltiosis in banana crossbreeding[J]. BMC Plant Biol, 2020, 20(1): 489.
[43]	Rouard M, Sardos J, Sempéré G, et al. A digital catalog of high-density markers for banana germplasm collections[J]. Plants People Planet, 2022, 4(1): 61-67.
[44]	Hou BH, Tsai YH, Chiang MH, et al. Cultivar-specific markers, mutations, and chimerisim of Cavendish banana somaclonal variants resistant to Fusarium oxysporum f. sp. cubense tropical race 4[J]. BMC Genomics, 2022, 23(1): 470.

编辑推荐 0

Metrics

阅读次数

全文

120

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	9	0	0	111

来源	本网站	其他网站

次数	111	9
比例	92%	8%

摘要

最新录用	在线预览	正式出版

0	0	73

	来源	本网站

	次数	73
	比例	100%