تعیین آللوتیپ ژن S-RNase و ناحیه پیشبر ژن MdMYB10 در چند ژنوتیپ سیب (Malus spp.)

نوع مقاله : مقالات پژوهشی

نویسندگان

• معصومه وکیلی قرطاول
• ناصر مهنا

گروه علوم باغبانی، دانشکده کشاورزی، دانشگاه تبریز، تبریز، ایران

https://doi.org/10.22067/jhs.2021.60821.0

چکیده

سیب (Malus spp.) یکی از مهم­ترین درختان میوه مناطق معتدله است. رنگ گوشت میوه سیب، از سفید تا قرمز متغیر است و تجمع مقدار بالایی از آنتوسیانین باعث قرمز بودن گوشت میوه آن می‌باشد، چنین افزایشی در مقدار آنتوسیانین در بسیاری از موارد به دلیل بیان بالای ژن MdMYB10 در بسیاری از بافت­ها از جمله گوشت میوه است، با این وجود،‌ در مواردی،‌ یک مکان ژنی پیوسته با آلل S₃ مکان ژنی S-RNase، به عنوان مسئول قرمزی گوشت میوه در بعضی از ژنوتیپ­ها پیشنهاد شده است. برای بررسی سازوکار قرمز بودن رنگ گوشت میوه در برخی ژنوتیپ­های محلی، استخراج DNA ژنومی از نمونه­های برگی بدست آمده از 9 ژنوتیپ سیب با گوشت قرمز و گوشت سفید انجام شد و آللوتیپ آنها از نظر ناحیه پیشبری ژن MdMYB10 و نیز وجود آلل S₃ در مکان ژنی S-RNase با استفاده از تکنیک واکنش زنجیره‌ای پلی‌مراز برای تکثیر با آغازگرهای اختصاصی هر ژن و الکتروفورز ژل آگارز قطعات تکثیر شده، مورد ارزیابی قرار گرفت. نتایج این پژوهش نشان داد که ژنوتیپ‌های گوشت سفید در ناحیه پیشبری ژن MdMYB10 فاقد آلل R₆ بوده و همگی هموزیگوت R₁R₁ تشخیص داده شدند. در حالی‌که، ارقام سیب گوشت قرمز دارای حداقل یک آلل R₆ در ناحیه پیشبری بوده و از نظر ژنوتیپ مکان ژنی خودناسازگاری S-RNase، آلل S₃ را دارا بودند. بنابراین، می­توان نمونه­های مذکور را هیبریدی از واریته ‘Niedzwetzkyana’ دانست. ممکن است گوشت قرمزی در یکی از ژنوتیپ­های مورد بررسی که برای ناحیه پیشبری R₁R₁ بود، مربوط به مکان ژنی پیوسته با آلل S₃ مکان ژنی S-RNase باشد. استفاده از این ژنوتیپ‌های گوشت قرمز برای اصلاح میوه­هایی با آنتوسیانین بالا توصیه می‌گردد.

کلیدواژه‌ها

• آنتوسیانین؛ سیب گوشت قرمز؛ ناحیه پیشبری ژن MdMYB10
• S-RNase

20.1001.1.20084730.1400.35.4.2.8

موضوعات

• بیوتکنولوژی و اصلاح درختان و گل های زینتی

عنوان مقاله [English]

Allelotyping of S-RNase Gene and Promoter Region of MdMYB10 Gene in some Genotypes of Apple (Malus spp.)

نویسندگان [English]

• Masumeh Vakili-Gartavol
• Naser Mahna

Department of Horticultural Sciences, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

چکیده [English]

Introduction: Red-fleshed apples (Malus spp.) are one of the rarest apple genotypes in the world and the accumulation of a high amount of anthocyanin, is the main cause of the redness of their fruit flesh. Anthocyanins are among important flavonoids and due to antioxidative activity, scavenge reactive oxygen species, and hence, are considered as one of the health-promoting nutraceuticals in the human diet. The amount of anthocyanins depends upon the expression of the transcription factors that are regulating their metabolic pathway. Among these transcription factors are the members of the MYB gene family. MdMYB10, belonging to this gene family in apple, has been shown to have a significant role in controlling the amount of anthocyanin production and redness in fruit flesh. The expression of MdMYB10 and consequently, the production of MdMYB10 proteins has positive feedback on its own expression. This happens due to a 23 bp microsatellite tandemly repeated 5 times in its promoter region (called allele R₆) which is a target sequence for MdMYB10 acting as a positive regulator. This structure invokes the overexpression of MdMYB10 which in turn increases the expression of anthocyanin producing enzymes and finally the amount of anthocyanin in all organs of the apple plant including fruit flesh. The apple Malus pumila var. Niedzwetzkyana and its derivatives have been reported to have such a structure in the promoter region of the MdMYB10 gene. The length of the R₆ allele is 496 bp, while the R₁ allele is only 392 bp long.
However, in some cases, a locus linked to the S₃ allele of the S-RNase gene has been proposed to be responsible for the redness of the fruit flesh in some genotypes. It has been reported that even the offspring of these plants have had red-fleshed fruits. 
Materials and Methods: To study the mechanism of the redness of the fruit flesh in some local genotypes, genomic DNA was extracted using the CTAB method from the leaf samples obtained from 9 red- and white-fleshed apple genotypes including Red Delicious, Golden Delicious, Miandoab, Makamik (Khalatpoushan), Bud 9, Varzighan, and Ivand. Then the allelotype of the promoter region of the MdMYB10 gene as well as the existence of S₃ allele at S-RNase locus was investigated using polymerase chain reaction. For amplification of the target sequences, MdMYB10 and S₃ specific primers were exploited and 1% agarose gel electrophoresis of the amplified fragments was used for observing and scoring the bands. All steps were repeated seven times.
Results and Discussion: The results in this research showed that the white-fleshed genotypes (Red Delicious, Golden Delicious, and Granny Smith) were lacking any R₆ allele at the promoter region of the MdMYB10 gene and were R₁R₁ homozygotes, while the red-fleshed genotypes (Miandoab, Makamik (Khalatpoushan), Bud 9, and Varzighan) had at least one R₆ allele at the mentioned promoter region as well as a S₃ allele in the self-incompatibility locus S-RNase. These results were in accordance with the previous reports. Therefore, these samples could be traced back to Malus pumila var. Niedzwetzkyana. Evaluating the S-RNase locus in these genotypes illustrated that Granny Smith (as positive control), Golden Delicious (as positive control), Makamik (Khalatpoushan), Miandoab, Varzeghan, Bud 9 and tissue culture sample, showing a band around 500 bp (smaller) had S₃ allele, while for Ivand and Red Delicious (as negative control) no S₃ band was obtained. For the tissue culture sample which was R₁R₁ at the promoter region and S₃ at S-RNase locus, it was postulated that flesh-redness may be due to the locus linked to the S₃ allele. We also got an unknown R band for the Ivand genotype when analyzing for the MdMYB10 promoter region. The sequencing of in the future studies, may help to unravel the mechanism by which shoot-redness happens in this genotype.   
Conclusion: The development of highly potent and novel cultivars for the fast-evolving market is indispensable in the plant breeding field. In this way, breeding apple plant, as an important temperate fruit with a long postharvest life, for redness of fruit flesh can be considered as a noticeable case. We could confirm in this research that in the endemic, red-fleshed apples, R₆ may be responsible for their high anthocyanin production. However, the S₃-RNase-linked locus should also be considered in marker-assisted breeding methods for this trait. Therefore, these red-fleshed genotypes are highly recommended to be employed in the national breeding programs for increasing the anthocyanin content of apple fruits.

کلیدواژه‌ها [English]

• Anthocyanin
• MdMYB10 gene promoter
• Red-fleshed apples
• S-RNase

مراجع

1. Allan A.C., Hellens R.P., and Laing W.A. 2008. MYB transcription factors that colour our fruit. Trends in Plant Science 13(3): 99-102.
2. Ban Y., Honda C., Hatsuyama Y., Igarashi M., Bessho H., and Moriguchi T. 2007. Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant and Cell Physiology 48(7): 958-970.
3. Broothaerts W. 2003. New findings in apple S-genotype analysis resolve previous confusion and request the re-numbering of some S-alleles. Theoretical and Applied Genetics 106(4): 703-714.
4. Broothaerts W., Janssens G.A., Proost P., and Broekaert W.F. 1995. cDNA cloning and molecular analysis of two self-incompatibility alleles from apple. Plant Molecular Biology 27(3): 499-511.
5. Chagné D., Carlisle C.M., Blond C., Volz R.K., Whitworth C.J., Oraguzie N.C., Crowhurst R.N., Allan A.C., Espley R.V., and Hellens R.P. 2007. Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple. BMC Genomics 8(1): 212.
6. Espley R.V., Brendolise C., Chagné D., Kutty-Amma S., Green S., Volz R., Putterill J., Schouten H.J., Gardiner S.E., and Hellens R.P. 2009. Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. The Plant Cell 21(1): 168-183.
7. Espley R.V., Hellens R.P., Putterill J., Stevenson D.E., Kutty‐Amma S., and Allan A.C. 2007. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. The Plant Journal 49(3): 414-427.
8. FARAGHER J.D. 1983. Temperature regulation of anthocyanin accumulation in apple skin. Journal of Experimental Botany 34(10): 1291-1298.
9. Forkmann G. 1991. Flavonoids as flower pigments: the formation of the natural spectrum and its extension by genetic engineering. Plant Breeding 106(1): 1-26.
10. Frankel R., and Galun E. 2012. Pollination mechanisms, reproduction and plant breeding. Vol. 2. Springer Science & Business Media.
11. Holton T.A., and Cornish E.C. 1995. Genetics and biochemistry of anthocyanin biosynthesis. The Plant Cell 7(7): 1071.
12. Honda C., Kotoda N., Wada M., Kondo S., Kobayashi S., Soejima J., Zhang Z., Tsuda T., and Moriguchi T. 2002. Anthocyanin biosynthetic genes are coordinately expressed during red coloration in apple skin. Plant Physiology and Biochemistry 40(11): 955-962.
13. Huang D.-J., Lin C.-D., Chen H.-J., and Lin Y.-H. 2004. Antioxidant and antiproliferative activities of sweet potato (Ipomoea batatas [L.] Lam ‘Tainong 57’) constituents. Bot. Bull. Acad. Sin. 45: 179-186.
14. Iversen C.K. 1999. Black currant nectar: effect of processing and storage on anthocyanin and ascorbic acid content. Journal of Food Science 64(1): 37-41.
15. Janssens G., Goderis I., Broekaert W., and Broothaerts W. 1995. A molecular method for S-allele identification in apple based on allele-specific PCR. Theoretical and Applied Genetics 91(4): 691-698.
16. Jin H., and Martin C. 1999. Multifunctionality and diversity within the plant MYB-gene family. Plant Molecular Biology 41(5): 577-585.
17. Kobel F. 1939. Weitere Untersuchungen uber die Befruchtungsverhaltnisse der Apfelund Birnsorten. Landw. Jahrb. Schweiz 53: 160-191.
18. Lin-Wang K., Bolitho K., Grafton K., Kortstee A., Karunairetnam S., McGhie T.K., Espley R.V., Hellens R.P., and Allan A.C. 2010. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biology 10(1): 50.
19. Lodhi M.A., Ye G.-N., Weeden N.F., and Reisch B.I. 1994. A simple and efficient method for DNA extraction from grapevine cultivars andVitis species. Plant Molecular Biology Reporter 12(1): 6-13.
20. Mahmoudi E., MOHAMMAD S.B., Yadollahi A., and Hosseini E. 2012. Independence of color intensity variation in red flesh apples from the number of repeat units in promoter region of the MdMYB10 gene as an allele to MdMYB1 and MdMYBA.
21. Martin C., and Paz-Ares J. 1997. MYB transcription factors in plants. Trends in Genetics 13(2): 67-73.
22. Mol J., Stuitje A., Gerats A., van der Krol A., and Jorgensen R. 1989. Saying it with genes: molecular flower breeding. Trends in Biotechnology 7(6): 148-153.
23. Sakurai K., Brown S.K., and Weeden N.F. 1997. Determining the self-incompatibility alleles of Japanese apple cultivars. HortScience 32(7): 1258-1259.
24. Sassa H., Hirano H., and Ikehashi H. 1992. Self-incompatibility-related RNases in styles of Japanese pear (Pyrus serotina Rehd.). Plant and Cell Physiology 33(6): 811-814.
25. Sassa H., Hirano H., and Ikehashi H. 1993. Identification and characterization of stylar glycoproteins associated with self-incompatibility genes of Japanese pear, Pyrus serotina Rehd. Molecular and General Genetics MGG 241(1-2): 17-25.
26. Sassa H., Hirano H., Nishio T., and Koba T. 1997. Style‐specific self‐compatible mutation caused by deletion of the S‐RNase gene in Japanese pear (Pyrus serotina). The Plant Journal 12(1): 223-227.
27. Schwinn K., Venail J., Shang Y., Mackay S., Alm V., Butelli E., Oyama R., Bailey P., Davies K., and Martin C. 2006. A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. The Plant Cell 18(4): 831-851.
28. Sekido K., Hayashi Y., Yamada K., Shiratake K., Matsumoto S., Maejima T., and Komatsu H. 2010a. Efficient breeding system for red-fleshed apple based on linkage with S3-RNase allele in ‘Pink Pearl’. HortScience 45(4): 534-537.
29. Sekido K., Yamada K., Shiratake K., Fukui H., and Matsumoto S. 2010b. MdMYB alleles responsible for apple skin and flesh color. Current Topics in Plant Biology Volume 11. 17-21.
30. Siegelman H., and Hendricks S. 1958. Photocontrol of anthocyanin synthesis in apple skin. Plant Physiology 33(3): 185.
31. Takos A.M., Jaffé F.W., Jacob S.R., Bogs J., Robinson S.P., and Walker A.R. 2006. Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiology 142(3): 1216-1232.
32. Umemura H., Shiratake K., Matsumoto S., Maejima T., and Komatsu H. 2011. Practical breeding of red-fleshed apple: cultivar combination for efficient red-fleshed progeny production. HortScience 46(8): 1098-1101.
33. van Nocker S., Berry G., Najdowski J., Michelutti R., Luffman M., Forsline P., Alsmairat N., Beaudry R., Nair M.G., and Ordidge M. 2012. Genetic diversity of red-fleshed apples (Malus). Euphytica 185(2): 281-293.
34. Walker A.R., Lee E., Bogs J., McDavid D.A., Thomas M.R., and Robinson S.P. 2007. White grapes arose through the mutation of two similar and adjacent regulatory genes. The Plant Journal 49(5): 772-785.