Development of Universal Two-color Fluorescent Quantitative PCR Marker for Wheat Subunit 7OE

doi:10.7668/hbnxb.20195855

Abstract

Abstract:

Although high-throughput KASP markers have been developed for the wheat quality subunit 7^OE,they are different from the KASP markers developed by SNP,the problem of not being able to effectively distinguish between homozygous and heterozygous remains.To clarify the issue of whether the 7^OE subunit is homozygous,this study used Jinqiang 6(containing 7^OE+8^* subunits)and Kenong 199(containing 7+9 subunits)and hybrid offspring as materials,and used the Waxy-D1 gene of wheat as an internal reference gene.The relative copy number of the 7^OE gene to the reference gene was detected by quantitative PCR using the universal dual-color fluorescence used in KASP markers to determine whether the 7^OE gene exists and whether it is homozygous,and the detection results were verified by a relevant molecular marker.The results showed that the relative copy number of the parent Jinqiang 6,with the 7^OE gene,was the highest,the relative copy number of the parent Kenong 199,without the 7^OE gene,was 0,and the relative copy number of their hybrid F₁ generation was intermediate,and the three types were easily separated.In its F₂ segregating population,the relative copy numbers of the 7^OE gene were also easily divided into high,medium and 0 three types.The genotypes that were detected as homozygous and heterozygous for the 7^OE gene were further detected by the PCR marker of the 9 subunit(which can detect 9 subunit and the 8^* subunit that are closely linked to the 7 subunit and the 7^OE subunit,respectively),and the results were completely consistent.The high-throughput 7^OE universal dual-color fluorescence quantitative PCR marker established in this study can accurately distinguish whether the 7^OE subunit is present,and whether it is homozygous,which has a positive effect on promoting the molecular marker-assisted selection of high-quality subunit 7^OE.

Key words: Wheat, High quality subunit, 7^OE, Dual color fluorescence, Quantitative PCR

CLC Number:

S512.1小麦

LIU Haichen, ZHANG Junmin, JIAO Bo, WANG Jiao, DONG Fushuang, YANG Fan, ZHAO Pu, MA Chunhong, CHAI Jianfang, ZHOU Shuo. Development of Universal Two-color Fluorescent Quantitative PCR Marker for Wheat Subunit 7^OE[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 20-25. doi: 10.7668/hbnxb.20195855.

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Figures/Tables 4

Fig.1 Two-color fluorescence quantitative PCR detection of relative copy numbers among 7^OE gene positive and negative parents and their F₁ hybrids A:P1.Jinqiang 6 with 7^OE gene,P2.Kenong 199 without 7^OE gene,F₁.The hybrids between parents Jinqiang 6 and Kenong 199;B:7^OE.Jinqiang 6,14,13 and 17.Xiaoyan 81,Jimai 20 and Yannong 19,H1,H2 and H3.The heterozygous DNAs obtained by artificially mixing Jinqiang 6 with Xiaoyan 81,Jimai 20 and Yannong 19 respectively.

Fig.2 Two-color fluorescent quantitative PCR detection of the 7^OE gene in the F₂ segregating population of Jinqiang 6 and Kenong 199 1—38.The 38 plants from the F₂ segregating population between Jinqiang 6 and Kenong 199.

Fig.3 PCR detection of 7^OE gene in the F₂ population of Jinqiang 6 and Kenong 199 M.Marker D2000;P1.Jinqiang 6;P2.Kenong 199;H.H₂O;1—21.The 21 plants from the F₂ segregating population of Jinqiang 6 and Kenong 199.

Fig.4 PCR detection of subunit 9 in the F₂ populations of Jinqiang 6 and Kenong 199 P1.Jinqiang 6;P2.Kenong 199;H.H₂O;1—29.The 29 plants from the F₂ segregating population of Jinqiang 6 and Kenong 199.

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