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Kiwifruit indel molecular marker and its screening method and application

A molecular marker and kiwifruit technology, applied in biochemical equipment and methods, recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of limited number of markers and limited polyploidy kiwifruit, and achieve accelerated breeding process, stable variation, The effect of simplifying steps

Active Publication Date: 2019-12-10
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of markers that have been developed is limited, and some markers have population limitations. For example, the sex-related markers developed in a certain group are not applicable in other groups; the research data of Qin Rui (2013) showed that SSR primers of Actinidia sinensis were used in jujube. 23% versatility in kiwi
Due to the influence of the ploidy of kiwifruit, the genetic linkage maps currently established are all based on diploids (Davide et al., 2015), which also limits the positioning of important quality and resistance traits in polyploid kiwifruit. Therefore, further development of kiwifruit molecular markers is needed

Method used

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  • Kiwifruit indel molecular marker and its screening method and application
  • Kiwifruit indel molecular marker and its screening method and application
  • Kiwifruit indel molecular marker and its screening method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The method for screening kiwifruit InDel molecular markers in the embodiment of the present invention includes: DNA extraction, InDel marker mining and primer design, PCR amplification and electrophoresis detection, and polymorphism analysis.

[0044] The specific description of each step is as follows:

[0045] (1) DNA extraction

[0046] For Actinidia jujuba 'RB-3', 'Kuiluxiong', 'Yongfeng No.1', '11-19xiong', the young leaves were harvested, stored in ice packs, and extracted by magnetic beads Leaf DNA was detected by agarose gel electrophoresis and spectrophotometer, and the DNA concentration was adjusted to 20ng / μl for later use.

[0047] Among them, the DNA extraction kit used can be purchased from Henan Huier Nano Technology Co., Ltd.

[0048] (2) InDel site mining and primer design

[0049] The illumina HiSeq4000 sequencing platform was used to re-sequence the whole genome of Actinidia jujuba 'RB-3', 'Kuiluxiong', 'Yongfeng No.1' and '11-19xiong'. Evaluate a...

Embodiment 2

[0060] An application of the InDel molecular marker screened in embodiment 1 in kiwifruit genetic diversity analysis, its application steps are as follows:

[0061] The materials used include 'RB-3', 'Kuiluxiong', 'RB-4', '11-19xiong', 'LD134', '11-19', 'Boshan Jasper', 'Hongyang', ' Hot16-A', 'Xu Xiang', 'Hayward'.

[0062] After extracting the DNA from the above materials, 14 pairs of InDel primers screened were used to carry out PCR on these 11 varieties, and polyacrylamide electrophoresis was used for detection. Figure 3-14 shown. Figure 3-14 In the middle, from left to right: 1:'RB-3', 2:'Kuilu Xiong',3:'Xu Xiang',4:'RB-4',5:'11-19 Xiong',6 :'Hort16A',7:'LD134',8:'11-19',9:'Boshan Jasper',10:'Hongyang',11:'Hayward'.

[0063] The genotype was determined according to the band positions amplified from 11 samples of each pair of primers, with a band marked as 1 and no band marked as 0, forming a 0, 1 matrix. The number of alleles, Shannon's information index I (Shannon'...

Embodiment 3

[0070] A kind of application in the InDel identification progeny authenticity that embodiment 1 is screened, its application steps are as follows:

[0071] The DNA of 22 offspring of 'Yongfeng No. 1' and '11-17' was extracted, and according to the polymorphism statistics results in Example 1, the InDel-44 primer was selected for the authenticity identification of the offspring.

[0072] Such as image 3 As shown, the first electrophoresis lane is the DL2000 marker, the second electrophoresis lane is the female parent, and the third electrophoresis lane is the male parent. The use of this primer can exclude the offspring without the paternal band pattern.

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Abstract

The invention discloses a kiwi fruit InDel molecular marker and a screening method and application thereof. Two different cold-proof Actinidia arguta genome DNAs are used as research subjects, resequencing of four variety genes is carried out, InDel primers are designed according to resequencing data, and PCR (polymerase chain reaction) amplification, agarose electrophoresis and non-denaturing polyacrylamide gel electrophoresis are carried out; polymorphism detection is carried in Actinidia arguta to screen out 14 pairs of InDel primers, the 14 pairs of InDel primers are applicable to the genetic diversity analysis for Actinidia chinensis, Actinidia deliciosa and the like and are also applicable to the researches, such as hybrid offspring authenticity identification, genetic map construction, and molecule-assisted breeding. The kiwi fruit InDel primers according to the embodiment of the invention has good mutation stability and low detection difficulty, allow InDel insertion / loss of large fragments, and allows agarose analysis, with steps that may be simplified.

Description

technical field [0001] The invention relates to molecular marker technology, in particular to a kiwifruit InDel molecular marker and its screening method and application. Background technique [0002] Actinidia Lindl belongs to Actinidiaceae and is a perennial dioecious plant. Kiwi fruit has attracted widespread attention for its unique flavor, rich in vitamin C, dietary fiber and various mineral nutrients, and has the effects of clearing the intestines and strengthening the stomach, and has become one of the important fruit types. [0003] The phenomenon of interspecific hybridization in kiwifruit is obvious in nature, and the chromosome ploidy is complex. The efficiency of genetic research using conventional methods is not high. The development of DNA molecular markers provides an effective way for kiwifruit research (Huang Hongwen, 2009; Dong Xiaoli et al., 2005). [0004] Molecular marker technologies such as RAPD, AFLP, SSR, etc. have been applied to the evaluation of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 林苗苗方金豹齐秀娟孙雷明钟云鹏
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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