SSR primer and method for identifying purity of hybrid seeds of Zaojia Luffa cylindrica

A technology for the purity and identification method of hybrid seeds, which is applied in the field of molecular detection, can solve the problems of cumbersome operations, cumbersome operation steps, and large area in the dyeing and color development steps of silver staining, and achieves high commercial application value and improves identification efficiency. , the effect of low cost

Pending Publication Date: 2020-11-20
HUNAN VEGETABLE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional purity detection of loofah hybrids mainly adopts morphological identification, which is mainly based on the morphological identification of the seeds and ovaries of the female parent and hybrids. This method is practical, but the identification time takes about 60 days, which has a long cycle and occupies an area of Defects such as large and greatly affected by the environment
At present, the use of molecular markers to identify the purity of hybrids has the advantages of good stability and large amount of information, and has been widely used in the detection of crop seed purity and quality. No. 2" and common loofah "Sisheng No. 2" hybrids were reported, but the primers and methods for the purity identification of Zaojia loofah have not been reported yet.
At the same time, at present, there are still problems in the identification of the purity of hybrids by SSR molecular markers in loofah: (1) Genome extraction adopts CTAB method or SOD method, and the operation steps are cumbersome and time-consuming; (2) The unoptimized PCR reaction system and procedures waste costs; (3) ) silver staining step dyeing and color development link operation is cumbersome, unstable results and other technical problems

Method used

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  • SSR primer and method for identifying purity of hybrid seeds of Zaojia Luffa cylindrica
  • SSR primer and method for identifying purity of hybrid seeds of Zaojia Luffa cylindrica
  • SSR primer and method for identifying purity of hybrid seeds of Zaojia Luffa cylindrica

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 is used for the screening of Zaojia loofah hybrid purity identification primer

[0028] According to the SSR primers of wax gourd genome, and using the genomic DNA of Zaojia loofah female parent 'S1' and male parent 'S2' as templates for primer screening, the marker S32 with a co-dominant band was obtained, and the corresponding primer pairs were SF32 and SR32, respectively. in

[0029] The nucleotide sequence of the upstream primer SF32 is: 5'-CATCACCACCACCACAACC-3' (SEQ ID NO: 1)

[0030] The nucleotide sequence of the downstream primer SR32 is: 5'-GCCACCAAAATCAGCTCTATG-3' (SEQ ID NO: 2).

[0031] The marking band is clear and repeatable. The primer pair S32 PCR amplification can produce a maternal-specific marker of about 100bp in length and a paternal-specific marker of about 80bp, such as figure 1 As shown, the 1-3 lanes in the figure are the female parent of Zaojia loofah, the 4-6 lanes are the male parent of Zaojia loofah, and the 7th lane is a hybri...

Embodiment 2

[0032] Example 2 Zaojia loofah hybrid seed purity identification method

[0033] Germinated seed hypocotyls are used as genomic DNA extraction samples in this embodiment

[0034] (1) Rapid extraction of genomic DNA from radicle roots of Zaojia loofah and its male and female parents

[0035] About 200 seeds were taken from each part of Zaojia loofah hybrid for the test, and 4-8 seeds were taken from each of the parents. After soaking the seeds for 5 hours, they were placed in a germination box lined with filter paper, kept at a constant temperature of 37°C and kept moist. About 6-7 days after germination, the hypocotyl or root tip of a single germinated seed is about 0.5 cm to 1 cm placed on a 96-well PCR plate, and its genomic DNA is extracted by rapid alkaline cooking. Put the extracted genomic DNA sample into a 96-well PCR plate, add 50 μl of 0.25 mol / L NaOH solution with a pipette gun, boil in boiling water for 3 minutes, and handle it with care to prevent boiling water fr...

Embodiment 3

[0046] Example 3 Verification of Zaojia Luffa Hybrid Purity Identification Primer

[0047] (1) Adopt the method of embodiment 2, utilize the specific primer described in embodiment 1, to 100 strains base field planting Zaojia loofah seedling stage sample detection, find 1 miscellaneous strain, purity is 99%, identification result and field identification result match. figure 2 It is the gel map of the purity identification of some Zaojia loofah hybrid seeds. The first lane in the figure is a 50bp marker, the second lane is the female parent, the third lane is the male parent, and the remaining lanes are samples to be tested.

[0048] (2) Adopt the method for embodiment 2 to sample the seeds of the Hunan Shuangfeng Seed Production Base, carry out purity identification, germinate 190 strains, adopt the simplified method to extract DNA samples and detect, the test results are all true hybrids, and the seed purity is 100%. , which is consistent with the field investigation resul...

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Abstract

The invention belongs to the technical field of molecular detection, and particularly relates to an SSR primer and method for identifying the purity of hybrid seeds of Zaojia Luffa cylindrica. The method disclosed by the invention can be used for detecting the purity of the hybrid seeds of the Zaojia Luffa cylindrica, and can effectively distinguish the hybrid seeds from a male parent and a femaleparent to quickly detect the purity of the hybrid seeds. The method has the advantages of simplicity, rapidness, accuracy, low cost, simplicity in operation and the like; by adopting the method, oneperson can complete the detection of nearly 500 samples in one day; and a traditional method for identifying the purity of the hybrid seeds of the Luffa cylindrica can be replaced, the operation is simple and convenient, labor is saved, land does not need to be occupied, a result is accurate and reliable, the identification efficiency is greatly improved, and the identification cost is reduced, sothat the method has relatively high commercial application value.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to an SSR primer and a method for identifying the purity of Zaojia luffa hybrid seeds. Background technique [0002] Loofah is a cultivated species in the genus Luffa of Cucurbitaceae, an annual climbing herb. There are two species of loofah cultivated in my country, namely common loofah (Luffa cylindrica (L.) M.J. Roem.) and ribbed loofah (Luffa acutangula (L.) Roxb.). Zaojia loofah belongs to the meat loofah in common loofah (Luffa cylindrica (L.) M.J.Roem.). In the seed production process of loofah hybrids, the method of detasseling the female parent is mainly used. That is, the parents are sown separately according to the growth period, and all the male flower buds of the female parent are manually removed before the female flower blooms. During the flowering period, the pollen of the male parent is collected and pollinated on the stigma of the female ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/156C12Q2600/13C12Q2565/125
Inventor 韩小霞胡新军李勇奇闵子扬袁祖华
Owner HUNAN VEGETABLE RES INST
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