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CN-121992136-A - SSR molecular marker of mulberry new variety 'Yunsang No. 9', core primer group, kit and application thereof

CN121992136ACN 121992136 ACN121992136 ACN 121992136ACN-121992136-A

Abstract

The invention discloses an SSR molecular marker of a new mulberry variety 'cloud mulberry No. 9', a core primer group, a kit and application thereof, and belongs to the technical field of crop variety identification. According to the invention, two pairs of core primers MASSR, 037 and MASSR and 063 are obtained by screening 200 pairs of SSR candidate primers, so that 'Yunsang No. 9' can be accurately identified from 48 control varieties (23 related variety resources and 25 nationwide common popularization varieties). The MASSR037 primer pair amplified two specific bands 157 bp and 169 bp, and the MASSR063 primer pair amplified three specific bands 279 bp, 288 bp and 294 bp. The invention has simple operation and strong specificity, and is suitable for variety authenticity identification, seedling purity detection and variety right protection of Yun Sang' No. Yun Sang.

Inventors

  • LI ZHENNAN
  • DING ZHIWEI
  • CHEN SONG
  • KANG XIAORU
  • TAO ZHENYANG
  • LI ZHENGANG
  • YANG QIHUI
  • RUAN MINGJU
  • FENG WEI
  • YANG JIAHU
  • HE JIAMEI
  • FENG YUMING
  • RAN RUIFA

Assignees

  • 云南省农业科学院蚕桑蜜蜂研究所

Dates

Publication Date
20260508
Application Date
20260211

Claims (10)

  1. 1. The SSR molecular marker for identifying the novel mulberry variety 'cloud mulberry No. 9' is characterized by comprising SSR molecular markers MASSR and/or MASSR063, wherein the repeated motif of the SSR molecular markers MASSR037 is (TAAA) n, n is more than or equal to 5, the 5 '-flanking sequence is shown as SEQ ID No.5, the 3' -flanking sequence is shown as SEQ ID No.6, the repeated motif of the SSR molecular markers MASSR063 is (ACA) n, n is more than or equal to 9, the 5 '-flanking sequence is shown as SEQ ID No.7, and the 3' -flanking sequence is shown as SEQ ID No. 8.
  2. 2. A core primer set for identifying an SSR molecular marker of a new variety 'yunssang 9', characterized by comprising a primer for an SSR molecular marker MASSR037 and/or a primer for an SSR molecular marker MASSR 063: The primer for SSR molecular marker MASSR and 037 is as follows: MASSR037-F:5'-ATGCCAGGTGGGTTTGTACG-3'; MASSR037-R:5'-TATGATGTTGGGCCCCACC-3'; the primer for SSR molecular marker MASSR063,063 is: MASSR063-F:5'-GGGCCCACCATCATCTTCAT-3'; MASSR063-R:5'-ATTGCCCGGAAATTCGAC-3'; Wherein, the 5' ends of MASSR037-F and MASSR063-F are marked with fluorescent report groups.
  3. 3. The core primer set of claim 2, wherein the fluorescent reporter group is FAM, HEX or TAMRA.
  4. 4. Use of the core primer set of claim 2 or 3 in the preparation of a kit for identifying a new variety of mulberry, 'yunssang No. 9'.
  5. 5. A kit for identifying a new variety of mulberry, 'yunssang No. 9', comprising the core primer set of claim 2 or 3.
  6. 6. A method for identifying a novel mulberry variety 'Yunsang No. 9' is characterized by comprising the steps of extracting genome DNA of a sample to be detected, carrying out PCR amplification by using the primer MASSR037-F/R and/or MASSR063-F/R as defined in claim 2 as a template to obtain a PCR product, carrying out acrylamide gel electrophoresis and capillary electrophoresis on the PCR product, and analyzing an electrophoresis band to identify whether the sample to be detected is the novel mulberry variety 'Yunsang No. 9'.
  7. 7. The method according to claim 6, wherein the analysis of the electrophoresis strip is performed to determine whether the sample is a new variety of mulberry, ' yunssang 9 ', specifically, whether two specific strips 157 bp and 169 bp are generated by PCR amplification using MASSR037-F/R as a primer, or whether three specific strips 279 bp, 288 bp and 294 bp are generated by PCR amplification using MASSR063-F/R as a primer, and if any one of the specific strips is generated, the sample is determined to be ' Yun Sang ', and if the specific strip is not generated, the sample is determined to be non-Yun Sang '.
  8. 8. The method of claim 6, wherein the PCR amplification reaction system is 10.0. Mu.L, including 20 ng/. Mu.L of DNA template 1.0. Mu.L, 2X TAQPCR MASTER Mix 5.0. Mu.L, 10. Mu.M upstream and downstream primers 0.5. Mu.L, ddH 2 O3.0. Mu.L.
  9. 9. The method of claim 6, wherein the PCR amplification is performed under conditions of 95℃for 5min, 95℃for 30 s,62℃to 52℃for 30 s,72℃for 30 s cycles, 95℃for 30 s,52℃for 30 s,72℃for 30 s cycles, 25 cycles, and 72℃for 20min cycles.
  10. 10. Use of the core primer set of claim 2 or 3 or the kit of claim 5 for identifying a new variety of mulberry, 'yunssang No. 9'.

Description

SSR molecular marker of mulberry new variety 'Yunsang No. 9', core primer group, kit and application thereof Technical Field The invention relates to the technical field of crop variety identification, in particular to an SSR molecular marker of a new mulberry variety 'cloud mulberry No. 9', a core primer group, a kit and application thereof. Background Leaves of Morus alba L are rich in various nutritional ingredients such as proteins, and are a main feed source for special economic animals and silkworms, and can also be used as feed sources for livestock and poultry such as cattle, sheep, chickens and the like, and mature clusters are important berries in life. In the development process of industry, the support of the excellent mulberry variety is not separated, and the large-scale popularization and application of the excellent variety are less than the identification right of the new variety. The new mulberry variety 'Yunnan mulberry 9' is a mulberry new variety cultivated by observing and testing adaptability and resistance of a single plant after hybridization of a common mulberry variety, namely, a mulberry 14 number serving as a male parent and a Yun Sang number serving as a female parent, through directional cultivation for 5 years, is proved to pass the approval of a new variety of Yunnan crops (namely, mulberry) 2023021 number in 2023, and is declared to be a dominant variety in Yunnan province at present, and the variety has high yield and excellent properties, is more suitable for cultivation in a higher altitude area than Yun Sang number, is a variety cultivated for adapting to stereoscopic climate in Yunnan province, has better brown spot resistance performance, is popular in the popularization process, but is difficult to separate from other seedlings in seedling stage, is not more reliable in identification means, and is blocked due to economic loss after misplacement. The variety is characterized by compact crown, multiple spring numbers, thick and long branches with slightly curved straight internodes, dark gray skin, dense internode spacing, 3/8 leaf sequence, large and mostly linear skin holes, strong anthocyanin development on the surface of young shoots, thick and strong terminal buds, long and triangular winter buds, brown and striated winter buds, heart-shaped leaves, dark green leaves, thick and soft leaves and strong luster. The leaf length is 26 cm-30 cm, and the leaf width is 20 cm-25 cm. The strain has strong discrimination, has the advantages of father and mother, has high yield, strong brown spot resistance, vigorous plant growth vigor, high water and fertilizer resistance, strong spring force, shearing resistance and early germination, belongs to early-maturing varieties in the late 1 month of the germination period of Mongolian from the city planting in Yunnan province, the early 2 month of the leaf opening period and the middle 4 month of the leaf maturation period, has wide weather adaptability, is suitable for cultivation in a 600-2000 m altitude silkworm production area in Yunnan province, and particularly has better performance in cold areas. In the popularization process of the new mulberry variety 'cloud mulberry No. 9', the external morphological characteristics of the seedling stage are difficult to distinguish from other varieties, so that effective supervision and arbitration are difficult to realize if the seedlings are on sale, and serious obstruction is brought to the popularization and utilization of the variety. Therefore, there is an urgent need to find a simple, rapid and effective identification means that is truly effective, not affected by the environment, and can accurately distinguish the varieties. The traditional identification of the new variety of the mulberry is mainly based on phenotypic character, and has the advantages of great environmental influence, poor stability and long test period, and seriously influences the effectiveness and authority of the identification of the new variety. The molecular marking technology has the characteristics of high polymorphism, short test period, no environmental influence and the like, and becomes an important means for variety identification and protection. The simple repeated sequence (Simple sequence repeat, SSR) has the advantages of co-dominance, good repeatability, easy detection, simple operation and the like, so that the SSR molecular marker can play a good role in the specific evaluation and identification of mulberry varieties. In the prior art, a specific molecular identification mark and a corresponding detection product aiming at a new mulberry variety 'cloud mulberry No. 9' are lacking, so that the seedling purity of the variety cannot be rapidly and accurately identified by a molecular biological method, and the technical requirements of variety right protection and seedling quality management are difficult to meet. Disclosure of Invention The invention aims to overcome the defect that the a