CRISPRCAS9 for hybrid maize plant in china

CRISPR-CAS9

1. KAHER’S DR. PRABHAKAR KORE BASIC SCIENCE RESEARCH CENTRE, BELAGAVI, KARNATAKA JOURNAL CLUB PRESENTATION

2. Expediting Next-Generation Hybrid Technology in Recalcitrant Maize Inbreds through In Vivo Targeted Activity of CRISPR/Cas9 Liudi Hou 1,2,†, Bing Xiao 3,4,†, Jinjie Zhu 2, Changlin Liu 2, Qingyu Wu 3 , Chuanxiao Xie 2, Huawen Zou 1,* and Xiantao Qi 2,*

3. Yangtze University Beijing, China Received: 30 April 2024 Revised: 23 May 2024 Accepted: 24 May 2024 Published: 27 May 2024

4. Introduction Importance of Maize: Maize is a crucial cereal crop for global food security, known for its heterosis, which boosts yield and vigor in hybrids. Seed Production Technology: Male sterility is essential for hybrid seed purity and efficiency. Cytoplasmic male sterility (CMS) and genic male sterility (GMS) are two main types, with GMS facing challenges in segregation. Photoperiod Thermosensitive GMS (PTGMS): Offers a solution where a single line can serve as both sterile and maintainer, with its function determined by environmental conditions.

5. Introduction conti … Seed Production Technology (SPT): Overcomes challenges in managing GMS lines by enabling distinct identification of male sterility and maintainer lines, expanding production methods. Gene Editing Revolution: CRISPR/Cas9 technology has enabled precise genetic enhancements in maize, including the development of the Manipulated GMS Maintainer (MGM) system. MGM System: Integrates CRISPR/Cas9 with SPT, facilitating efficient creation of sterile genes. It includes the MS26∆E5 editor T-DNA and MGM T-DNA vectors, enabling non-transgenic sorting of maintainer and sterile seeds, streamlining hybrid seed production.

6. Aim of the study The aim of this study is to improve hybrid seed production in commercial maize by integrating the MGM system into the Z372 inbred line. This involves using CRISPR/Cas9 to target and delete the Ms26 gene in Z372, aiming for high genetic background restoration.

7. Materials and Methods

8. 1. GENOMIC DNA EXRACTION Plant genomic DNA was extracted from leaves of maize using CTAB method and was used in detection of CAS9 gene and mutation analysis of MS26 gene Z372 MGM plants were screened using DsRed fluorescence as a reliable indicator to select MGM positive kernels and exclude GMS negative kernels 2. DETECTION OF GENETICALY MODIFIED COMPONENTS The genetically modified components of CRISPR-Cas9 in both MGM and GMS material was identified by performing PCR reaction using Cas9-F and Cas9-R primers The mutations in ms26 gene target in both MGM and GMS sample was detected by PCR using MS26-F and MS26-R Primers

9. 3. THE IDENTIFICATION OF MGM TRANSGENIC COPY NUMBER The previously extracted genomic DNA was digested with Hind-III enzyme Addition of predesign primers and probes to the reaction system containing DNA Addition of Bio-Rad droplet generation oil into the reaction mixture and loaded it into a droplet generator cartridge to create droplets These generated droplets were carefully then transformed to a 96-well PCR plate and PCR reaction was carried out Then the plate was transferred to a droplet reader for analysis of droplet count and finally the measurement of transgenic copy number of generated using Bio-Rad QuantaSoft TM software

10. 4. COPY NUMBER CALCULATING Using QuantaSoft TM software, the copy number of target gene relative to an invariant reference gene was ascertained by calculating the ratio of target molecule concentration to the reference molecule concentration, multiplied by copy number of the reference species in the genome. The formula for calculation is expressed as CN=A/BXNB 5. WHOLE-GENOME SNP GENOTYPING Gene chip detection was performed using the gene sequencer. Where initial data processing involved Background marker filtering Selecting only homozygous and inconsistent loci between parental lines. M arker integrity filtering was then performed by extracting the SNP with the lowest missing proportion in each mSNP region as the representative SNP marker for that region

11. 6 . WHOLE GENOME SNP ANALYSIS AND GENOTYPE CORRECTION USING SMOOTH STATISTICAL METHOD Genotype correction and imputation conducted using smooth software which leverages the principal that recombination events tend to occur in specific fragments The smooth statistical methods used for whole-genome SNP analysis facilitated the correction of potential errors in progeny genotypes at specific loci and the imputation of partially missing sites The background recovery rate was calculated by using the filtered markers

12. 7. POLLENS STAINING WITH IDOINE-POTASSIUM IODIDE (KI/I 2 ) To visualize the pollen grains iodine potassium iodide solution was utilized A mature anther was placed on a microscope slide to which 1-2 drops of Lugol’s solution was applied for staining The slide was observed under an optical microscope

13. 8. IMAGING AND FLOURESCENCE OBSERVATION The stained pollen grains were observed through a Nikon stereomicroscope at a 10X magnification The identification of MGM maize ears and kernels was done using a fluorescent flashlight equipped with 550 nm excitation wavelength and DsRed -specific filter glasses. To observe the longitudinal sections of MGM-positive seeds, they used the stereomicroscope again, with 500 nm excitation wavelength and DsRed -specific filter glasses. 9. Chi-Square ANALYSIS To ascertain the ratio of viable to non-viable pollen in the anthers of Z372-MGM2, the chi-square (x 2 ) test was employed to measure the deviation between observed and expected values of the sample. 10. FIELD EXPERIMENTS AND TRAIT MEASUREMENTS The experimental setup consisted of plots measuring 4 meter in length and 1.2 meter in width . To mitigate the potential effect of edge effects on our findings only plants situated in the centre of each plot were selected for sampling

14. The experiment encompassed cultivation and pollination of materials across around five generation in the initial generation which is designated as parental generation eighteen individuals of the ZC01-3a-7 and Z372 were planted in separate rows Select a healthy individual for cross pollination, where ZC0137 serving as female parent and Z372 serving as a male parent, yielding F1 generation Plant Z372 Row along side 20 F1 Seeds DNA was extracted from leaf samples for genotyping MGM, Cas9 , MS26 genes BC1F1 generation. BC2F1 generation. BC2F2 generation. 11. PLANTING AND MANAGEMENT OF MATERIALS ACROSS GENERATION

15. RESULTS

16. Generation of ms26∆E5 mutations using in VIVO Cas9 activity among MGM and GMS progeny the Z372 line.

17. Whole-genome SNP analysis of background restoration rate in Z372-MGM and Z372-GMS

18. Discussion Advances in Maize Breeding with CRISPR/Cas9 Technology - Combining CRISPR/Cas9 gene editing with backcross breeding has made it easier to develop GMS and MGM lines in maize. Efficient Strategy for Developing GMS Lines - In this study, researchers edited the Ms26 gene in maize (Z372) using genetic editing, adding only the desired mutation (ms26∆E5) and maintaining the plant's original characteristics. Preventing Linked Drag Transmission - The MGM strain's ZmAA1 gene, controlled by the PG47 promoter, prevents linked drag segments from being transmitted to GMS lines through male gametes, ensuring Z372 GMS plants are free of linked drag, preserving desirable traits' purity. Implications and Future Directions - The GMS lines enable efficient hybrid seed production and commercialization. Future research aims to expand this method to more maize varieties and other plants, promoting sustainable agriculture.

19. Conclusion They developed an efficient method to create genic male sterility (GMS) and maintainer lines in maize using CRISPR/Cas9 technology and backcross breeding. This approach achieves high background recovery rates by the BC2F2 generation and produces transgene-free GMS lines, ideal for commercial hybrid seed production. This research shows the potential of gene editing for quickly and effectively developing GMS and MGM lines in various maize varieties and other plants. Patents : A Chinese patent corresponding to this study has been authorized. Patent no. ZL201710223233.1

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