Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9

Zhang, Zhihui; Wang, Jie; Kuang, Huaqin; Hou, Zhihong; Gong, ****; Bai, Mengyan; Zhou, Shaodong; Yao, **aolei; Song, Shikui; Yan, Long; Guan, Yuefeng

doi:10.1007/s42994-022-00071-8

Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9

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Published: 07 March 2022

Volume 3, pages 110–114, (2022)
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Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9

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Zhihui Zhang¹^na1,
Jie Wang^1,2^na1,
Huaqin Kuang²^na1,
Zhihong Hou³^na1,
**** Gong²,
Mengyan Bai⁴,
Shaodong Zhou¹,
**aolei Yao¹,
Shikui Song²,
Long Yan⁵ &
…
Yuefeng Guan ORCID: orcid.org/0000-0002-1436-3828²

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Abstract

Pod shattering can lead to devastating yield loss of soybean and has been a negatively selected trait in soybean domestication and breeding. Nevertheless, a significant portion of soybean cultivars are still pod shattering-susceptible, limiting their regional and climatic adaptabilities. Here we performed genetic diagnosis on the shattering-susceptible trait of a national registered cultivar, Huachun6 (HC6), and found that HC6 carries the susceptible genotype of a candidate Pod dehiscence 1 (PDH1) gene, which exists in a significant portion of soybean cultivars. We next performed genome editing on PDH1 gene by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). In T₂ progenies, several transgene-free lines with pdh1 mutations were characterized without affecting major agronomic traits. The pdh1 mutation significantly improved the pod shattering resistance which is associated with aberrant lignin distribution in inner sclerenchyma. Our work demonstrated that precision breeding by genome editing on PDH1 holds great potential for precisely improving pod shattering resistance and adaptability of soybean cultivars.

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Conventional breeding has been playing a fundamental role in crop improvement in the past century. Nevertheless, incorporating beneficial genetic variations while excluding unfavorable alleles remains a major challenge, impacted by recombination rates, population size, allelic variations, and effectiveness of phenotypic selection (Lyzenga et al. 2021). As a result, an elite cultivar may take 5–10 years to develop, yet still carry substantial unfavorable allelic variations. Genome editing technologies, particularly clustered regularly interspaced short palindromic repeats (CRISPR)/associated (Cas) nucleases (CRISPR/Cas), can facilitate knock-out, knock-in, and base-editing of target genes, opening up new possibilities to precise improvement of crops (Cai et al. 2020; Chen et al. 2019; Li et al. Full size image

To diagnose the genetic basis of pod shattering susceptibility in HC6, we performed QTL map** with a recombinant inbred line (RIL) population of HC6 and pod shattering-resistant JD12. A reproducible major QTL controlling shattering resistance was mapped to chromosome 16, which overlapped with the previously reported qPDH1 QTL (Fig. 1C). The putative PDH1 gene was proposed as Glyma16g25580 (Wm82.a1.v1) encoding a dirigent (DIR) family protein expressed in the inner sclerenchyma of pod walls in shattering susceptible varieties (Funatsuki et al. 2014). We sequenced Glyma16g25580 and found a SNP in JD12 (chr16-29944393, A/T (HC6/JD12); Wm82.a2.v1) leading to a nonsense variant, consistent with a previous report that Glyma16g25580 exists as a truncated gene in shattering-resistant cultivars (Funatsuki et al. 2014). According to the nonsense SNPs A-T, we surveyed the haplotypes of PDH1 gene among resequencing data from 1080 soybean cultivars. The proposed shattering-resistant H-T haplotype is largely fixed in regions with low relative humidity and mechanic harvesting, including 94.50% in northeast China and 85.17% in HHH region (Fig. 1D). In contrast, the shattering-susceptible H-A haplotype is retained in areas with relatively high humidity and/or manual harvesting, including 59.72% of south China, 83.64% of Japan, 81.51% of Korea, and 65.71% of southeast Asia cultivars (Fig. 1D). This result suggested that the presence of Glyma16g25580 gene is highly associated with the relative humidity and harvesting mode shaped pod shattering trait.

Genome editing technologies, particularly clustered regularly interspaced short palindromic repeats (CRISPR)/associated (Cas) nucleases (CRISPR/Cas), can facilitate knock-out, knock-in, and base-editing of target genes, creating an avenue for elimination of maladapted genetic variations in germplasm (Cai et al. 2020; Chen et al. 2019; Li et al. 2020; Tang et al. 2019; Wang et al. 2020b). The Glyma16g25580 (designated PDH1 thereafter) open reading frame is mainly responsible for the pod shattering susceptible trait. We then sought to generate mutation of PDH1 by CRISPR/Cas9 in HC6. We designed three sgRNAs, cloned into pGES701 vector individually, and pooled for Agrobacterium-mediated transformation (Fig. 1E–F). Among 23 T₀ transgenic plants, 17 lines contained sgRNA, 5 lines contained 2 sgRNAs, and 1 contained all sgRNAs. Hi-TOM (Liu et al. 2019) and sanger sequencing analysis showed that 10 of the 23 T₀ transgenic plants carried mutations in at least one target locus. In T₁ progenies, we characterized homozygous mutant plants from two lines, HC6^pdh1−5 with a 221 bp deletion and HC6^pdh1−9 with 1 bp deletion, respectively (Fig. 1G). qRT-PCR showed that the expression of PDH1 gene was diminished by homozygous mutations in both lines (Fig. 1H). In T₂ progenies, homozygous mutant plants without transgene were characterized in both lines (data not shown).

In a heat dried assay, HC6 pods displayed a high ratio of shattering (Ratio Pod Shattering = 63.75%, n = 8; Fig. 1I–J). In contrast, HC6^pdh1−5 and HC6^pdh1−9exhibited significant resistance to pod shattering (RPS = 1.25%, n = 8; Fig. 1I–J). The dehisced pod walls of HC6^pdh1−5 and HC6^pdh1−9 exhibited much lower degrees of torsion than those of HC6, which is consistent with previous finding that qPDH1 locus was associated with curling of pod walls (Funatsuki et al. 2014). We then analyzed the anatomical characteristics of HC6^pdh1−5, HC6^pdh1−9and HC6 pods. We found that the lignin layer in inner sclerenchyma of HC6 tends to be thicker and looser. In contrast, the lignin layer in HC6^pdh1−5 and HC6^pdh1−9 appeared to be thinner and compact (Fig. 1L). This result demonstrated that genome editing of the PDH1 gene may affect the pod shattering resistance of HC6 by influencing the deposition of lignin layer in inner sclerenchyma. In 2021 summer, we performed a field trail at Shijiazhuang, Heibei, China. When harvesting was delayed for 2 weeks, HC6 exhibited significant pod shattering that caused substantial yield losses. In contrast, HC6^pdh1−5 and HC6^pdh1−9 barely showed shattering at the same condition (Fig. 1K). Meanwhile the genome editing of PDH1 did not significantly affect other agronomic traits, including plant height, branch number, pod number per plant, seed number per plant, 100-seed weight, and seed yield per plant (as determined before pod shattering occurs in HC6) (Fig. 1M).

Here we showcased the genetic diagnosis and “gene therapy” of the pod shattering trait of soybean by CRISPR/Cas9 genome editing. In comparison with introgression breeding, genome editing approach could rapidly and precisely improve a trait, and is not limited by genetic diversity of breeding populations (Chen et al. 2019; Li et al. 2020; Manghwar et al. 2019). Therefore, genome editing can be integrated as a routine part of a breeding cycle to eliminate unfavorable alleles (such as PDH1) to facilitate the generation of a genetically superior cultivar.

Materials and methods

Genomic editing of soybean PDH1 by CRISPR/Cas9

CRISPR/Cas9 mutations of PDH1 in HC6 was performed using the protocol published previously (Bai et al. 2020), and a new CRISPR/Cas9 vector, pGES701 (Fig. 1E), was used for genome editing. The mixed Agrobacterium solution was transformed into the soybean cultivar HC6 via A. tumefaciens-mediated transformation, as described previously (Bai et al. 2020).

RNA extraction and real-time qPCR

To measure the expression of PDH1 gene in WT plants and mutants, real-time qPCR was performed using total RNA extracted from pod wall samples (3 weeks after flowering). Total RNA extraction, cDNA synthesis, and data analysis were performed as previously described (Wang et al. 2020a).

Evaluation of pod dehiscence percentage

The pod dehiscence percentage of WT and mutations was evaluated by heat treatment: ten fully matured pods of each plant were collected and kept in a circulation drier at 60 °C for 6 h and then counted the number of dehisced pods, respectively. Eight plants per genotype were sampled. Fully matured pods of WT and mutations were examined for pod-wall lignification. Soybean pod was embedded in 7% agarose and cross-sections (80 μm thick) were stained with 10% toluidine blue, and observed under a microscope (Eclipse Ni-U, Nikon, Japan).

References

Bai M et al (2020) Generation of a multiplex mutagenesis population via pooled CRISPR–Cas9 in soya bean. Plant Biotechnol J 18:721–731. https://doi.org/10.1111/pbi.13239
Article CAS Google Scholar
Cai Y et al (2020) Target base editing in soybean using a modified CRISPR/Cas9 system. Plant Biotechnol J 18:1996–1998. https://doi.org/10.1111/pbi.13386
Article Google Scholar
Chen K, Wang Y, Zhang R, Zhang H, Gao C (2019) CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol 70:667–697. https://doi.org/10.1146/annurev-arplant-050718-100049
Article CAS Google Scholar
Funatsuki H et al (2014) Molecular basis of a shattering resistance boosting global dissemination of soybean. Proc Natl Acad Sci 111:17797–17802. https://doi.org/10.1073/pnas.1417282111
Article CAS Google Scholar
Li J, Li H, Chen J, Yan L, **a L (2020) Toward precision genome editing in crop plants. Mol Plant 13:811–813. https://doi.org/10.1016/j.molp.2020.04.008
Article CAS Google Scholar
Liu Q et al (2019) Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems. Sci China Life Sci 62:1–7. https://doi.org/10.1007/s11427-018-9402-9
Article CAS Google Scholar
Lyzenga WJ, Pozniak CJ, Kagale S (2021) Advanced domestication: harnessing the precision of gene editing in crop breeding. Plant Biotechnol J 19:660–670. https://doi.org/10.1111/pbi.13576
Article CAS Google Scholar
Manghwar H, Lindsey K, Zhang X, ** S (2019) CRISPR/Cas system: recent advances and future prospects for genome editing. Trends Plant Sci 24:1102–1125. https://doi.org/10.1016/j.tplants.2019.09.006
Article CAS Google Scholar
Tang X et al (2019) Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnol J 17:1431–1445. https://doi.org/10.1111/pbi.13068
Article CAS Google Scholar
Wang J et al (2020a) Generation of seed lipoxygenase-free soybean using CRISPR–Cas9. Crop J 8:432–439. https://doi.org/10.1016/j.cj.2019.08.008
Article Google Scholar
Wang M et al (2020b) Targeted base editing in rice with CRISPR/ScCas9 system. Plant Biotechnol J 18:1645–1647. https://doi.org/10.1111/pbi.13330
Article Google Scholar
Zhang J, Singh AK (2020) Genetic control and geo-climate adaptation of pod dehiscence provide novel insights into soybean domestication. G3 (bethesda) 10:545–554. https://doi.org/10.1534/g3.119.400876
Article CAS Google Scholar

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Acknowledgements

We appreciate Professor Hong Liao for providing RIL population for analysis. We thank the Cytology Core of FAFU-UCR Joint Center for the assistance on microscopy. This work was supported by Innovative Research Groups of the Natural Science Foundation of Hebei province (C2020301020).

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Zhihui Zhang, Jie Wang, Huaqin Kuang and Zhihong Hou contribute equally to this work.

Authors and Affiliations

College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian, China
Zhihui Zhang, Jie Wang, Shaodong Zhou & ** Gong, Shikui Song & Yuefeng Guan
College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing, 163319, Heilongjiang, China
Zhihong Hou
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian, China
Mengyan Bai
The Key Laboratory of Crop Genetics and Breeding of Hebei, Institute of Cereal and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang, 050035, China
Long Yan

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Zhihui Zhang
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Jie Wang
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Huaqin Kuang
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Zhihong Hou
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**** Gong
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Mengyan Bai
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Shaodong Zhou
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**aolei Yao
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Shikui Song
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Long Yan
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Yuefeng Guan
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Contributions

YG designed research; ZZ, JW, HK, ZH, DK, PG, MB, SZ, XY, LC, HK, and SS performed experiments; ZZ, JW, HK, ZH, LY, and YG analyzed data; YG, LY, ZZ, and JW wrote the manuscript.

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Correspondence to Yuefeng Guan.

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Zhang, Z., Wang, J., Kuang, H. et al. Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9. aBIOTECH 3, 110–114 (2022). https://doi.org/10.1007/s42994-022-00071-8

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Received: 20 December 2021
Accepted: 09 February 2022
Published: 07 March 2022
Issue Date: June 2022
DOI: https://doi.org/10.1007/s42994-022-00071-8

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Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9

Abstract

Materials and methods

Genomic editing of soybean PDH1 by CRISPR/Cas9

RNA extraction and real-time qPCR

Evaluation of pod dehiscence percentage

References

Acknowledgements

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