Double haploid (DH) technology, based on in vivo haploid induction, enables the fixation of recombinant haplotypes within two generations, thereby greatly increasing crop breeding efficiency (Jacquier et al., 2020). Although haploid plants can be produced from some legumes via an in vitro anther/microspore culture approach (Croser et al., 2006), an in vivo (seed-based) haploid induction system has not yet been established for this family, hindering the application of DH technology. Here, we report the successful generation of haploid plants through seeds by editing DMP (DOMAIN OF UNKNOWN FUNCTION 679) homologues in Medicago truncatula, a well-characterized model legume.

Mutations in ZmDMP were shown to enhance haploid induction in maize (Zea mays) when combined with mutations in MTL/NLD/ZmPLA1 (Gilles et al., 2017; Kelliher et al., 2017; Liu et al., 2017; Zhong et al., 2019). Although MTL/NLD/ZmPLA1 is not conserved in dicots, DMP is conserved in both monocots and dicots (including legumes), and loss of function ZmDMP orthologues in the dicot Arabidopsis (Arabidopsis thaliana) trigger maternal haploid induction (Zhong et al., 2020), opening the possibility of applying the DMP-triggered in vivo haploid induction system to leguminous plants. In agreement with previous reports (Zhong et al., 2019, 2020), phylogenetic analysis showed that ZmDMP has homologues in several legumes, including soybean (Glycine max), alfalfa (Medicago sativa) and M. truncatula (Figure 1a). Using M. truncatula, we explored whether the mutation of DMP homologues might be used for haploid induction in legumes.

We searched the M. truncatula genome (v4.0) using a Basic Local Alignment Sequence Tool for Protein (BLASTP) analysis and ZmDMP as query. When using a minimum protein sequence identity of 40%, we identified six putative DMP-like proteins. Phylogenetic analysis showed that MtDMP8 (Medtr7g010890) and MtDMP9 (Medtr5g044580), which are most similar to ZmDMP (63.9% and 62.8% sequence identity, respectively), cluster together with ZmDMP in a separate subclade that includes Arabidopsis DMP8 and DMP9 (Figure 1a). MtDMP8 and MtDMP9 both contained four putative transmembrane domains. Consistent with this prediction, both proteins colocalized with the PIP2A (At3g53420)-based plasma membrane marker pm-GFP (Zhu et al., 2020) when MtDMP8 and MtDMP9 were transiently expressed as red fluorescent protein (RFP) fusions in Arabidopsis leaf protoplasts (Figure 1b). RT-qPCR analysis revealed that both MtDMP8 and MtDMP9 are highly expressed in mature anthers and pollen, with MtDMP9 being more highly expressed, suggesting that MtDMP8 and MtDMP9 function during the late stages of gametophyte development (Figure 1c).

To assess the role of MtDMP8 and MtDMP9 in haploid induction in M. truncatula, we generated single and double knockout mutants in MtDMP8 or MtDMP9 (Figure 1d) using the pDIRECT_22C vector of the CRISPR-Cas9 toolkit (Cermak et al., 2017) and two pairs of specific guide RNA sequences (gRNAs, each pair targeting one gene). After Agrobacterium (Agrobacterium tumefaciens)-mediated transformation of M. truncatula accession R108 (Zhu et al., 2020), CRISPR mutants with deletions and insertions that led to translational frame shifts were found at MtDMP8 and/or MtDMP9 in the T₀ generation (Figure 1d). Pollen development was normal in the T₁ progeny of mtdmp8 and mtdmp9 single mutants, but pollen viability was reduced in mtdmp8 mtdmp9 double mutants (Figure 1e). Furthermore, seed set was slightly reduced in both mtdmp8 and mtdmp9 single mutants, but mtdmp8 mtdmp9 double mutants showed drastically reduced seed set (Figure 1f), confirming previously reported defects in seed set and putative roles for MtDMP8 and MtDMP9 in fertilization. Haploid M. truncatula plants, which exhibit typical haploid characteristics of reduced stature, as well as small ovules and sterile pollen, were identified amongst the self-pollinated progenies of mtdmp8 mtdmp9 mutants (Figure 1g–i). The average haploid induction rate (HIR) ranged from 0.29% to 0.82% among the T₂ progeny of mtdmp8 mtdmp9 mutant lines (Figure 1j). However, not a single haploid plant was identified among the T₂ progeny from selfing mtdmp8 and mtdmp9 single mutants or wild-type plants (Figure 1j). To investigate whether mtdmp8 mtdmp9 mutants could induce haploid embryos in different female parents, the M. truncatula ecotype Jemalong A17 was pollinated with pollen from mtdmp8 mtdmp9-1. We identified three haploids among 550 plants from this crossing, whereas no haploids were found among the 620 plants resulting from the cross using wild-type R108 as pollen donor (Figure 1j). The haploid plants were morphologically similar to the female parent A17 (Figure 1k). Thus, the simultaneous inactivation of MtDMP8 and MtDMP9 can trigger in vivo maternal haploid induction in M. truncatula.

Our successful haploid induction in M. truncatula provides a promising starting point for legume haploid gene editing and mechanistic studies of haploid induction in legumes. Future work will extend the range of applications of DMP-triggered in vivo haploid induction to crops and forages such as soybean and alfalfa, paving the way for the deployment of DH technology in legume breeding.

双单倍体（DH）技术，基于体内单倍体诱导，能够在两代内固定重组单倍体，从而大大提高作物育种效率（Jacquier et al ., 2020）。尽管一些豆科植物可以通过体外花药/小孢子培养方法生产单倍体植物（Croser et al ., 2006），但尚未建立该科的体内（基于种子）单倍体诱导系统，阻碍了应用DH技术。在这里，我们报告了通过编辑DMP ( DOMAIN OF UNKNOWN FUNCTION 679)通过种子成功生成单倍体植物) Medicago truncatula中的同源物，这是一种特征良好的模型豆科植物。

当与MTL / NLD / ZmPLA1中的突变结合时， ZmDMP中的突变可增强玉米（ Zea mays）中的单倍体诱导（Gilles等人，2017；Kelliher等人，2017；Liu等人，2017；Zhong等人） ., 2019 年）。虽然 MTL/NLD/ZmPLA1 在双子叶植物中不保守，但 DMP 在单子叶植物和双子叶植物（包括豆科植物）中都是保守的，双子叶植物拟南芥 ( Arabidopsis thaliana ) 中ZmDMP直系同源物的功能丧失触发母体单倍体诱导 (Zhonget al ., 2020 )，开启了将 DMP 触发的体内单倍体诱导系统应用于豆科植物的可能性。与之前的报道一致（Zhong et al ., 2019 , 2020），系统发育分析表明ZmDMP在几种豆科植物中具有同源物，包括大豆（Glycine max）、紫花苜蓿（Medicago sativa） 和M. truncatula (图 1a)。使用M。 truncatula，我们探讨了DMP同源物的突变是否可用于豆科植物的单倍体诱导。

我们搜索了M。 truncatula基因组 (v4.0) 使用用于蛋白质 (BLASTP) 分析的基本局部比对序列工具和 ZmDMP 作为查询。当使用 40% 的最小蛋白质序列同一性时，我们确定了六种推定的 DMP 样蛋白质。系统发育分析表明，与 ZmDMP 最相似的 MtDMP8 (Medtr7g010890) 和 MtDMP9 (Medtr5g044580)（分别为 63.9% 和 62.8% 的序列同一性）与 ZmDMP 聚集在一个单独的亚进化枝中，包括拟南芥 DMP8 和 DMP9（图 1a） . MtDMP8 和 MtDMP9 都包含四个假定的跨膜结构域。与这一预测一致，这两种蛋白质都与基于 PIP2A (At3g53420) 的质膜标记物 pm-GFP 共定位（Zhu et al ., 2020) 当 MtDMP8 和 MtDMP9 在拟南芥叶原生质体中瞬时表达为红色荧光蛋白 (RFP) 融合体时 (图 1b)。RT-qPCR 分析显示MtDMP8和MtDMP9在成熟的花药和花粉中均高表达，而MtDMP9的表达更高，表明MtDMP8和MtDMP9在配子体发育的后期发挥作用（图 1c）。

为了评估MtDMP8和MtDMP9在M. truncatula单倍体诱导中的作用，我们使用 CRISPR-Cas9 工具包的 pDIRECT_22C 载体在MtDMP8或MtDMP9（图 1d）中产生了单敲除和双敲除突变体（Cermak et al ., 2017）和两对特定的指导 RNA 序列（gRNA，每对靶向一个基因）。经过农杆菌（Agrobacterium tumefaciens）介导的M转化。 truncatula加入 R108 (Zhu et al ., 2020)，在 T _0代的MtDMP8和/或MtDMP9中发现了具有导致翻译移码的缺失和插入的 CRISPR 突变体（图 1d）。在mtdmp8和mtdmp9单突变体的 T ₁后代中花粉发育正常，但在mtdmp8 mtdmp9双突变体中花粉活力降低（图1e）。此外， mtdmp8和mtdmp9单突变体的种子组略有减少，但mtdmp8 mtdmp9双突变体的种子组显着减少（图 1f），证实了先前报道的种子组缺陷和MtDMP8的假定作用和MtDMP9在受精中。在mtdmp8 mtdmp9突变体的自花授粉后代中鉴定出单倍体M. truncatula植物，其表现出典型的矮身高单倍体特征以及小胚珠和不育花粉（图1g-i）。mtdmp8 mtdmp9突变系的T ₂后代的平均单倍体诱导率（HIR）范围为0.29％至0.82％（图1j）。然而，在来自自交mtdmp8和mtdmp9单突变体或野生型植物的 T ₂后代中没有鉴定出单个单倍体植物（图 1j）。调查是否mtdmp8 mtdmp9突变体可以在不同母本中诱导单倍体胚胎，蒺藜苜蓿生态型Jemalong A17用来自mtdmp8 mtdmp9 -1的花粉授粉。我们在来自该杂交的 550 株植物中鉴定了三个单倍体，而在使用野生型 R108 作为花粉供体的杂交产生的 620 株植物中未发现单倍体（图 1j）。单倍体植物在形态上与母本 A17 相似（图 1k）。因此，MtDMP8和MtDMP9的同时失活可以在M中引发体内母体单倍体诱导。 蒺藜_

我们在M中成功的单倍体诱导。 truncatula为豆科植物单倍体基因编辑和豆科植物单倍体诱导机制研究提供了一个有希望的起点。未来的工作将把 DMP 触发的体内单倍体诱导的应用范围扩大到大豆和苜蓿等作物和牧草，为 DH 技术在豆科植物育种中的应用铺平道路。