An in planta haploid induction system in Brassica napus

Doubled haploid technology is widely used to accelerate plant breeding,but its use in the important oilseed crop Brassica napus L.is limited because B.napus haploids could only be obtained through in vitro anther or microspore cultures.Recently,maize(Zea mays)lines containing mutations in Domain of unknown function 679 membrane protein(DMP)were used as haploid inducer lines.This new haploid induction mechanism has been extended to several other plants,including the dicots Arabidopsis thaliana,tomato(Solanum lycopersicum),and tobacco(Nicotiana tabacum).Here,we knocked out four BnaDMP genes in the B.napus cultivar Westar using a clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 vector with an enhanced green fluorescent protein expression cassette.Plants with DMP mutations in B.napus in the T_(0),T_(1),and T_(2) generations exhibited a haploid induction rate up to 2.53%.These results suggest that targeting BnaDMP could be useful for haploid induction in B.napus.

Haploids can be induced in knockout mutants of OsPLA1,but not OsDMP3 or OsDMP6,in rice

Doubled haploid(DH)technology is an important tool in crop breeding because it can significantly accelerate the breeding process.ZmPLA1/MATL/NLD and ZmDMP are two key genes controlling haploid induction(HI)in maize,exhibiting a synergistic effect.However,it is unknown whether knock out of ZmDMP orthologs can stimulate HI in rice.In this study,a ZmPLA1 ortholog(OsPLA1)and two ZmDMP orthologs(OsDMP3 and OsDMP6)were identified in rice.All three genes encode plasma membrane-localized proteins and were highly expressed in mature anthers.Knockout of OsPLA1 in both Minghui 63 and Nipponbare resulted in reduced seed setting rate(SSR)and caused HI.The osdmp3,osdmp6 and the double mutant failed to trigger HI independently,nor increased the haploid induction rate(HIR)when combined with ospla1.Repeated pollinations operations of QX654A with the ospla1 mutant significantly improve SSR,while reducing HIR.RNA-seq profiling of mature ospla1 mutant anthers indicated that a large number of differentially expressed genes(DEGs)were enriched in redox homeostasis and lipid metabolic GO terms,plant hormone signal transduction,and MAPK signaling pathways.These findings provide important insights towards construction of an efficient DH breeding technology and study of the molecular mechanism of HI in rice.

A female in vivo haploid-induction system via mutagenesis of egg cell-specific peptidases

Crop breeding schemes can be significantly accelerated by using(doubled)haploid plants.In vivo haploid induction has been applied in plant breeding for decades but is still not available for all crops and genotypes,and haploidization rates are generally very low.Therefore,methodological improvements to and new concepts for haploidization are required.Here,we report a novel system for the induction of haploid plants by mutating genes encoding egg cell-specific aspartic endopeptidases(ECSs).We show that after successful sperm–egg cell fusion,ECSs play a critical role to ensure male and female nucleus fusion after fertilization.The ecs1 ecs2 double mutant can induce haploids by both selfing and hybridization in Arabidopsis and ECS mutation is also capable of producing haploids in rice.In summary,our study develops a novel approach for maternal haploidization and provides new insights into the molecular basis of fertilization.

High temperature increases centromeremediated genome elimination frequency and enhances haploid induction in Arabidopsis

Double haploid production is the most effective way to create true-breeding lines in a single generation.In Arabidopsis,haploid induction via mutation of the centromere-specific histone H3(cenH3)has been shown when the mutant is outcrossed to the wild-type,and the wild-type genome remains in the haploid progeny.However,factors that affect haploid induction are still poorly understood.Here,we report that a mutant of the cenH3 assembly factor Kinetochore Null2(KNL2)can be used as a haploid inducer when pollinated by the wild-type.We discovered that short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction 10-fold.We also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines,suggesting that haploidinducing lines in crops can be identified in a naturally occurring or chemically induced mutant population,avoiding the generic modification(GM)approach at any stage.Furthermore,a cenh3-4 mutant functioned as a haploid inducer in response to short-term heat stress,even though it did not induce haploids under standard conditions.Thus,we identified KNL2 as a new target gene for the generation of haploid-inducer lines and showed that exposure of centromeric protein mutants to high temperature strongly increases their haploid induction efficiency.

Doubled haploid technology and synthetic apomixis:Recent advances and applications in future crop breeding

Doubled haploid(DH)technology and synthetic apomixis approaches can considerably shorten breeding cycles and enhance breeding efficiency.Compared with traditional breeding methods,DH technology offers the advantage of rapidly generating inbred lines,while synthetic apomixis can effectively fix hybrid vigor.In this review,we focus on(i)recent advances in identifying and characterizing genes responsible for haploid induction(Hl),(ii)the molecular mechanisms of Hl,(ili)spontaneous haploid genome doubling,and(iv)crop synthetic apomixis.We also discuss the challenges and potential solutions for future crop breeding programs utilizing DH technology and synthetic apomixis.Finally,we provide our perspectives about how to integrate DH and synthetic apomixis for precision breeding and de novo domestication.

A reactive oxygen species burst causes haploid induction in maize

Haploid induction (HI) is an important tool in crop breeding. Phospholipase A1 (ZmPLA1)/NOT LIKE DAD (NLD)/MATRILINEAL (MTL) is a key gene controlling HI in maize;however, the underlying molecular mechanism remains unclear. In this study, to dissect why loss of ZmPLA1 function could mediate HI we performed a comprehensive multiple omics analysis of zmpla1 mutant anthers by integrating transcriptome, metabolome, quantitative proteome, and protein modification data. Functional classes of significantly enriched or differentially abundant molecular entities were found to be associated with the oxidative stress response, suggesting that a reactive oxygen species (ROS) burst plays a critical role in HI. In support of this, we further discovered that a simple chemical treatment of pollen with ROS reagents could lead to HI. Moreover, we identified ZmPOD65, which encodes a sperm-specific peroxidase, as a new gene controlling HI. Taken together, our study revealed a likely mechanism of HI, discovered a new gene controlling HI, and created a new method for HI in maize, indicating the importance of ROS balance in maintaining normal reproduction and providing a potential route to accelerate crop breeding.

Construction of homozygous diploid potato through maternal haploid induction

Reinventing the tetraploid potato into a seed-propagated,diploid,hybrid potato would significantly accelerate potato breeding.In this regard,the development of highly homozygous inbred lines is a prerequisite for breeding hybrid potatoes,but self-incompatibility and inbreeding depression present challenges for developing pure inbred lines.To resolve this impediment,we developed a doubled haploid(DH)technology,based on mutagenesis of the potato DOMAIN OF UNKNOWN FUNCTION 679 membrane protein(StDMP)gene.Here,we show that a deficiency in StDMP allows the generation of maternal haploids for generating diploid potato lines.An exercisable protocol,involving hybridization,fluorescent marker screening,molecular and flow cytometric identification,and doubling with colchicine generates nearly 100%homozygous diploid potato lines.This dmp-triggered haploid induction(HI)system greatly shortens the breeding process and offers a robust method for generating diploid potato inbred lines with high purity.

AtRKD5 inhibits the parthenogenic potential mediated by AtBBM

Parthenogenesis,the development of unfertilized egg cells into embryos,is a key component of apomixis.AtBBM(BABY BOOM),a crucial regulator of embryogenesis in Arabidopsis,possesses the capacity to shift nutritional growth toward reproductive growth.However,the mechanisms underlying AtBBM-induced parthenogenesis remain largely unexplored in dicot plants.Our findings revealed that in order to uphold the order of sexual reproduction,the embryo-specific promoter activity of AtBBM as well as repressors that inhibit its expression in egg cells combine to limiting its ability to induce parthenogenesis.Notably,AtRKD5,a RWPRK domain-containing(RKD)transcription factor,binds to the 3'end of AtBBM and is identified as one of the inhibitory factors for AtBBM expression in the egg cell.In the atrkd5 mutant,we successfully achieved enhanced ectopic expression of AtBBM in egg cells,resulting in the generation of haploid offspring via parthenogenesis at a rate of 0.28%.Furthermore,by introducing chimeric Arabidopsis and rice BBM genes into the egg cell,we achieved a significant 4.6-fold enhancement in haploid induction through the atdmp8/9 mutant.These findings lay a strong foundation for further exploration of the BBM-mediated parthenogenesis mechanism and the improvement of haploid breeding efficiency mediated by the dmp8/9 mutant.