Haploid Induction Research Articles

Influence of Culture Media and Their Compositions on Haploid Induction in Indian Short Day Onion

Two media, Gamborg’s medium (B5) and Murashige and Skoog’s medium (MS) with various combinations, were used to study their effect on haploid induction efficiency from unpollinated flowers or ovaries in tropical short day onion. In B5 medium, highest induction efficiency (1.9%) was observed on HAP18 followed by HAP17 and HAP05 whereas in MS medium, HAP40 recorded highest induction frequency (5.0%) followed by HAP32. Kinetin, meta-topolin, thidiazuron did not influence embryo induction. Sucrose at 7.5% in B5 medium and at 10% in MS medium was found to be optimum for induction. Days for plantlet induction were 98.9 ± 3.9 days in B5 medium and 83.1 ± 2.7 days in MS medium. Induction rate of 0.51% and survival of 27.8% in B5 medium and induction rate of 0.72% and survival of 33% in MS medium were observed. Three haploids, 1 mixoploid and 1 diploid in B5 medium and 2 haploids, one diploid in MS medium were obtained. Flow cytometry and cytology confirmed the status of haploid plants. MS medium was found to be better than B5 medium and newer combinations for haploid induction were identified. Plant Preservative Mixture (PPM™) at 1 mg/l was found to be efficient for inhibiting contamination from field grown flower buds. These combinations will be helpful in devising new strategies for faster and higher rate of haploid induction in tropical short day Indian cultivars.

Haploid identification using tropicalized haploid inducer progenies in maize

Abstract The aim of this study was to identify maize haploid plants and compare the efficiency of identification of maize haploid plants using the R1-nj morphological marker, plant vigor, flow cytometry, chromosome counting, and microsatellite molecular markers under tropical conditions. We also established a protocol for chromosome duplication in maize haploid plants. Fourteen S0:1 and seven S2:3 haploid inducer progenies were crossed with GNZ9501 in 2012/2013 and 2014/2015, respectively. Through use of the R1-nj trait, we were able to identify 552 putative haploid seeds in 2012/2013 and 260 putative haploid seeds in 2014/2015. Only 1.84% were true positives according to flow cytometry in 2012/2013. In 2014/2015, 75% of the putative haploids were true negatives according to molecular markers. Plant vigor had a high proportion of true negatives. Molecular markers and flow cytometry are more efficient in classifying plant ploidy level. Chromosome duplication was efficient in all plants.

Detection of Chromothripsis in Plants.

Chromothripsis, or chromosome shattering, occurs after chromosomes missegregate, are pulverized and subsequently repaired erroneously, leading to highly complex structural rearrangements. In plants, chromothripsis has been observed as a result of mitotic malfunction connected with the incomplete loss of haploid inducer chromosomes during uniparental genome elimination. Uniparental genome elimination, a process that results in haploid induction, is a phenomenon that typically results in the loss of an entire parental chromosome set in early embryos, resulting in haploid plants. In Arabidopsis thaliana, genome elimination can be achieved via the manipulation of the centromere-specific histone H3 variant, CENH3. Genomic characterization of F1 progeny resulting from CENH3-mediated genome elimination crosses in Arabidopsis revealed haploids (~39%), diploids (~25%), and aneuploids (~37%). Within the aneuploid class, ~11% show evidence for chromothripsis. Here, we present a protocol to identify Arabidopsis aneuploids that have inherited chromothriptic chromosomes during genome elimination crosses and describe in detail how to perform in silico reconstructions for individuals with chromothripsis using the somatic mutation finder (SMuFin) tool.

Improved Procedure for Induction of the Androgenetic Doubled Haploids in Zebrafish.

Androgenesis is useful for induction of doubled haploids from male genetic resources and contributes to the restoration of individuals from cryopreserved sperm. Here, we determined the suitable conditions for egg in vitro preservation and the suitable dose of UV irradiation for genetic inactivation of the egg nucleus, and established an improved procedure for induction of androgenetic-doubled haploids in zebrafish. The suitable solution for egg preservation was evaluated by the fertilization rate using different types of solutions or conditions. Hank's solution with 0.5% bovine serum albumin (pH8.0) was suitable for the preservation of zebrafish eggs. In addition, we discovered an improvement of fertilization rates by temporal preservation of ovulated eggs in the suitable solution. UV irradiation of eggs at 50-75 mJ/cm2 induced haploid embryos. Microsatellite genotyping using eight loci revealed the paternity and homozygosity of the putative androgenetic doubled haploids. The yield rate of androgenetic doubled haploids, which were induced by UV irradiation and heat shock, ranged from 0.4% to 10.7%.

Single nucleus sequencing reveals spermatid chromosome fragmentation as a possible cause of maize haploid induction

Production of maternal haploids using a conspecific haploid inducer is routine and highly efficient in maize. However, the underlying mechanism of haploid induction (HI) is unclear. We develop a method to isolate three nuclei from a pollen grain and four microspores from a tetrad for whole-genome sequencing. A high rate of aneuploidy is observed at the three-nucleus stage (6/22 pollens) rather than at the tetrad stage (1/72 microspores) in one HI line CAU5. Frequent aneuploidy is also observed in another two inducer lines, but not in two regular lines, which implies that HI may be associated with pollen aneuploidy. We further sequence the individual embryos and endosperms of 88 maize kernels crossing between regular and inducer lines. Genome-wide elimination of the CAU5-derived chromosome is identified in eight of 81 embryos. Together, these results suggest that continuous chromosome fragmentation occurring post meiosis in the gametophyte may cause haploidy of the embryo.

Haploid induction in plants

Gilles et al. introduce the technique of haploid induction in plant breeding.

Novel technologies in doubled haploid line development

Summaryhaploid inducer line can be transferred (DH) technology can not only shorten the breeding process but also increase genetic gain. Haploid induction and subsequent genome doubling are the two main steps required for DH technology. Haploids have been generated through the culture of immature male and female gametophytes, and through inter‐ and intraspecific via chromosome elimination. Here, we focus on haploidization via chromosome elimination, especially the recent advances in centromere‐mediated haploidization. Once haploids have been induced, genome doubling is needed to produce DH lines. This study has proposed a new strategy to improve haploid genome doubling by combing haploids and minichromosome technology. With the progress in haploid induction and genome doubling methods, DH technology can facilitate reverse breeding, cytoplasmic male sterile (CMS) line production, gene stacking and a variety of other genetic analysis.

Maternal doubled haploid production in interploidy hybridization between Brassica napus and Brassica allooctaploids.

We found a new in vivo route to produce maternal doubled haploid of Brassica napus . The pollen donor, an allooctaploid rapeseed, acts as a DH inducer. Inbred line has a powerful advantage in cultivar breeding and genetic analysis. Compared to the traditional breeding methods, doubled haploid production can save years off the breeding process. Though genotype-dependent tissue culture methods are widely used in the Brassica crops, seed-based in vivo doubled haploid developing systems are rare in nature and in the laboratory. As interspecific cross and interploid hybridization play an important role in genome evolution and plant speciation, we created a new Brassica artificial hybrid, a Brassica allooctaploid (AAAACCCC, 2n=8×=76), by interspecific crossing and genome doubling. A homozygous line was observed at the third self-generation of a synthesized Brassica allohexaploid (AAAACC, 2n=6×=58). Crosses between B. napus as female and Brassica allooctaploid as pollen donor were conducted, which yielded maternal doubled haploid B. napus that were identified based on phenotype, ploidy, and molecular analysis. The Brassica octaploid acted as a maternal doubled haploid inducer and had a relatively high induction rate. Our research provides a new insight for generation of homozygous lines in vivo using a single-step approach, as well as promotes the understanding in breeding programs and genetic studies involving the Brassicas.

Identification of in vivo induced maternal haploids in maize using seedling traits

In vivo haploid induction in high frequency followed by efficient identification of haploids are important components of deriving completely homozygous doubled haploid (DH) lines in maize. Several genetic marker systems were proposed and/or used for identification of in vivo maternal haploids in maize, such as R1-nj (Navajo), high oil, red root and transgenic markers. In this study, we propose a new method of haploid/diploid identification based on natural differences in seedling traits of haploids and diploids, which can be used in any induction cross independently of the genetic marker systems. Using confirmed haploids and diploids from five different populations, the study established that haploid and diploid seedlings exhibit significant differences for seedling traits, particularly radicle length (RL), coleoptile length (CL), and number of lateral seminal roots (NLSR). In six populations that exhibited complete inhibition of the commonly used R1-nj (Navajo) marker, we could effectively differentiate haploids from diploids by visual inspection of the seedling traits. In the haploid seed fraction identified based on R1-nj marker in ten populations, false positives were reduced several-fold by early identification of haploids at seedling stage using the seedling traits. We propose that seedling traits may be integrated at the haploid identification stage, especially in populations that are not amenable to use of genetic markers, and for improving the efficiency of DH line production by reducing the false positives.

New insight into the mechanism of heterofertilization during maize haploid induction

To study the influence of both pollen competition ability (PCA) and haploid induction rate (HIR) on heterofertilization, we used maize haploid inducers with different HIRs as male parents. For these experiments, maize plants underwent dual pollination, first by the inducers and then by self-pollination at different time intervals. We found that PCA strongly influenced heterofertilization, as measured by the resulting ratio of hybrid kernels to selfed kernels. When log10 (hybrid kernel/selfed kernel) was close to 0, i.e., when pollen from the different parents arrived at the embryo sac nearly simultaneously, the heterofertilization rate (HFR) was high. Haploid inducers with a high HIR, such as CAU5 and CAUB73, led to not only more haploids and embryo aborted kernels but also more heterofertilized kernels, as compared with a non-inducer or with a haploid inducer with a low HIR. We thus conclude that PCA is an important factor that affects HFR through polytubey. In addition, during maize haploid induction, abnormal fertilization could also contribute to heterofertilization, which may be due to fertilization recovery of kernels that otherwise would have been aborted at early stage.

Inheritance patterns of the response to in vitro doubled haploid induction in perennial ryegrass (Lolium perenne L.)

The ability to produce doubled haploid (DH) plants has found broad application in research and breeding. For major crop species such as maize (Zea mays L.) and barley (Hordeum vulgare L.), routine large-scale production of DHs has enabled the acceleration of breeding processes, for example through efficient generation of homozygous lines. However, in forage crops such as perennial ryegrass (Lolium perenne L.), low and genotype-specific responses to in vitro anther culture (AC) still limit wide-spread use of DHs. Here, we report the responses of nine bi-parental populations, segregating for microspore embryogenesis and plant regeneration capacity, to an effective AC protocol. Genotypes of exceptionally high androgenic ability, producing over 200 green plants per 100 anthers cultured, could be selected. Continuous and distinctly shaped distributions for the evaluated traits were indicative of quantitative polygenic control and the presence of different alleles in each population. An insignificant association of embryo production with plant regeneration, as well as a low correlation between green and albino plant yield (ρ = 0.20), suggested that different genes influence these traits. The populations evaluated here provide a rich source of alleles needed for the introgression of high levels of androgenic capacity into recalcitrant material. Moreover, this germplasm is ideally suited for use in future genotyping and mapping studies so that the genetic control of androgenic capacity in perennial ryegrass can be elucidated. Ultimately, our results will help to realize the potential of DH induction in one of the world’s most important forage crop species.

Dissection of a major QTL qhir1 conferring maternal haploid induction ability in maize

Key messageAmong the qhir11 and qhir12 sub-regions of a major QTL qhir1, only qhir11 has significant effect on maternal haploid induction, segregation distortion and kernel abortion.In vivo haploid induction in maize can be triggered in high frequencies by pollination with special genetic stocks called haploid inducers. Several genetic studies with segregating populations from non-inducer x inducer crosses identified a major QTL, qhir1, on chromosome 1.04 contributing to in vivo haploid induction. A recent Genome Wide Association Study using 51 inducers and 1482 non-inducers also identified two sub-regions within the qhir1 QTL region, named qhir11 and qhir12; qhir12 was proposed to be mandatory for haploid induction because the haplotype of qhir11 was also present in some non-inducers and putative candidate genes coding for DNA and amino acid binding proteins were identified in the qhir12 region. To characterize the effects of each sub-region of qhir1 on haploid induction rate, F2 recombinants segregating for one of the sub-regions and fixed for the other were identified in a cross between CML269 (non-inducer) and a tropicalized haploid inducer TAIL8. To quantify the haploid induction effects of qhir11 and qhir12, selfed progenies of recombinants between these sub-regions were genotyped. F3 plants homozygous for qhir11 and/or qhir12 were identified, and crossed to a ligueless tester to determine their haploid induction rates. The study revealed that only the qhir11 sub-region has a significant effect on haploid induction ability, besides causing significant segregation distortion and kernel abortion, traits that are strongly associated with maternal haploid induction. The results presented in this study can guide fine mapping efforts of qhir1 and in developing new inducers efficiently using marker assisted selection.

Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize.

Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.

Tapping the genetic diversity of landraces in allogamous crops with doubled haploid lines: a case study from European flint maize.

Using landraces for broadening the genetic base of elite maize germplasm is hampered by heterogeneity and high genetic load. Production of DH line libraries can help to overcome these problems. Landraces of maize (Zea mays L.) represent a huge reservoir of genetic diversity largely untapped by breeders. Genetic heterogeneity and a high genetic load hamper their use in hybrid breeding. Production of doubled haploid line libraries (DHL) by the in vivo haploid induction method promises to overcome these problems. To test this hypothesis, we compared the line per se performance of 389 doubled haploid (DH) lines across six DHL produced from European flint landraces with that of four flint founder lines (FFL) and 53 elite flint lines (EFL) for 16 agronomic traits evaluated in four locations. The genotypic variance ([Formula: see text]) within DHL was generally much larger than that among DHL and exceeded also[Formula: see text] of the EFL. For most traits, the means and [Formula: see text] differed considerably among the DHL, resulting in different expected selection gains. Mean grain yield of the EFL was 25 and 62% higher than for the FFL and DHL, respectively, indicating considerable breeding progress in the EFL and a remnant genetic load in the DHL. Usefulness of the best 20% lines was for individual DHL comparable to the EFL and grain yield (GY) in the top lines from both groups was similar. Our results corroborate the tremendous potential of landraces for broadening the narrow genetic base of elite germplasm. To make best use of these "gold reserves", we propose a multi-stage selection approach with optimal allocation of resources to (1) choose the most promising landraces for DHL production and (2) identify the top DH lines for further breeding.

Discrimination of haploid and diploid maize kernels via multispectral imaging

AbstractThe use of doubled haploids (DHs) in maize has become ubiquitous in maize breeding programmes as it allows breeders to go from cross to evaluation in as little as 2 years. Two important aspects of the in vivo DH system used in maize are as follows: (i) the identification of haploid progeny and (ii) doubling of the haploid genome to produce fertile inbred lines. This study is focused on the first step. Currently, identification of maize haploid progeny is performed manually using the R1‐nj seed colour marker. This is a labour‐intensive and time‐consuming process; a method for automated sorting of haploids would increase the efficiency of DH line development. In this study, six inbred lines were crossed with the maternal haploid inducer ‘RWS/RWK‐76’ and a sample of seed was sorted manually for each line. Using the VideometerLab 3 system, spectral imaging techniques were applied to discriminate between haploids and hybrids. Using DNA markers to confirm the haploid/diploid state of the tested seed, for the majority of genotypes haploid identification was possible with over 50% accuracy.

MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction.

Sexual reproduction in flowering plants involves double fertilization, the union of two sperm from pollen with two sex cells in the female embryo sac. Modern plant breeders increasingly seek to circumvent this process to produce doubled haploid individuals, which derive from the chromosome-doubled cells of the haploid gametophyte. Doubled haploid production fixes recombinant haploid genomes in inbred lines, shaving years off the breeding process. Costly, genotype-dependent tissue culture methods are used in many crops, while seed-based in vivo doubled haploid systems are rare in nature and difficult to manage in breeding programmes. The multi-billion-dollar maize hybrid seed business, however, is supported by industrial doubled haploid pipelines using intraspecific crosses to in vivo haploid inducer males derived from Stock 6, first reported in 1959 (ref. 5), followed by colchicine treatment. Despite decades of use, the mode of action remains controversial. Here we establish, through fine mapping, genome sequencing, genetic complementation, and gene editing, that haploid induction in maize (Zea mays) is triggered by a frame-shift mutation in MATRILINEAL (MTL), a pollen-specific phospholipase, and that novel edits in MTL lead to a 6.7% haploid induction rate (the percentage of haploid progeny versus total progeny). Wild-type MTL protein localizes exclusively to sperm cytoplasm, and pollen RNA-sequence profiling identifies a suite of pollen-specific genes overexpressed during haploid induction, some of which may mediate the formation of haploid seed. These findings highlight the importance of male gamete cytoplasmic components to reproductive success and male genome transmittance. Given the conservation of MTL in the cereals, this discovery may enable development of in vivo haploid induction systems to accelerate breeding in crop plants.

Brassica juncea Lines with Substituted Chimeric GFP-CENH3 Give Haploid and Aneuploid Progenies on Crossing with Other Lines.

Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.

Efficiency of double haploid production in wheat through wide hybridization and embryo rescue

The effect of haploid induction in wheat F1s by Zea mays, Imperata cylindrica and growth environments was investigated. Doubled Haploid (DH) plant production via maize and I. cylindrica technique from field grown LWH x VL616 F1 plants was equally efficient. In second environment, four F1 hybrids viz. LWH x VL616, HPW266 x Yr15 (CN25087), HS490 x HI1563 and Local Red x WHD938 were grown in greenhouse conditions and pollinated with maize. Efficiency of DH production was considerably high from greenhouse grown F1s as compared to field grown F1s. We also report an efficient protocol for DH production in bread wheat.

The Generation of Doubled Haploid Lines for QTL Mapping.

Recombinant inbred lines (RILs) are an essential tool for quantitative trait locus (QTL) mapping in Arabidopsis thaliana. Conventionally, the development of these lines is a time-consuming and tedious process requiring six to eight generations of selfing. Here, we describe an alternative approach: the rapid generation of RILs in A. thaliana via the creation of doubled haploids. In this method, F1 plants are crossed to an engineered haploid inducer to produce haploid plants. The chromosomes of these haploids then spontaneously double, generating immortalized homozygous F2 lines called doubled haploid RILs (DH RILs). Finally, DH RILs are genotyped using low-coverage whole-genome sequencing and are ready to be used for QTL mapping.

A study on natural recovery of tassel fertilization and doubling method in maize haploids.

Doubling method is the technical barriers in maize haploid breeding. It was very important to establish the independent intellectual property rights for doubling method. In this experiment, the maize haploid inducer, TG15, was used for producing maternal haploids. Also, haploids were obtained from two kinds of maternal genotypes involved in the experiment, including high-oil type and common type. Significant differences were observed among offspring of various genotypes in the recovery of haploid fertilization. In 21 hybrid offspring haploids, the average powder rate was 8.28%, and the seed setting rate was 4.98%. The experimental results showed that when the hybrids were treated with 0.08% colchicine, the average powder rate and seed setting rate of offspring haploids were 35.53 and 20.30%, respectively, which were significantly higher than the hybrids with natural recovery ability. This study primarily established the doubling method of haploids called "bud seedling method" in China which was very practicably in maize doubled haploid breeding.