Haploid Induction Rate Research Articles

In vivo maternal haploid induction based on genome editing of DMP in Brassica oleracea.

In vivo maternal haploid induction based on genome editing of DMP in Brassica oleracea.

Investigating the Effect of the Interaction of Maize Inducer and Donor Backgrounds on Haploid Induction Rates.

Doubled haploid technology is a feasible, fast, and cost-efficient way of producing completely homozygous lines in maize. Many factors contribute to the success of this system including the haploid induction rate (HIR) of inducer lines, the inducibility of donor background, and environmental conditions. Sixteen inducer lines were tested on eight different genetic backgrounds of five categories in different environments for the HIR to determine possible interaction specificity. The HIR was assessed using the R1-nj phenotype and corrected using the red root marker or using a gold-standard test that uses plant traits. RWS and Mo-17-derived inducers showed higher average induction rates and the commercial dent hybrid background showed higher inducibility. In contrast, sweet corn and flint backgrounds had a relatively lower inducibility, while non-stiff stalk and stiff stalk backgrounds showed intermediate inducibility. For the poor-performing donors (sweet corn and flint), there was no difference in the HIR among the inducers. Anthocyanin inhibitor genes in such donors were assumed to have increased the misclassification rate in the F1 fraction and, hence, result in a lower HIR.

Development of Multiple-Heading-Date mtl Haploid Inducer Lines in Rice

In vivo doubled haploid (DH) production based on crossing heterozygous germplasm with mtl haploid inducer lines promises to transform modern rice (Oryza sativa) breeding. However, this technology is limited, as haploid inducers and pollen acceptors have asynchronous heading dates. To address this obstacle, we developed a panel of multiple-heading-date mtl haploid inducer lines that produce pollen for more than 35 days. We edited the MTL gene in a hybrid rice with the CRISPR-Cas9 system. We then selected transgene-free homozygous mutants in the T1 generation and reproduced to T4 generation by single-seed descent method. We obtained 547 mtl haploid inducers with diverse heading dates (from 73 to 110 days) and selected 16 lines comprising a core population with continuous flowering. The seed-setting rate and haploid induction rate (HIR) of the core panel were 4.0–12.7% and 2.8–12.0%, respectively. Thus, our strategy of using multiple-heading-date mtl haploid inducers could accelerate the use of in vivo DH technology in rice breeding.

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 T0 , T1 , and T2 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.

Effects of TaMTL-Edited Mutations on Grain Phenotype and Storage Component Composition in Wheat

Wheat nutrition and processing-quality are primarily based on the endosperm ingredients. However, the effect of embryos on grain traits and components remains unclear. In this study, we found that in the cross-pollinated and self-pollinated progenies of the four wheat mtl mutants (mtl-A, mtl-AD, mtl-BD, and mtl-ABD) the haploid induction rates were 0–15.6% and 0–14.1%, and the embryo abortion rates were 0–27.4% and 0–24.1%, respectively, in which mtl-A had no effect on haploid induction and embryo development. The embryoless grains (ELG) were comparable to the normal grains (NG) from mtl-AD, mtl-BD, and mtl-ABD in grain length, grain width and thousand-kernel weight, but the grain traits were significantly less than those in NG from mutant mtl-A. During grain filling period, mtl-ABD had similar ELG ratio and amount of starch granule (SG) and protein body (PB) in ELG and NG. At maturity stage, the morphological features of A-type and B-type SG in ELG were similar to those in NG in mtl mutants; however, amylose, gliadin, and glutenin contents were higher in ELG, and total starch, albumin and globulin contents were higher in NG. Our results clarified the effect of the wheat mtl mutants on haploid induction and grain traits and nutrition composition in this crop, and provided new clues for studying the development of embryo and endosperm and their interaction in plants.

Overexpression of Modified CENH3 in Maize Stock6-Derived Inducer Lines Can Effectively Improve Maternal Haploid Induction Rates.

Maize (Zea mays) doubled haploid (DH) breeding is a technology that can efficiently generate inbred lines with homozygous genetic backgrounds. Haploids are usually produced through in vivo induction by haploid inducer lines in maize. Currently, two approaches are usually used to develop maize haploid inducer lines. One is through the conventional breeding improvement based on the Stock6 germplasm, and this strategy is extensively used to induce maternal haploids in commercial maize DH breeding. Another strategy, newly developed but less utilized so far, is by genetic manipulation of the Centromeric Histone3 (CENH3) in regular lines. However, whether both approaches can be combined to develop the haploid inducer line with higher maternal haploid induction rate (HIR) has not been reported. In this study, we manipulated the Stock6-derived inducer lines by overexpressing maize CENH3 fused with different fluorescent protein tags and found that the engineered Stock6-derived lines showed an obvious increase in the maternal HIR. Intriguingly, this above strategy could be further improved by substituting a tail-altered CENH3 for the full-length CENH3 in the tagged expression cassette, resulting in a maternal HIR up to 16.3% that was increased by ~6.1% than Stock6-derived lines control. These results suggested that integration of two in vivo haploid induction methods could rapidly and effectively improve the maternal HIRs of maize Stock6-derived inducer lines, and provided a potentially feasible solution for further optimizing the process of commercial maize DH breeding.

Selection Gain of Maize Haploid Inducers for the Tropical Savanna Environments.

Lacking elite haploid inducers performing high haploid induction rate (HIR) and agronomic performance is one of fundamental factors hindering the rapid adoption of doubled haploid technology in maize hybrid breeding, especially under tropical savanna climate. Breeding haploid inducers for specific agro-ecology, thus, is indispensable yet challenging. We used temperate inducer Stock6 as genetic source for haploid induction ability and eight tropical maize genotypes as principal donors for agronomic adaptation. Three cycles of modified ear-to-row with 5% intra-family selection were applied in a population set of 78 putative haploid inducer families emphasized on agronomic performance, R1-nj anthocyanin intensity, and inducer seed set. Genetic gains, variance components, and heritability on given traits were estimated. Hierarchical clustering based on five selection criteria was performed to investigate the phenotypic diversity of putative families. Cycle effect was predominant for all observed traits. Realized genetic gain was positive for HIR (0.40% per cycle) and inducer seed set (30.10% or 47.30 seeds per ear per cycle). In this study, we reported the first haploid inducers for regions under tropical savanna climate. Three inducer families, KHI-42, KHI-54, and KHI-64, were promising as they possessed HIR about 7.8% or 14 haploid seeds per tester ear and inducer seed rate about 95.0% or 208 inducer seeds per ear. The breeding method was effective for enhancing the seed set and the expression of R1-nj anthocyanin marker of inducers, yet it showed a low effectiveness to improve haploid induction rate. Introgression of temperate inducer Stock6 into tropical gene pool followed by phenotypic selections through modified ear-to-row selection on inducer seed set and R1-nj marker did not compromise the agronomic traits of tropical inducer families. Implications and further strategies for optimizing genetic gain on HIR are discussed.

Loss-of-function alleles of ZmPLD3 cause haploid induction in maize

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR–Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

In vivo haploid induction potential of Himalayan maize (Zea mays) and cogon grass (Imperata cylindrica) gene pools in different segregational cycles of intra and inter-generic crosses of wheat

AbstractMaize and Imperata cylindrica have been utilized globally as a pollen source for induction of haploids in wheat through chromosome elimination technique. Pollen parents with a higher haploid induction rate are desired for recovering the high frequency of haploids in wheat and related species. The present investigation was carried out with the aim to assess haploid induction efficiency of diverse germplasm of maize and I. cylindrica in different generations of intra and intergeneric crosses of hexaploid and tetraploid wheat and triticale-wheat derivatives. Crosses of twenty-six lines (female) with each of two I. cylindrica and twenty-one maize genotypes (testers) were evaluated for four haploid induction parameters viz., pseudoseed formation frequency (PFF), embryo formation frequency (EFF), haploid regeneration frequency (HRF) and haploid formation frequency (HFF). I. cylindrica outperformed maize in haploid induction rate with a frequency of embryos formed with I. cylindrica (18.39%) were significantly higher as compared to maize (4.08%). In the case of I. cylindrica genotype Ic-ye identified best with mean EFF of 30.55, 14.48 and 25.43% for hexaploids, tetraploids and triticale × wheat derivatives, respectively whereas in the case of maize genotype HPMC-60 performed best with EFF of 12.61% for hexaploids, HPMC-58 (12.58%) for tetraploids and HPMC-16 for triticale × wheat derivatives with EFF of 8.91%. I. cylindrica genotype Ic-ye and maize genotypes HPMC-14, HPMC-53, HPMC-60, HPMC-64 with significantly positive GCA for haploid induction parameters may be utilized as efficient pollen parents for recovering higher frequency of haploids in wheat.

Development of high-oil maize haploid inducer with a novel phenotyping strategy

Doubled haploid (DH) technology is important in modern maize breeding. Haploid inducers determine the efficiency of both haploid induction and identification. It has taken decades to improve the efficiency, haploid induction rate (HIR), from the ∼2% of the ancestor haploid inducer, stock6, to the ∼10% of modern haploid inducers. Improvement of kernel oil content (KOC) would further enhance haploid identification efficiency. Using molecular marker-assisted selection, in combine with the number of haploids per ear as phenotypic criterion, we developed a new high-oil haploid inducer line, CHOI4, with a mean HIR of 15.8% and mean KOC of 11%. High KOC of CHOI4 can achieve a mean accuracy greater than 90% in identification of haploids of different backgrounds, with reduced false discovery rates and false negative rates in comparison with the previous high-oil haploid inducer line, CHOI3. Comparison of phenotypic selection strategies suggested that the number of haploids per ear can be used as a phenotyping criterion during haploid inducer line development. CHOI4 could further increase the efficiency of large-scale DH breeding programs with lower cost.

Effects of Donor x Inducer Interaction on the Success of Haploid Induction and Comparison of Haploid Seed Identification Methods in the In vivo Maternal Haploid Technique in Maize

This study was conducted to investigate the effect of donor and inducer lines on the haploid induction rate in the in vivo maternal doubled haploid technique in maize and to compare the methods used in haploid seed separation. In the study, three donor materials were used for induction crossing with two inducer lines. Root tip cutting (Method 1) and stem injection (Method 2) techniques were used as chromosome doubling methods. In discrimination techniques, seed classes, visual discrimination results, spectral data and digitized data of images were used. Data were modeled using support vector machine (SVM) method and created models were evaluated over the complexity matrix. The results of the research revealed that the haploid induction rate varies depending on the donor genotypes and inducers, and the genotype responses against the applied chromosome folding method were also different. Differences were observed in the success of the three methods compared for haploid and diploid seed separation. The successful classification rates were %87, %83, and %79 in visual, spectral and image processing models, respectively. Results showed that both spectral techniques and image processing techniques can be used to distinguish haploid / diploid seeds in in vivo maternal haploid technique.

PL-4 (CIP596131.4): an Improved Potato Haploid Inducer

Dihaploid production from elite tetraploid cultivars is key to both traditional and novel breeding approaches that seek to simplify potato genetics. For this purpose, efficient and widely compatible haploid inducers (HIs) are needed. We compared PL-4, a new HI developed at the International Potato Center, to known HIs IvP101 and IvP35. By pollination of elite tetraploid breeding lines, we showed that PL-4 performed significantly better and had a homogeneous response regardless of the genetic background of the pistillate parents, on the most important efficiency traits—number of dihaploids per 100 fruits and haploid induction rate. Moreover, PL-4 exhibited a reduced proportion of hybrid seeds, a convenient trait for efficient screening. In this context, we recommend PL-4 as an enhanced HI for the potato breeding community.

Development of late temperate in vivo haploid inducers

In vivo doubled haploid technique has been widely used in advanced maize breeding programs due to cost, labor and time advantages and increase in efficiency. However, the number of available inducer lines in the world is sufficient. Six BC1 breeding populations including RWS and RWK-76 haploid inducer lines and late temperate ADK-451, ADK-737 and ADK-455 lines were developed by Sakarya Maize Research Institute (MRI) in Turkey. The RWS and RWK-76 haploid inducer lines were used as donors. Pedigree method was employed to develop the inducer lines. Anthocyanin coloration of plant, tassel length, branch number of tassel, plant height, days to flowering, embryo-endosperm colorfulness and haploid induction rate (HIR) were determined. The genotypes with the best characteristics were selected. The families from BC1F3 to BC1F7 were hybridized to liguleless line to determine the HIR and families with HIR over 8% were selected from BC1 populations. The HIR, plant height and days to tassel flowering values of in-1021 and in-1076 candidate haploid inducer lines were 10.5 and 12.3%, 195 and 200 cm, and 69 and 68 days, respectively. The HIR value of RWS donor haploid inducer ranged from 8.9 to 11.3% and for RWK-76 from 7.3 to 9.8%. Simple Sequence Repeats (SSRs) markers were used to identify genetic similarity between late temperate haploid inducer lines and donors. The similarity rates of in-1021 and in-1076 inducer lines to the RWS donor were 38 and 15%, and to the RWK-76 donor were 23 and 27%. The similarity rate between the two candidate inducer lines was 30%. The results indicated that the late temperate haploid inducer lines developed will increase the efficiency of maize breeding.

Rice Haploid Inducer Development by Genome Editing.

The current method to induce haploids in rice is anther culture, which is time-consuming and labor intensive and only works for some varieties. Here we describe a seed-based haploid induction system created by CRISPR/Cas9 technology. By editing OsMATL, we generate rice haploid inducer lines with a 2-5% haploid induction rate in different germplasms.

Doubled haploid production in maize under Sub-montane Himalayan conditions using R1-nj-based haploid inducer TAILP1

The use of in vivo haploid induction system makes the doubled haploid (DH) technology easier to adopt for the conventional maize breeders. However, despite having played an important role in the initial developmental phases of DH technology, Indian maize research has yet to harvest its benefits. Haploid Inducer Lines (HILs) developed by CIMMYT are being widely used in maize breeding programmes in many countries including India. There, however, is no published information on the efficiency of DH line production using CIMMYT HILs in Indian maize breeding programmes. In the present study, the efficiency of DH production using CIMMYT’s tropically adapted inducer line TAILP1 was investigated with eight source populations including two of sweet corn. The average haploid induction rate (HIR) of TAILP1 was 5.48% with a range of 2.01 to 10.03%. Efficiency of DH production ranged from 0.14 to 1.87% for different source populations with an average of 1.07%. The information generated will be useful for maize breeders intending to use DH technology for accelerated development of completely homozygous lines.

MYO, a Candidate Gene for Haploid Induction in Maize Causes Male Sterility.

Doubled haploid technology is highly successful in maize breeding programs and is contingent on the ability of maize inducers to efficiently produce haploids. Knowledge of the genes involved in haploid induction is important for not only developing better maize inducers, but also to create inducers in other crops. The main quantitative trait loci involved in maize haploid induction are qhir1 and qhir8. The gene underlying qhir1 has been discovered and validated by independent research groups. Prior to initiation of this study, the gene associated with qhir8 had yet to be recognized. Therefore, this research focused on characterizing positional candidate genes underlying qhir8. Pursuing this goal, a strong candidate for qhir8, GRMZM2G435294 (MYO), was silenced by RNAi. Analysis of crosses with these heterozygous RNAi-transgenic lines for haploid induction rate revealed that the silencing of MYO significantly enhanced haploid induction rate by an average of 0.6% in the presence of qhir1. Recently, GRMZM2G465053 (ZmDMP) was identified by map-based gene isolation and shown to be responsible for qhir8. While our results suggest that MYO may contribute to haploid induction rate, results were inconsistent and only showing minor increases in haploid induction rate compared to ZmDMP. Instead, reciprocal crosses clearly revealed that the silencing of MYO causes male sterility.

Development of in Vivo Haploid Inducer Lines for Screening Haploid Immature Embryos in Maize.

Doubled haploid technology is widely applied in maize. The haploid inducer lines play critical roles in doubled haploid breeding. We report the development of specialized haploid inducer lines that enhance the purple pigmentation of crossing immature embryos. During the development of haploid inducer lines, two breeding populations derived from the CAU3/S23 and CAU5/S23 were used. Molecular marker-assisted selection for both qhir1 and qhir8 was used from BC1F1 to BC1F4. Evaluation of the candidate individuals in each generation was carried out by pollinating to the tester of ZD958. Individuals with fast and clear pigmentation of the crossing immature embryos, high number of haploids per ear, and high haploid induction rate were considered as candidates. Finally, three new haploid inducer lines (CS1, CS2, and CS3) were developed. The first two (CS1 and CS2) were from the CAU3/S23, with a haploid induction rate of 8.29%–13.25% and 11.54%–15.54%, respectively. Meanwhile, the CS3 was from the CAU5/S23. Its haploid induction rate was 8.14%–12.28%. In comparison with the donor haploid inducer lines, the 24-h purple embryo rates of the newly developed haploid inducer lines were improved by 10%–20%, with a ~90% accuracy for the identification of haploid immature embryos. These new haploid inducer lines will further improve the efficiency of doubled haploid breeding of maize.

Development and innovation of haploid induction technologies in plants.

Haploid induction is one of the main techniques for breeding new varieties of major crops, and its key steps are improving the haploid induction rate and simplifying the induction procedure. With the development and innovation of plant haploid induction technologies, haploid breeding has been widely used in varietal improvement of important crops, showing the advantages of rapid homozygosity of heterozygous genes, shortening breeding period, and improving breeding efficiency. The combination of haploid breeding with crossing breeding, mutation breeding, reverse breeding, and molecular marker-assisted selection will greatly improve the effectiveness of crop breeding. Haploids and doubled haploids have demonstrated their usefulness in production of genetic populations, characterization of gene functions, and transgenic and cytological studies in plants. In this review, we summarize the progress of haploid induction technologies in view of various haploid induction techniques and applications of haploids and double haploids. In particular, the advances on the haploid induction in several major crops by genome editing were briefly described. Finally, we discuss current issues and future perspectives in this field, so as to promote the application of the haploid induction techniques, especially the techniques of creating haploid inducer lines by genome editing in crop breeding.

A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis

Doubled haploid technology using inducer lines carrying mutations in ZmPLA1/MTL/NLD and ZmDMP1-4 has revolutionized traditional maize breeding. ZmPLA1/MTL/NLD is conserved in monocots and has been used to extend the system from maize to other monocots5-7, but no functional orthologue has been identified in dicots, while ZmDMP-like genes exist in both monocots and dicots4,8,9. Here, we report that loss-of-function mutations in the Arabidopsis thaliana ZmDMP-like genes AtDMP8 and AtDMP9 induce maternal haploids, with an average haploid induction rate of 2.1 ± 1.1%. In addition, to facilitate haploid seed identification in dicots, we established an efficient FAST-Red fluorescent marker-based haploid identification system that enables the identification of haploid seeds with >90% accuracy. These results show that mutations in DMP genes also trigger haploid induction in dicots. The conserved expression patterns and amino acid sequences of ZmDMP-like genes in dicots suggest that DMP mutations could be used to develop in vivo haploid induction systems in dicots.

Development of doubled haploid maize lines by using in vivo haploid technique

The doubled haploid technology is now an integral component of modern maize breeding programs. In this study, the maternal haploid induction (gynogenesis) method was used to derive Doubled-Haploid (DH) lines from elite maize germplasm adapted to Turkey. Temperate haploid inducers (RWS, RWK-76, RWS x RWK-76 and WS14) were used as pollinators, and a set of 30 single-crossses (in FAO 650-700 maturity groups) were used as source materials. Putative haploid seeds were selected based on expression of R1-nj anthocyanin color marker. Highest haploid induction rate (20.42%) was recorded by using RWK-76 as inducer line, and the lowest haploid induction rate (17.75%) was obtained through WS14. Putative haploid seeds were germinated and seedlings were treated with 0.06% colchicine + 0.5% dimethylsulfoxide solution. Following transfer of seedlings into the field, 2178 D0 plants were obtained out of a total of 3012 treated haploids. Live plants were from 89% of 2178 seedlings which are planted to the field. Fertile plants were formed 57% of live plants. Inbreeding was succeeded in 31.23% of fertile plants and only 7.8% of inbreeding plants were able to produce seeds. Consequently, 27 doubled haploid lines were developed.