Haploid Induction Rate Research Articles

Maternal Haploids Are Preferentially Induced by CENH3-tailswap Transgenic Complementation in Maize.

Doubled haploid plants are invaluable breeding tools but many crop species are recalcitrant to available haploid induction techniques. To test if haploid inducer lines can be engineered into crops, CENH3−∕− and CENH3:RNAi lines were complemented by AcGREEN-tailswap-CENH3 or AcGREEN-CENH3 transgenes. Haploid induction rates were determined following testcrosses to wild-type plants after independently controlling for inducer parent sex and transgene zygosity. CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility. CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids. In contrast, many of the complemented CENH3−∕− lines produced haploids at low frequencies. The rate of gynogenic haploid induction reached a maximum of 3.6% in several hemizygous individuals when backcrossed as males. These results demonstrate that CENH3-tailswap transgenes can be used to engineer in vivo haploid induction systems into maize plants.

Fine mapping of qhir8 affecting in vivo haploid induction in maize.

The QTL qhir8 affecting in vivo haploid induction in maize was mapped to a 789kb region, embryo abortion rate and segregation ratios were analyzed, linkage markers for MAS were developed. The doubled-haploid (DH) technology has become an important tool for line development in modern maize breeding. However, the genetic basis of haploid induction remains elusive. In previous QTL mapping research, qhir8 besides qhir1 significantly affected haploid induction rate (HIR). Our objective was to fine map qhir8 and assess its effect on HIR, segregation distortion (SD) and embryo abortion (EmA). A total of 3989 F2 plants from the cross of inducers CAUHOI and UH400 were screened for recombinants in the qhir8 region. F2 plants and F3 plants from selfing progenies of 34 recombinant F2 plants were evaluated for HIR, SD and EmA. In parallel, we developed 31 new markers providing good coverage of the qhir8 region. We confirmed that qhir8 has an increasing effect on HIR and EmA, but not on SD. Moreover, we successfully narrowed down the qhir8 locus to a 789kb region flanked by markers 4292232 and umc1867.

Controlling Misclassification Rates in Identification of Haploid Seeds from Induction Crosses in Maize with High‐Oil Inducers

ABSTRACTIn vivo production of double haploid (DH) lines in maize (Zea mays L.) requires reliable identification of haploid (H) seeds. A new method for achieving this goal is production of induction crosses with high‐oil (HO) inducers and sorting the resulting H and diploid crossing (C) seeds based on their oil content (OC). Balancing the false discovery rate (FDR) and false negative rate (FNR) by choice of a suitable proportion α of selected seeds represents an unsolved problem with this method. We investigated solutions by applying mixture distribution (MD) analysis to the OC of induction crosses for estimating the means and standard deviation (μH, μC, and σ) of H and C seeds and the haploid induction rate κ. Moreover, we developed formulas and software for calculating the FDR and FNR from these estimates. Using several induction crosses with HO inducer UH600, parameter estimates from (i) MD analysis in different environments and (ii) gold standard classification (GSC) of plants in the field agreed well for μH and μC, but only moderately for σ and κ. Parameter estimates from the MD provided meaningful guidelines for calculating the expected FDR and FNR. Selecting the α = 7.5% proportion of seeds with lowest OC was optimal for most induction crosses and balanced the FDR and FNR. In conclusion, induction crosses with HO inducers hold great promise for promoting the DH technology in maize, but an automated high‐throughput platform for sorting the seeds from the MD into several distinct classes with increasing OC is recommended to take full advantage of this novel approach.

Marker-assisted selection and evaluation of high oil in vivo haploid inducers in maize

Doubled haploid technology, which is used to rapidly purify genetic resources, is one of the key technologies in modern maize breeding. In a previous study, the major quantitative trait locus qhir1, which influences in vivo haploid induction, was narrowed down to a 243-kb region, which made it feasible to use marker-assisted selection (MAS) for inducer development. Recently, a new method was developed for haploid identification using oil content (OC). The objective of this study was to develop high oil inducer lines using MAS of the qhir1 locus. We constructed an F2 population, two backcross populations that were backcrossed to the inducer CAU5 (BC1F1-CAU5) and the high oil inbred line GY923 (BC1F1-GY923), respectively, which was derived from the cross GY923 × CAU5, and subjected continuous selfing to develop high oil inducer lines. In each cycle, three different parameters including kernel OC, marker genotype at qhir1 and haploid induction rate (HIR) were used for pedigree selection. Three candidate high oil inducer lines were developed, with an OC of approximately 8.5 %, an HIR of approximately 8 % and superior agronomic performance, which are suitable values for the application of these lines to haploid identification by OC. Our results confirm the notion that HIR selection combined with MAS for qhir1 is an effective approach to haploid inducer breeding. In addition, we determined that the accuracy of haploid identification by OC is influenced by the female germplasm resource and the high oil inducer and that appropriate critical points for OC can balance the false discovery rate and false negative rate.

Mapping of maternal QTLs for in vivo haploid induction rate in maize (Zea mays L.)

Haploid technology can significantly shorten the time required for inbred line improvement, accelerate the breeding process, and reduce breeding costs. The production of haploids is not only dependent on the genetics of the paternal haploid inducer, but it is also affected by the genetic background of the maternal donor during the process of haploid production. To address the maternal genetic contribution to haploid production, we pollinated a mapping population consisting of 186 F2:3 family lines derived from a cross between Zheng58 and Chang7-2 with the inducer line CAU5 and selected haploid kernels using R1-nj kernel markers. Two quantitative trait loci (QTLs), qmhir1 and qmhir2, which contribute to the maternal genetics of haploid induction, were detected on chromosomes 1 and 3, respectively. The qmhir1 locus is located between the flanking marker loci umc1292 and bnlg1014, and it explained 14.70 % of the phenotypic variation. The qmhir2 locus is located between marker loci umc1844 and umc2277 and it explained 8.42 % of the phenotypic variation. The genetic effect of both QTLs is partial dominance.

Fine mapping of qhir1 influencing in vivo haploid induction in maize

Production of haploids by the in vivo haploid induction method has now become routine for generating new inbred lines in maize. In previous studies, a major quantitative trait locus (QTL) (qhir1) located in bin 1.04 was detected, explaining up to 66% of the genotypic variance for haploid induction rate (HIR). Our objectives were to (1) fine-map qhir1 and (2) identify closely linked markers useful for marker-assisted breeding of new inducers. For this purpose, we screened a mapping population of 14,375 F2 plants produced from a cross between haploid inducer UH400 and non-inducer line 1680 to identify recombinants. Based on sequence information from the B73 reference genome, markers polymorphic between the two parents were developed to conduct fine mapping with these recombinants. A progeny test mapping strategy was applied to accurately determine the HIR of the 14 recombinants identified. Furthermore, F3 progeny of recombinant F2 plants were genotyped and in parallel evaluated for HIR. We corroborated earlier studies in that qhir1 has both a significantly positive effect on HIR but also a strong selective disadvantage, as indicated by significant segregation distortion. Altogether, we were able to narrow down the qhir1 locus to a 243 kb region flanked by markers X291 and X263.

Development of in vivo haploid inducers for tropical maize breeding programs

Lack of adapted haploid inducers currently impedes adoption of the doubled haploid technology in tropical maize breeding programs. Our objective was to generate inducers with improved adaptation to tropical conditions. We developed segregating generations from crosses between temperate inducers having haploid induction rates (HIR) of 8–10 % and tropical CIMMYT maize lines (CML; HIR = 0 %) and evaluated these for HIR and agronomic performance under tropical lowland field conditions. The applied pedigree breeding scheme comprising mass selection on individual F2 plants for highly heritable and visually scorable traits, followed by family-based selection for HIR and other agronomic characteristics in advanced selfing and backcross (BC) generations seems highly suitable for breeding improved haploid inducers with adaptation to different agroecologies. The most advanced tropical inducer candidates (TIC) combine HIR of up to 10 % with improved pollen production, disease resistance, and plant vigor compared to temperate inducers under tropical conditions. Agronomic characteristics were significantly improved in the BC to CML compared to BC to inducers, while mean HIR of both populations were similar, indicating that backcrossing to the adapted parent was suitable to improve adaptation of new inducers without sacrificing high HIR. When screening random open-pollinated maize accessions, HIR of up to 3 % were observed, suggesting that novel genetic variation may be present in maize accessions that could be exploited to improve HIR in maize. In conclusion, tropical inducer development proceeds well, but evaluation of TIC in multi-environment trials needs to be completed before large-scale dissemination can commence.

New Insights into the Genetics ofin VivoInduction of Maternal Haploids, the Backbone of Doubled Haploid Technology in Maize

Haploids and doubled haploid (DH) inbred lines have become an invaluable tool for maize genetic research and hybrid breeding, but the genetic basis of in vivo induction of maternal haploids is still unknown. This is the first study reporting comparative quantitative trait locus (QTL) analyses of this trait in maize. We determined haploid induction rates (HIR) in testcrosses of a total of 1061 progenies of four segregating populations involving two temperate haploid inducers, UH400 (HIR = 8%) and CAUHOI (HIR = 2%), one temperate and two tropical inbreds with HIR = 0%, and up to three generations per population. Mean HIR of the populations ranged from 0.6 to 5.2% and strongly deviated from the midparent values. One QTL (qhir1) explaining up to p = 66% of the genetic variance was detected in bin 1.04 in the three populations involving a noninducer parent and the HIR-enhancing allele was contributed by UH400. Segregation ratios of loci in bin 1.04 were highly distorted against the UH400 allele in these three populations, suggesting that transmission failure of the inducer gamete and haploid induction ability are related phenomena. In the CAUHOI × UH400 population, seven QTL were identified on five chromosomes, with qhir8 on chromosome 9 having p > 20% in three generations of this cross. The large-effect QTL qhir1 and qhir8 will likely become fixed quickly during inducer development due to strong selection pressure applied for high HIR. Hence, marker-based pyramiding of small-effect and/or modifier QTL influencing qhir1 and qhir8 may help to further increase HIR in maize. We propose a conceptual genetic framework for inheritance of haploid induction ability, which is also applicable to other dichotomous traits requiring progeny testing, and discuss the implications of our results for haploid inducer development.

Doubled Haploids in Tropical Maize: I. Effects of Inducers and Source Germplasm on in vivo Haploid Induction Rates

ABSTRACTThe adoption of the doubled haploid (DH) technology in tropical maize (Zea mays L.) breeding programs is lagging behind that of temperate programs due to a lack of tropical haploid inducers and reliable information on the performance of temperate inducers under nontemperate conditions. The objective of this study was to determine the in vivo haploid induction ability of three temperate inducers crossed to a diverse set of tropical maize source germplasm under tropical conditions. Three experiments were conducted employing inducers as male parents to pollinate 120 source germplasm in three environments in Mexico. Haploid induction rates (HIR) obtained under field conditions were determined with two different haploid identification systems. Highly significant genotypic differences were detected among inducers and source germplasm for HIR but no interactions were observed between the two factors. Mean HIR under tropical conditions were similar to those reported for evaluations under temperate conditions indicating that temperate inducers can be employed for initiation of DH breeding programs in the tropics. Misclassification of diploids as haploids resulted in inflated HIR, particularly in highly variable source germplasm such as landraces or when expression of the identification marker was weak. We conclude that induction of haploidy is not a limiting factor for DH line production in tropical maize, but there is a need for the development of well‐adapted tropical inducers.

Inducer line generated double haploid seeds for combined waxy and opaque 2 grain quality in subtropical maize (Zea mays. L.)

The implementation of modern inducer lines in maize breeding can substantially decrease the time required to create elite inbred lines. In industrialized countries, this method has already largely replaced conventional backcross methods. However, the application of in vivo gynogenesis for inducing doubled haploids is still limited to European and US maize germplasms and has still to be adapted for exotic plant material. The reliability of three modern European inducer lines from the University of Hohenheim (Germany) was investigated for the production of haploid progenies from subtropical specialty maize. Three Chinese hybrids heterozygous for waxy maize and opaque 2 were used as maternal donor material, as maize double recessive for waxy and opaque 2 may improve the nutrition of ethnic minorities in Southeast Asia. However, many false positives were detected by flow cytometry among putative haploid seeds based on anthocyanin pigmentation because the color expression was inhibited in almost 50% of the induced seeds from this maternal plant material. Based on flow cytometry, the haploid induction rates were high with 10.2–12.3%, and the chromosome doubling rates were around 50%; therefore the principal potential of producing DH was confirmed for subtropical maize. However inducer lines for the precise and fast recognition of truly induced haploid seeds still need to be developed.

Effect of source germplasm and season on the in vivo haploid induction rate in tropical maize

For in vivo production of doubled haploid (DH) lines in maize, the rate of haploid induction is of crucial importance. Maternal haploid induction depends primarily on the inducer used as a pollinator. However, the source germplasm used as a maternal parent and the environmental conditions for induction may also influence haploid induction and these aspects have not been examined in tropical maize so far. The objectives of our study were to (i) monitor the variation for haploid induction rate (HIR) among diverse source germplasm in tropical maize, (ii) determine the relative importance of general (GCA) and specific (SCA) combining abilities for HIR, and (iii) investigate the influence of summer and winter seasons and genotype × season interactions on this trait. Ten inbreds were mated in a half diallel design. The resulting 45 F1 single crosses were pollinated with the haploid inducer hybrid RWS × UH400 during the summer 2008 and winter 2009 seasons in a lowland tropical environment in Mexico. HIR of the single crosses averaged over seasons ranged from 2.90 to 9.66% with an overall mean of 6.74%. Mean HIR was significantly (P < 0.01) higher during the winter (7.37%) than summer season (6.11%). Significant (P < 0.01) variation was observed due to GCA effects of parental inbreds of single crosses but not for SCA, GCA × season and SCA × season interactions. Our study underpins that a higher HIR in tropical maize can be obtained by selecting appropriate source germplasm and undertaking pollination under favorable environmental conditions.

Hybrid maize breeding with doubled haploids: III. Efficiency of early testing prior to doubled haploid production in two-stage selection for testcross performance

Early testing prior to doubled haploid (DH) production is a promising approach in hybrid maize breeding. We (1) determined the optimum allocation of the number of S(1) families, DH lines, and test locations for two different breeding schemes, (2) compared the maximum selection gain achievable under both breeding schemes, and (3) investigated limitations in the current method of DH production. Selection gain was calculated by numerical integration in two-stage breeding schemes with evaluation of testcross progenies of (1) DH lines in both stages (DHTC), or (2) S(1) families in the first and DH lines within S(1) families in the second stage (S(1)TC-DHTC). Different assumptions were made regarding the budget, variance components, and time of DH production within S(1) families. Maximum selection gain in S(1)TC-DHTC was about 10% larger than in DHTC, indicating the large potential of early testing prior to DH production. The optimum allocation of test resources in S(1)TC-DHTC involved similar numbers of test locations and test candidates in both stages resulting in a large optimum number of S(1) families in the first stage and DH lines within the best two S(1) families in the second stage. The longer cycle length of S(1)TC-DHTC can be compensated by haploid induction of individual S(1) plants instead of S(1) families. However, this reduces selection gain largely due to the current limitations in the DH technique. Substantial increases in haploid induction and chromosome doubling rates as well as reduction in costs of DH production would allow early testing of S(1) lines and subsequent production and testing of DH lines in a breeding scheme that combines high selection gain with a short cycle length.