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.

Elite,transformable haploid inducers in maize

The introduction of alleles into commercial crop breeding pipelines is both time consuming and costly.Two technologies that are disrupting traditional breeding processes are doubled haploid(DH)breeding and genome editing(GE).Recently,these techniques were combined into a GE trait delivery system called HI-Edit(Haploid Inducer-Edit).In HI-Edit,the pollen of a haploid inducer line is reprogrammed to deliver GE traits to any variety,obviating recurrent selection.For HI-Edit to operate at scale,an efficient transformable HI line is needed,but most maize varieties are recalcitrant to transformation,and haploid inducers are especially difficult to transform given their aberrant reproductive behaviors.Leveraging marker assisted selection and a three-tiered testing scheme,we report the development of new Iodent and Stiff Stalk maize germplasm that are transformable,have high haploid induction rates,and exhibit a robust,genetically-dominant anthocyanin native trait that may be used for rapid haploid identification.We show that transformation of these elite‘‘HI-Edit”lines is enhanced using the BABYBOOM and WUSCHEL morphogenetic factors.Finally,we evaluate the HI-Edit performance of one of the lines against both Stiff Stalk and non-Stiff Stalk testers.The strategy and results of this study should facilitate the development of commercially scalable HI-Edit systems in diverse crops.

Exceptionally high genetic variance of the doubled haploid(DH)population of poplar

Doubled haploid(DH)plants have been widely used for breeding and biological research in crops.Pop ulus spp.have been used as model woody plant species for biological research.However,the induction of DH poplar plants is onerous,and limited biological or breeding work has been carried out on DH individuals or populations.In this study,we provide an effective protocol for poplar haploid induction based on an anther culture method.A total of 96 whole DH plant lines were obtained using an F1hybrid of Populus simonii×P.nigra as a donor tree.The phenotypes of the DH population showed exceptionally high variance when compared to those of half-sib progeny of the donor tree.Each DH line displayed distinct features compared to those of the other DH lines or the donor tree.Additionally,some excellent homozygous lines have the potential to be model plants in genetic and breeding studies.

Generation of a human haploid neural stem cell line for genome-wide genetic screening

BACKGROUND Haploid embryonic stem cells(haESCs)have been established in many species.Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens.AIM To derive human haploid neural stem cells(haNSCs)to carry out lineage-specific screens.METHODS Human haNSCs were differentiated from human extended haESCs with the help of Y27632(ROCK signaling pathway inhibitor)and a series of cytokines to reduce diploidization.Neuronal differentiation of haNSCs was performed to examine their neural differentiation potency.Global gene expression analysis was conducted to compare haNSCs with diploid NSCs and haESCs.Fluorescence activated cell sorting was performed to assess the diploidization rate of extended haESCs and haNSCs.Genetic manipulation and screening were utilized to evaluate the significance of human haNSCs as genetic screening tools.RESULTS Human haESCs in extended pluripotent culture medium showed more compact and smaller colonies,a higher efficiency in neural differentiation,a higher cell survival ratio and higher stability in haploidy maintenance.These characteristics effectively facilitated the derivation of human haNSCs.These human haNSCs can be generated by differentiation and maintain haploidy and multipotency to neurons and glia in the long term in vitro.After PiggyBac transfection,there were multiple insertion sites in the human haNSCs’genome,and the insertion sites were evenly spread across all chromosomes.In addition,after the cells were treated with manganese,we were able to generate a list of manganese-induced toxicity genes,demonstrating their utility as genetic screening tools.CONCLUSION This is the first report of a generated human haploid somatic cell line with a complete genome,proliferative ability and neural differentiation potential that provides cell resources for recessive inheritance and drug targeted screening.

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.

Methods for Producing Maize(Zea mays L.) Haploids and Their Application in Maize Breeding

This article summarizes the research progress inthe breeding of maize haploids, including the production and identification of maize haploids, especially the methods to produce doubled haploids and the selection of basic materials, as well as the applications of the maize haploids in maize breeding. Finally, this article pro- poses several issues researchers should pay attention to, and the prospects of hap- Ioids in maize breeding.

Study on Haploid Induction Rates in Different Maize Inducers

[Objective] The aim was to analyze the differences in haploid induction rates of different inducers. [Method] Six maize inducers with purple spot and purple color were selected as the male parents to pollinate six inbred lines. [Result] The mean haploid induction rates were significantly different among the inducers: KMS-3 >WY-1 >PR-2 >YP-13 >KMS-2 >KMS-1. The haploid induction rates of the different hybrid materials were significantly different: K410 >105A >103A >104A >107A >D271 >106A>L73>N21>KZ58. [Conclusion] The haploid inducer line PR-2, which had high haploid induction rate and low variation coefficient, was an elite haploid inducer.

Research and Breeding Application Progress of the Technique of Producing Double Haploid of Wheat by Wide Hybridization between Wheat and Maize

The technique of producing doublehaploid of wheat by distant hybridization between wheat and maize has characterized with better inducing effect, shorter in- ducing period, easy operation, and so on. At present, it is the most efficient and has great potential of application in breeding of wheat. This article reviewed princi- ple and production process of the technique, research situation of the three key in- dicators of the technology（embryo rate, seedling rate and success rate of doubling）in recent years, and application of the technology in breeding, genetics, germplasm improvement of wheat. At last, both the achievements and the direction of further improvement and development of the technology in our program were discussed.

Doubling Efficiency of Maize Haploids Treated with Different Methods

Haploid seedlings were inducted from different maize materials.At 2-3-leaf stage,maize haploids were treated with 0.06% colchicine and 2.0% dimethyl sulfoxide（DMSO） by dipping root,dripping heart leaf and acupuncturing growing point,respectively.The doubling rate and mortality rate in different treatments were analyzed by variance analysis and multiple comparisons.The result showed that growing point acupuncturing method exhibited the highest doubling efficiency with an average doubling rate of 23%,seed-setting rate of 21.4%,and mortality rate of 16.3%.Composed with other two chemical doubling methods,growing point acupuncturing method significantly improved the doubling rate of maize haploids with a lower application dose of colchicine.This study laid the foundation for industrial application of haploid breeding techniques.

Study on Haploid Induction in Different Maize Genotypes

This study aimed to investigate the induction effect on different genotypes of maize of haploid inducers RWS, Stock6, CAU Inducer and hybridization-derived breeding lines. The results showed that 2005 had the highest induction rate （2.85%） when using inducer Stock6 as male parent material; haploid induction rates were significantly different among different genotypes of maize when using RWS, Stock6, CAU Inducer and their crossbreeding materials as the male parents; inducers 2061, 2062, 2058 and 2059 led to relatively high induction rates, which could be emphatically utilized. Furthermore, during the screening of induction materials, different female parent materials should be selected based on the breeding objective.

Study on Chromosome Doubling for Haploid Produced by Wheat×Maize Crossing

There is no spontaneous chromosome doubling in haploid plants produced by wheat X maize crossing. In order to obtain doubled haploid, two chromosome doubling methods were used. Results showed that: After adding colchicine solution directly into a medium for young embryos that had been cultured 7 days, frequencies of embryo germination in colchicine concentrations of 50mg/L, 100mg/L and 200mg/L were 32.1% , 26.4% and 16.3% , respectively, and frequencies of chromosome doubling were 85.3% , 100% and 50.0% , respectively. But in the control without colchicine, the frequency of embryo germination was 67.4% and no seed was setting. As the time of colchicine treatment increased from 24 to 72 hours, the frequency of embryo germination was reduced, and 24 hours had better results. After soaking seeding crowns and roots with colchicine solution of 500mg/L, 750mg/L and 1 000mg/L for 5 hours, the frequencies of doubling were 89.6%, 76.0% and 73.3%, respectively. By soaking crowns and roots of strong seedings with 500mg/L colchicine solution, the frequency and efficiency of doubling were 98.2% and 93.2% , respectively.

Mapping QTLs with epistatic effects and QTL×environment interactions for plant height using a doubled haploid population in cultivated wheat

Quantitative trait loci （QTLs） for plant height in wheat （Triticum aestivum L.） were studied using a set of 168 doubled haploid （DH） lines, which were derived from the cross Huapei 3/Yumai 57. A genetic linkage map was constructed using 283 SSR and 22 EST-SSR markers. The DH population and the parents were evaluated for wheat plant height in 2005 and 2006 in Tai＇an and 2006 in Suzhou. QTL analyses were performed using the software of QTLNetwork version 2.0 based on the mixed linear model. Four additive QTLs and five pairs of epistatic effects were detected, which were distributed on chromosomes 3A, 4B, 4D, 5A, 6A, 7B, and 7D. Among them, three additive QTLs and three pairs of epistatic QTLs showed QTL×environment interactions （QEs）. Two major QTLs, Qph4B and Qph4D, which accounted for 14.51% and 20.22% of the phenotypic variation, were located similar to the reported locations of the dwarfing genes Rhtl and Rht2, respectively. The Qph3A-2 with additive effect was not reported in previous linkage mapping studies. The total QTL effects detected for the plant height explained 85.04% of the phenotypic variation, with additive effects 46.07%, epistatic effects 19.89%, and QEs 19.09%. The results showed that both additive effects and epistatic effects were important genetic bases of wheat plant height, which were subjected to environmental modifications, and caused dramatic changes in phenotypic effects. The information obtained in this study will be useful for manipulating the QTLs for wheat plant height by molecular marker-assisted selection （MAS）.

Phenotypic Expression of Whitebacked Planthopper Resistance in the Newly Established japonica/indica Doubled Haploid Rice Population

A new doubled haploid (DH) rice population was established from a cross between WBPH-resistant japonica Chunjiang 06 (CJ-06) and susceptible indica TN1. Sucking inhibitory and ovicidal resistance of the DH rice lines were evaluated on the basis of non-preference response of WBPH immigrants and honeydew excretion by WBPH females, and appearance of watery lesions in the necrotic discoloration of leaf sheaths ovipositied by WBPH,respectively. Both the major gene resistance to WBPH, sucking inhibitory and ovicidal resistance, showed 1 (resistant): 1 (susceptible) segregation ratio in the DH population. Relative density of WBPH populations and damage scores in the DH population indicated combined functions of both the major resistance genes as well as QTLs affecting the host plant response to WBPH infestations. Thus, the newly developed CJ-06/TN1 DH population could be a useful material to analyze major genes and QTLs for WBPH resistance in japonica rice.

Weighted Correlation Network Analysis(WGCNA) of Japanese Flounder(Paralichthys olivaceus) Embryo Transcriptome Provides Crucial Gene Sets for Understanding Haploid Syndrome and Rescue by Diploidization

Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.

Differential Expression of Whitebacked Planthopper Resistance in the japonica/indica Doubled Haploid Rice Population under Field Evaluation and Seedbox Screening Test

Whitebacked planthopper (WBPH) -resistance in a japonica / indica doubled haploid (DH) rice population established from a cross between WBPH-resistant japonica Chun]iang 06 and susceptible indica TN1, was comparatively evaluated through a field experiment based on the WBPH immigrant density and standardized seedbox screening test (SSST). All the susceptible DH lines in the field experiment behaved accordingly in SSST. However, 35 of resistant 66 lines (53%) in the field, were categorized to susceptible groups in SSST. Likewise, there were no significant differences in WBPH immigrant densities among 70 DH lines that were highly resistant to susceptible in SSST. The results revealed that SSST could not evaluate properly WBPH resistance in the DH lines. Four QTLs for WBPH-resistance phenotyped by the immigrant density were detected on chromosomes 2, 3, 4, and 11. Of them, the QTL on chromosome 4 was the most effective (LOD 21.8, variance 78%). Five QTLs associated with seedling mortality were mapped on chromosomes 2, 3, 4, 5 and 6. In addition to the QTL (LOD 10.5, variance 68%) on chromosome 4, there was another major QTL (LOD 12.7, variance 71%) located on chromosome 5, which was SSST-specific but might be irrespective of the WBPH resistance traits.

Haploid Induction via In vitro Gynogenesis in Tomato(Solanum lycopersicum L.)

In order to determine the potential for haploid induction via in vitro gynogenesis in tomato, the ovules and protoplasts of embryo sacs from the hybrids Zhongza 101 and Zhongza 105 were cultured. An efficient method of ovule isolation was established in this study. Using this method, 100-150 ovules could be isolated from one ovary. Isolated ovules were cultured on three induction media to induce gynogenesis in vitro. During culture, ovules were enlarged markedly, with opaque white color. When observed microscopically, there were cell divisions and cell clumps in embryo sacs. Subsequently, the cell clumps in embryo sacs ceased growth, likely because the integument grew faster than embryo sacs did and hindered the fiarther development of embryo sacs. Therefore, subsequent callus morphogenesis might be originated from the integument. Thousands ofcalli from the two tomato varieties were obtained. Five diploid plants were regenerated after 15 months of subculturing. To eliminate the hindering effect of integument on embryo sac cells, the protoplasts of embryo sacs were prepared and cultured. After 48 hours of culture, the protoplasts of embryo sacs doubled in size and gradually formed clusters of cells. These results suggested that gynogenesis might be a potential way for haploid induction in tomato.

In vitro anther culture and Agrobacterium-mediated transformation of the AP1 gene from Salix integra Linn. in haploid poplar(Populus simonii × P. nigra)

A reliable,efficient anther culture system,the dominant technique for generating haploid plants in breeding programs,that can be used for generating transgenic poplar plants has been needed.In the present study,therefore,an anther culture system was developed using isolated mid-and late-uninucleate anthers of poplar(Populus simonii x P.nigra).From a combination of SSR and ploidy analyses,six double haploid and two haploid lines were characterized from 86 plants grown from 16 regenerated anther cultured lines.After 48 months of development,two plant lines from the regenerated plants maintained their haploid level in vitro for over 2 years.A number of haploid plants from the different lines weretransferred to soil.The leaves of these transplants were then used as explants for transformation with the APETALA1(AP1) gene using Agrobacterium tumefaciens.Overexpression of AP1 in haploid poplar induced early flowering with obvious petals when ectopically expressed.To our knowledge,this is the first report on changes in flowering time in AP1-trangenic poplar,which is important for elucidating the regulatory mechanism of tree flower development.

Detection of QTLs with Additive Effects, Epistatic Effects, and QTL × Environment Interactions for Zeleny Sedimentation Value Using a Doubled Haploid Population in Cultivated Wheat

In order to understand the genetic basis for Zeleny sedimentation value （ZSV） of wheat, a doubled haploid （DH） population Huapei 3 × Yumai 57 （Yumai 57 is superior to Huapei 3 for ZSV）, and a linkage map consisting of 323 marker loci were used to search QTLs for ZSV. This program was based on mixed linear models and allowed simultaneous mapping of additive effect QTLs, epistatic QTLs, and QTL x environment interactions （QEs）. The DH population and the parents were evaluated for ZSV in three field trials. Mapping analysis produced a total of 8 QTLs and 2 QEs for ZSV with a single QTL explaining 0.64-14.39% of phenotypic variations. Four additive QTLs, 4 pairs of epistatic QTLs, and two QEs collectively explained 46.11% of the phenotypic variation （PVE）. This study provided a precise location of ZSV gene within the Xwmc 93 and GluD1 interval, which was designated as Qzsv-1D. The information obtained in this study should be useful for manipulating the QTLs for ZSV by marker assisted selection （MAS） in wheat breeding programs.

Mapping QTLs for Drought Tolerance at Seedling Stage in Rice Using Doubled Haploid Population

QTLs for drought tolerance at the rice seedling stage were analyzed using a doubled haploid （DH） population consisted of 251 lines from the cross between a japonica parent Maybelle and an indica parent Baiyeqiu. A genetic linkage map with 226 SSR marker loci was constructed. Single-locus analysis following composite interval mapping （CIM） detected a total of five QTLs located on five different chromosomes of rice. Four QTLs were also detected following two-locus analysis, resolving two pairs of epistatic QTLs （E-QTI_s） with positive and additive genetic effects. The results indicated that the alleles from the parent Baiyeqiu contributed DH population to improve drought tolerance at the seedling stage.

Development and Characterization of Elite Doubled Haploid Lines from Two Indica Rice Hybrids

Despite significant yield advantage over inbred rice, the adoption rate of hybrid rice in India is very low due to the high seed cost and poor quality of the produce. To alleviate the problem, we initiated a doubled haploid （DH） breeding approach to develop new lines from two elite indica rice hybrids （CRHR5 and CRHR7） through rapid fixation of homozygosity in the recombinants. In vitro culture of the rice anthers resulted in 243 and 186 fertile DH lines of CRHR5 and CRHR7, respectively. Flow cytometric and pollen fertility analyses confirmed the DH ploidy status of the regenerations. Morpho-agronomic evaluation revealed 100% uniformity and stability for all the characters in the DH lines of both hybrids. Nineteen promising DH lines of each hybrid were advanced to A2 generation for yield evaluation. The yield levels of the DH lines ranged from 5 097-6 965 kg/hm^2 for CRHR5 and 5 141-7 235 kg/hm^2 for CRHR7, which were at par or higher than the parental hybrids. Physico-chemical characterization and cooking quality analyses revealed significant and acceptable values for grain length and width, alkali spreading value, amylose content and water uptake ratio of the selected DH lines. Three DH lines, CR5-10, CR5-49, CR5-61 from CRHR5, and four DH lines, CR7-5, CR7-7, CR7-12 and CR7-52 from CRHR7, showed significant grain yield and quality characteristics and have been recommended for multi-location trials for subsequent release as new indica doubled haploid rice varieties.