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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Mapping resistant QTLs for rice sheath blight disease with a doubled haploid population

Sheath blight（SB） disease,caused by Rhizoctonia solani Kuhn,is one of the most serious diseases causing rice（Oryza sativa L.） yield loss worldwide.A doubled haploid（DH） population was constructed from a cross between a japonica variety CJ06 and an indica variety TN1,and to analyze the quantitative trait loci（QTLs） for SB resistance under three different environments（environments 1-3）.Two traits were recorded to evaluate the SB resistance,namely lesion height（LH） and disease rating（DR）.Based on field evaluation of SB resistance and a genetic map constructed with 214 markers,a total of eight QTLs were identified for LH and eight QTLs for DR under three environments,respectively.The QTLs for LH were anchored on chromosomes 1,3,4,5,6,and 8,and explained 4.35-17.53%of the phenotypic variation.The SB resistance allele of qHNLH4 from TN1 decreased LH by 3.08 cm,and contributed to 17.53%of the variation at environment 1.The QTL for LH（qHZaLH8） detected on chromosome 8 in environment 2 explained 16.71%of the variation,and the resistance allele from CJ06 reduced LH by 4.4 cm.Eight QTLs for DR were identified on chromosomes 1,5,6,8,9,11,and 12 under three conditions with the explained variation from 2.0 to 11.27%.The QTL for DR（qHZaDR8）,which explained variation of 11.27%,was located in the same interval as that of qHZaLH8,both QTLs were detected in environment 2.A total of six pairs of digenic epistatic loci for DR were detected in three conditions,but no epistatic locus was observed for LH.In addition,we detected 12 QTLs for plant height（PH） in three environments.None of the PH-QTLs were co-located with the SB-QTLs.The results facilitate our understanding of the genetic basis for SB resistance in rice.

Nuclear Fusion during Early Stage of Microspore Embryogenesis Indicates Chromosome Doubling in Wheat (Triticum aestivum)

Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective cell-type. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as “small” are likely haploids and nuclei classified as “large” are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.

In vitro Regeneration of First-generation Inbred Progenies of Cucurbita pepo L. Double Haploids

Using seeds of inbred progenies of Cucurbita pepo L. DH12-11409 double haplolds as experimental materials, the effects of different hormone combinations, explant types and seedling ages on adventitious bud regeneration and rooting of C. pepo L. were investigated. According to the results, inoculating cotyledonary nodes of yellow-green cotyledons from 5-d-old C. pepo L. double haploids to MS ＋ 30 g/L Suc ＋ 8 g/L Agar ＋ 2.0 mg/L 6-BA ＋ 0.1 mg/L NAA led to the best results with the induction frequency of 90.0% and differentiation coefficient of 8.5. MS medium with addition of 0.05 or 0.1 mg/L NAA led to the highest rooting rate. Regenerated seedlings with 5 - 6 true leaves exhibited the highest survival rate of 90.0%, which was the optimal period for domestication and transplanting of regenerated seedlings. This study laid a solid foundation for high-efficiency utilization of heterosis of C. pepo L.

A New Approach to Select Doubled Haploid Rice Lines under Salinity Stress Using Indirect Selection Index

This study determined the indirect selection index of doubled haploid(DH)rice using a multivariate analysis approach to select lines adaptive to salinity stress,comprising three experiments.The first experiment involved the selection of good agronomic characters in a field experiment conducted at an experimental station in Bogor,Indonesia.The second experiment involved salinity tolerance screening through hydroponic cultures using 0 and 120 mmol/L NaCl,conducted at a greenhouse in Bogor.The third experiment involved the validation of the indirect adaptability selection index(IASI)through a field experiment in Sukra(saline area).Field experiments followed a randomized complete block design(RCBD),whereas an RCBD nested factorial design was used for the greenhouse experiment.The first and second experiments used 56 DH lines and four check varieties with three replications.In the second experiment,Pokkali and IR29 varieties were also added as tolerant and sensitive checks of salinity,respectively.The third experiment used 28 selected DH lines,Inpari 29 and one sensitive DH line.The good agronomic index(GAI)was 0.465 yield+0.433 number of productive tillers+0.31 number of filled grains.This generated 24 DH rice lines with good agronomic traits.The salinity tolerance index(SaTI)was developed through the average of standardized salinity tolerance score and salinity selection index based on discriminant analysis.This generated 34 DH rice lines with good salinity stress tolerance.The IASI(IASI=GAI–SaTI)selected 28 DH rice lines adaptive to salinity stress and it was considered effective by Sukra validation.

Resistance Performance to Whitebacked Planthopper in Different Phenotypes of Japonica/Indica Doubled Haploid Rice Lines

Field performance of whitebacked planthopper （WBPH）-resistance of four phenotypes was evaluated in Chunjiang 06 （C J-06） / TN1 DH rice lines, which were expressed by different combinations of sucking inhibitory and ovicidal traits inherited independently from C J-06. WBPH established the highest populations in susceptible DH lines that had neither sucking inhibitorynor ovicidal resistance. Both immigration and subsequent population levels were kept below the damage-causing density in the sucking inhibitory DH lines even under a WBPH outbreak. WBPH could not build up populations in the DH lines having both the sucking inhibitory and ovicidal resistance. Although WBPH immigrated preferentially to non-sucking inhibitory DH lines with ovicidal resistance, subsequent population buildup was significantly suppressed. It was concluded that the differential performance to WBPH-resistance in CJ-06 / TN1 DH lines was primarily due to the sucking inhibitory trait, and complementarity to the ovicidal trait.

Yield Stability and Adaptability of Elite Doubled Haploid (DH) Rice Genotypes in Different Locations in Central Clay Plains of the Sudan

Stability analysis of grain yield is an efficient tool for the selection of varieties adapted to fairly wide cultivation zone. A study aims to determine adaptability and stability for grain yield and agronomic performance of 10 elite Doubled Haploid (DH) rice genotypes generated through another culture technique, along with local cultivar Umgar as a check under irrigated lowland conditions in three different environments in the central clay plains of the Sudan;during two cropping seasons 2019 and 2020. The trials were conducted in a randomized complete block design with three replications. Grain yield stability was studied, using the Additive Main effect and Multiplicative Interaction (AMMI) model. Bi-plots were developed following GGE bi-plot methodology over the six environments. The combined analysis of variance revealed significant to highly significant (P < 0.001) effects of genotypes, environments, and genotype by environment interaction. Moreover, the analysis of variance based on AMMI indicated significant genotypes, environments, and GE interaction with a total variation of 43.16%, 44.26% and 12.58% respectively. A biplot-AMMI analysis and yield stability index incorporating the AMMI stability value and yield in a single non-parametric index were used to identify the genotypes with the highest and stable yield. The overall mean for grain yield was 5.69 t/ha, with a range of 5.77 to 4.12 t/ha. The genotypes k150147, k150870, k150032, k150912, and k150307 out yielded the local check Umgar by 40%, 38%, 34%, 34% and 33% respectively. The most genotypes were mid-early maturities which were harvested at 107, 113, 108, 109 and 112 days after sowing, respectively. These five genotypes also showed grain yield stability along with their high mean yield performance according to the AMMI analysis and widely adaptable to the tested locations. Therefore, the five out yielding genotypes will be considered for cultivation under irrigated system condition in central clay in the Sudan.

Genetic Diversity Analysis of Elite Doubled Haploid Rice Genotypes for Yield Attributing Traits in White Nile State, Sudan

The development of new rice varieties is highly dependent on genetic diversity in desirable agronomic traits. Therefore, this study aimed to identify potential genotypes having the characters of Korean varieties (Tongil-type) and japonica developed through doubled haploid (DH) technology to apply in our breeding materials. 35 elite DH lines derived from another culture of Korean and African rice along with two local checks were planted in a randomized complete block design with three replications during the two seasons of 2019 and 2020. All evaluated genotypes exhibited a wide and significant variation in the ten measured traits. The highest heritability related to high genetic advance was recorded for the number of tiller/plant, grain yield t/ha, number of filled grain per panicle, and thousand grain weights (g). Genotypic coefficient of variation and genetic advance were recorded for number of tiller/plant, number of filled grain per panicle, 1000 grain weight and grain yield t/ha in both seasons. Moreover, there was a highly significant and positive correlation of grain yield with number of filled grain per panicle (0.65), number of tiller/plant (0.64) and number of panicle per m2 (0.54). Cluster analysis based on grain yield components trait grouped the 37 rice genotypes into four clusters. Cluster B was the largest and consisted of 13 genotypes. Finally, it could be concluded that, based on number of productive tillers, number of filled grain/panicle, number of panicle per m2, and grain yield, the lines KF170506, KF170509, KF170542, KF170530, KF170543, KF170500 and KF170510 were high potential for further selection for new type of irrigated rice. In addition, hybridization of these 7 high-yielding could be used to achieve higher heterosis among the genotypes. Furthermore, this evaluation could be useful in developing reliable selection indices for improving rice breeding programs.