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.

大白菜抗根肿病DH系的创制与鉴定

为了快速获得纯合稳定的抗根肿病大白菜DH系,为大白菜抗根肿病育种提供基础材料,以5个大白菜抗根肿病基因型为试材进行游离小孢子培养,并结合分子标记、形态观察和根肿菌人工接种对获得的小孢子再生植株进行了表型和抗性鉴定。结果表明,5个基因型均诱导出胚,出胚率变异在0.02~1.72个胚/蕾,3个基因型20aCR12、21aCR6、21aCR12获得再生植株,再生植株率分别为27.09%,1.45%,26.07%。抗性标记鉴定表明,与CRa、CRb^(kato)连锁的分子标记在获得的50个小孢子再生植株均扩增出了抗性条带。表型调查表明,50个小孢子株系生殖期基生叶片形状、颜色、抽薹早晚、育性等差异很大,自交获得的29个DH系营养期株型、叶片性状及结球性状表现出多样性。抗性接种表明,11份DH系对根肿菌4号和1号生理小种侵染病情指数均小于33.33,表现为抗病或耐病,其中2种菌侵染病情指数均小于5.0的高抗DH系有3份,分别为20aCR12-23、20aCR12-29和21aCR12-39。研究表明,单倍体培养结合分子标记辅助鉴定可快速获得纯合的抗根肿病DH系。

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.

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.

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.

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.

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.

小麦DH高效生产技术体系在云南的研究与应用

双单倍体(DH)技术可使杂合育种材料在一个世代纯合稳定,被广泛用于作物遗传育种。小麦×玉米杂交是产生小麦DH的主要途径之一,但小麦和玉米一般在不同季节种植,制约了该技术的广泛应用。春性和经春化处理的半冬性、冬性小麦材料在云南昆明自然条件下一年四季均可播种、收获,为每年4月至12月进行小麦×玉米杂交提供了便利条件。本团队前期建立了平均得胚率为25%(15%~70%)、成苗率为62%(50%~80%)以及加倍率为62%(50%~90%)的小麦DH批量生产技术规程。并于2015年以来,利用国内外共1900余份不同遗传背景的春性、半冬性和冬性小麦材料进行验证,共获得10.5×10^(4)个小麦DH株系;构建了64个DH遗传群体;育成了云麦110、云麦112等小麦新品种;创制了24个优良小麦温光敏核不育系及一批抗病优良品系,初步实现了该DH技术在小麦育种中的应用。后续还需继续完善规模化DH生产技术规程,提高得胚率、成苗率、加倍率等关键技术指标和DH生产效率,降低成本,促进该技术在小麦遗传育种中更广泛地应用。

普通小麦(浙农林12×CASL7AS)DH系株高的分析

小麦株高与植株倒伏密切相关,影响小麦产量。为挖掘小麦株高QTL位点和候选基因,从遗传和分子水平上解释株高分子机理,本研究将野生二粒小麦染色体臂置换系CASL7AS为母本,课题组自选株系浙农林12(简称ZNL12)为父本,杂交F 1经过花培得到178个DH群体。通过4年2个种植点株高数据,利用55K SNP芯片构建高密度遗传图谱,对小麦株高性状进行QTL分析。该遗传连锁图谱包含3655个SNP标记,长度为4738.45 cM,标记间平均遗传距离为1.30 cM,覆盖了小麦21条染色体。其中,A、B、D染色体组分别含有标记1466、1492和697个。QTL分析共检测出53个QTL位点,分布于1A、3A、5A、7A、1B、3B、4B、5B、7B、2D、4D、6D和7D染色体上,可解释2.80%~38.50%表型变异。其中,稳定主效QTL有2个,分别为QPh.zafu.4B-1和QPh.zafu.4D-1,其贡献率分别为25.18%~38.50%和20.67%。经细胞生物学分析,基因型为(0,2)矮秆植株胚芽鞘的表皮细胞长度显著短于基因型为(2,0)的高秆植株,基因型为(0,0)、(2,2)中间型的植株胚芽鞘表皮细胞长度无显著差异,由此推测小麦株高的变异由细胞长度因素决定。QPh.zafu.4B-1候选基因Rht-B1b基因编码区矮秆株系,第904核苷酸处发生碱基“C-T”的突变,形成终止密码子(CAG-TAG);而QPh.zafu.4D-1候选基因Rht-D1b基因编码区矮秆株系,第781核苷酸处碱基突变“G-T”,编码氨基酸中缬氨酸变为天冬氨酸。结果为小麦株高候选基因的筛选和QTL定位提供了优异的遗传位点和候选基因,未来可用于小麦抗倒伏相关的分子标记辅助育种。

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.

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.

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.

DH血缘对玉米单倍体诱导率和自然加倍率的影响

通过对4份Lancaster种质进行双列杂交和统计分析,研究含有DH系的基础材料对单倍体诱导率、散粉率和自然加倍率的影响。结果表明,不同基因型材料的诱导率为3.6%~11.4%,散粉率为11.4%~40.0%,自然加倍结实率为3.4%~14.1%;来源不同的单倍体株系的诱导率、散粉率以及结实率均表现出相同的趋势,由高到低排序依次为双亲都为DH系的基础材料、双亲有1个为DH系的基础材料、双亲无DH系的基础材料。以含有DH系的基础材料具有较高的诱导率、散粉率和结实率。将优秀自交系与选育的DH系进行杂交,有助于提高诱导效率和加倍效率,使优良基因充分聚合,实现轮回上升式改良种质的目标,旨在为提高单倍体育种效率提供理论依据。

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.

Gametic selection in a doubled haploid population derived from anther culture of indica/japonica hybrid of Oryza Sativa L.

It is one of the key problems for application ofanther culture in hybrid breeding, geneticanalysis, and molecular mapping whether thedoubled haploid (DH) population derived fromanther culture of rice crosses represents a ran-dom array of the microspore population, i.e.whether gametic selection occurs in androgene-sis. A DH population including 132 lines de-

芥蓝游离小孢子培养技术优化及其在育种中的应用

以16个芥蓝品种(F1)为供体材料,比较分析了优化花蕾的消毒、小孢子的游离释放以及过滤等环节对芥蓝游离小孢子培养效果的影响。结果表明,16份参试芥蓝供体材料都具有胚胎发生能力,并全部获得了不同类型的再生植株,实现了较大规模、批量地获得芥蓝双单倍体(DH)。经田间综合评价筛选,应用DH系育成了不同熟性的芥蓝一代杂种20M4芥蓝、21M2芥蓝和22M11芥蓝。