Analyses and identifications of quantitative trait loci and candidate genes controlling mesocotyl elongation in rice

Rice direct seeding has the significant potential to save labor and water,conserve environmental resources,and reduce greenhouse gas emissions tremendously.Therefore,rice direct seeding is becoming the major cultivation technology applied to rice production in many countries.Identifying and utilizing genes controlling mesocotyl elongation is an effective approach to accelerate breeding procedures and meet the requirements for direct-seeded rice(DSR) production.This study used a permanent mapping population with 144 recombinant inbred lines(RILs) and 2 828 bin-markers to detect quantitative trait loci(QTLs) associated with mesocotyl length in 2019 and 2020.The mesocotyl lengths of the rice RILs and their parents,Lijiangxintuanheigu(LTH) and Shennong 265(SN265),were measured in a growth chamber at 30°C in a dark environment.A total of 16 QTLs for mesocotyl length were identified on chromosomes 1(2),2(4),3(2),4,5,6,7,9,11(2),and 12.Seven of these QTLs,including qML1a,qML1b,qML2d,qML3a,qML3b,qML5,and qML11b,were reproducibly detected in both years via the interval mapping method.The major QTL,qML3a,was reidentified in two years via the composite interval mapping method.A total of 10 to 413 annotated genes for each QTL were identified in their smallest genetic intervals of 37.69 kb to 2.78 Mb,respectively.Thirteen predicted genes within a relatively small genetic interval(88.18 kb) of the major mesocotyl elongation QTL,qML3a,were more thoroughly analyzed.Finally,the coding DNA sequence variations among SN265,LTH,and Nipponbare indicated that the LOC_Os03g50550 gene was the strongest candidate gene for the qML3a QTL controlling the mesocotyl elongation.This LOC_Os03g50550 gene encodes a mitogen-activated protein kinase.Relative gene expression analysis using qRT-RCR further revealed that the expression levels of the LOC_Os03g50550 gene in the mesocotyl of LTH were significantly lower than in the mesocotyl of SN265.In conclusion,these results further strengthen our knowledge about rice’s genetic mechanisms of mesocotyl elongation.This investigation’s discoveries will help to accelerate breeding programs for new DSR variety development.

Screening for Rice Germplasms with Specially-Elongated Mesocotyl

The lengths of mesocotyl in the seedlings of 84 lowland rice varieties and 12 upland rice varieties were measured following the treatments of daylight and darkness during germination. The elongation of mesocotyl in the varieties tested was inhibited under daylight condition, and the mesocotyl of all the varieties elongated variably under darkness condition. The elongated lengths of the mesocotyl in upland rice, ranging from 0.36 cm to 1,61 cm with an average of 0.81 cm, was obviously longer than those in lowland rice, ranging from 0.12 cm to 1.56 cm with an average of 0.42 cm. Among 14 rice varieties with over 1 cm of mesocotyl length, five belonged to upland rice, and nine to lowland rice. The possible utilization of the elongated-mesocotyl rice germplasm in varietal imorovement, direct-seeded plantina and seed ouritv testina were discussed.

Integrated transcriptome, small RNA, and degradome analysis to elucidate the regulation of rice seedling mesocotyl development during the passage from darkness to light

The mesocotyl,a structure located between the basal part of the seminal root and the coleoptile node of seedlings,contributes to pushing the shoot tip through the soil surface,a function that is essential for the uniform emergence of direct-seeded rice.Its elongation is inhibited by light and induced in darkness.This investigation of an indica rice(P25)with vigorous mesocotyl elongation was aimed at identifying the"omics"basis of its lightinduced growth inhibition.A transcriptomic comparison between mesocotyl tissues that had developed in the dark and then been exposed to light identified many differentially expressed genes(DEGs)and differentially abundant micro RNAs(mi RNAs).Degradome sequencing analysis revealed 27 negative mi RNA-target pairs.A co-expression regulatory network was constructed based on the mi RNAs,their corresponding targets,and DEGs with a common Gene Ontology term.It suggested that auxin and light,probably antagonistically,affect mesocotyl elongation by regulating polyamine oxidase activity.

水稻已克隆中胚轴伸长基因效应解析及优异单倍型鉴定

旱直播是未来水稻生产的重要发展方向,在南亚和东南亚籼稻种植区已有较大面积,然而热带和亚热带地区粳稻种植多采用传统育苗移栽。中胚轴长度(Mesocotyl length,ML)是影响水稻旱直播出苗和幼苗活力的重要因素。基于分子标记辅助选择(Molecular marker assisted selection,MAS)育种,培育长中胚轴种质是促进水稻旱直播推广最为经济、高效的方式。迄今,已有4个水稻中胚轴伸长基因报道,即OsGSK2、GY1、OsPAO5和OsSMAX1。笔者选择3K重测序项目中TROP和TEMP粳稻亚群开展分析,测定中胚轴长度并鉴定OsGSK2、GY1、OsPAO5和OsSMAX1基因的优异单倍型。结果表明,TROP和TEMP亚群中胚轴长度呈典型的连续正态分布。OsGSK2、GY1、OsPAO5和OsSMAX1分别包括3、3、3和6种单倍型;同一基因在TROP和TEMP亚群中单倍型分布频率也存在差异。TROP亚群中OsGSK2-Hap1、GY1-Hap2、OsPAO5-Hap3、OsSMAX1-Hap2和OsSMAX1-Hap3为优异单倍型;TEMP亚群优异单倍型为OsGSK2-Hap1、OsPAO5-Hap2和OsSMAX1-Hap2。另外,TROP和TEMP亚群中多数中胚轴优异单倍型材料所对应的苗高要高于非优异单倍型,在破土出苗时易形成生长优势。鉴定到的优异单倍型在TROP和TEMP亚群中均呈现较显著的加性效应,可用于MAS育种。本研究为不同地区直播粳稻的选育提供参考,促进旱直播技术的快速推广。

Targeted mutagenesis of POLYAMINE OXIDASE 5 that negatively regulates mesocotyl elongation enables the generation of direct-seeding rice with improved grain yield

Under conditions of labor or resource scarcity,direct seeding,rather than transplantation,is the preferred mode of rice(Oryza sativa)cultivation.This approach requires varieties that exhibit uniform seedling emergence.Mesocotyl elongation(ME),the main driver of rapid emergence of rice seedlings from soil,is enhanced by darkness and inhibited by light.Plant polyamine oxidases(PAOs)oxidize polyamines(PAs)and release H2O2,Here,we established that OsPAO5 expression in rice seedlings is increased in the presence of light and inhibited by darkness.To determine its role in ME,we created OsPAO5 mutants using CRISPR/Cas9.Compared with the wild type,pao5 mutants had longer mesocotyls,released less H2O2,and synthesized more ethylene.The mutant seedlings emerged at a higher and more uniform rate,indicating their potential for use in direct seeding.Nucleotide polymorphism analysis revealed that an SNP(PAO5-578G/A)located 578 bp upstream of the OsPAO5 start codon alters its expression,and was selected during rice mesocotyl domestication.The PAO5-578G genotype conferring a long mesocotyl mainly exists in wild rice,most Aus accessions,and some Geng(Japonica)accessions.Intriguingly,knocking out OsPAO5 can remarkably increase the grain weight,grain number,and yield potential.In summary,we developed a novel strategy to obtain elite rice with higher emergence vigor and yield potential,which can be conveniently and widely used to breed varieties of direct-seeding rice.

Downregulation of Rice DWARF 14 LIKE Suppress Mesocotyl Elongation via a Strigolactone Independent Pathway in the Dark

Strigolactones （SLs） are a class of plant hormones that control plant development in response to environmental conditions. In rice, mesocotyl elongation is regulated by SLs in the dark, while mesocotyls are longer in SL deficient or insensitive mutants. SLs are perceived by DWARF14 （D14）, which is a member of a small gene family. In this study, we examined the function of another D14 family gene in rice, D14 LIKE （D14L）, focusing on mesocotyl growth. The mesocotyls of D14L RNAi lines are longer than those of WT in the dark. This phenotype is enhanced when the D14L RNAi lines are combined with the d14 mutation, suggesting that D14 and D14L work independently to inhibit mesocotyl elongation. This phenotype is alleviated by the exogenous supply of GR24, a synthetic SL, suggesting that D14L is not necessary for SL signaling. D14L mRNA is predominantly expressed in vascular bundles and crown root primordia. Our results suggest that D14L and D14 confer their effects via an SL independent pathway and an SL signaling pathway respectively.

Effects of gibberellin priming on seedling emergence and transcripts involved in mesocotyl elongation in rice under deep direct-seeding conditions

Mesocotyl elongation is a key trait influencing seedling emergence and establishment in direct-seeding rice cultivation.The phytohormone gibberellin(GA)has positive effects on mesocotyl elongation in rice.However,the physiological and molecular basis underlying the regulation of mesocotyl elongation mediated by GA priming under deep-sowing conditions remains largely unclear.In the present study,we performed a physiological and comprehensive transcriptomic analysis of the function of GA priming in mesocotyl elongation and seedling emergence using a direct-seeding japonica rice cultivar ZH10 at a5-cm sowing depth.Physiological experiments indicated that GA priming significantly improved rice seedling emergence by increasing the activity of starch-metabolizing enzymes and compatible solute content to supply the energy essential for subsequent development.Transcriptomic analysis revealed 7074 differentially expressed genes(false discovery rate of<0.05,|log2(fold change)|of≥1)after GA priming.Furthermore,gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analyses revealed that genes associated with transcriptional regulation,plant hormone biosynthesis or signaling,and starch and sucrose metabolism were critical for GA-mediated promotion of rice mesocotyl elongation.Further analyses showed that the expression of the transcription factor(TF)genes(v-myb avian myeloblastosis viral oncogene homolog(MYB)alternative splicing 1(MYBAS1),phytochrome-interacting factors 1(PIF1),Oryza sativa teosinte branched 1/cycloidea/proliferating cell factor 5(Os TCP5),slender 1(SLN1),and mini zinc finger 1(MIF1)),plant hormone biosynthesis or signaling genes(brassinazole-resistant 1(BZR1),ent-kaurenoic acid oxidase-like(KAO),GRETCHEN HAGEN 3.2(GH3.2),and small auxin up RNA 36(SAUR36)),and starch and sucrose metabolism genes(α-amylases(AMY2 A and AMY1.4))was highly correlated with the mesocotyl elongation and deep-sowing tolerance response.These results enhance our understanding of how nutrient metabolism-related substances and genes regulate rice mesocotyl elongation.This may facilitate future studies on related genes and the development of novel rice varieties tolerant to deep sowing.

水稻中胚轴伸长相关候选基因的关联分析

旱直播技术可有效提升水稻生产效率。中胚轴长度(ML,mesocotyl length)是影响旱直播水稻出苗和幼苗活力的重要性状。选育长中胚轴品种是促进旱直播技术推广最为经济、有效的方式。旱直播在南亚和东南亚地区的籼稻种植区已有一定推广面积,在粳稻种植区推广面积较少。前人已发现一批中胚轴伸长相关候选基因,但其可靠性及适用性尚待验证。基于已报道的97个中胚轴伸长相关候选基因,在不同来源的TROP和TEMP两个粳稻自然群体开展候选基因关联分析,鉴定到4个显著候选基因,解释4.7%~6.3%和5.4%~6.7%的遗传变异。其中,LOC_Os01g44130、LOC_Os03g50560和LOC_Os05g27790在TROP和TEMP群体中均显著关联,而LOC_Os11g10990和LOC_Os10g20860分别在TROP和TEMP群体中显著关联。候选基因所编码蛋白主要参与植物激素合成与代谢、信号转导和植物生长进程。进一步在TROP群体(LOC_Os05g27790-Hap3和LOC_Os05g27790-Hap6、LOC_Os03g50560-Hap1、LOC_Os01g44130-Hap1和LOC_Os11g10990-Hap1和LOC_Os11g10990-Hap3)和TEMP群体(LOC_Os05g27790-Hap6、LOC_Os01g44130-Hap1和LOC_Os10g20860-Hap5)中分别鉴定到6个和3个可用于分子标记辅助育种的优异单倍型。本研究鉴定到的显著关联候选基因及其优异单倍型可应用于水稻长中胚轴分子育种实践中。

糯高粱顶土能力与幼苗器官形态关系的研究

[目的]高粱是传统的特色杂粮作物,具有食用、酿酒、饲用、能源、青贮用等多种用途。然而,高粱种子出苗率低直接影响其大面积推广种植。通过研究高粱顶土能力与幼苗器官形态的关系,旨在为提高高粱种质出苗率以及高粱新品种选育成效提供理论依据。[方法]本研究选用3个糯高粱品种晋糯3号(10480A_(2)×L17)、晋糯4号(11476A_(2)×L2R)、红糯16号(11494A_(2)×L17)及对应的父母本共8份材料,采用培养皿无阻力发芽、黑暗水培发芽、3个不同播深沙培发芽及大田出苗率调查试验4种发芽试验,分析中胚轴和胚芽鞘伸长特性与出苗率之间的关系。[结果]无阻力条件下,高粱种子发芽试验结果表明不同种质发芽率和发芽势具有明显差异,杂交种的发芽率和发芽势大于其对应的父母本。黑暗水培处理下,杂交种与母本中胚轴伸长达到显著或极显著差异。进一步模拟大田环境沙培发芽试验发现,不同种质顶土能力对播种深度的敏感程度不同,11476A_(2)、10480A_(2)对播种深度有一定的耐受力,顶土能力较强,而11494A_(2)、L17则对播种深度较敏感,顶土能力较弱;杂交种的顶土能力基本与其对应的母本相一致。不同播种深度处理下,种质胚芽鞘长度变化不明显,而中胚轴显著伸长,说明高粱顶土能力与中胚轴伸长直接相关。大田试验出苗率与各性状指标相关分析结果表明,出苗率与中胚轴呈显著正相关,说明中胚轴的伸长能力是高粱种质出苗率变化的重要因素,可作为今后评价高粱种质出苗率的指标。本研究发现,不同播种深度条件下种质11476A_(2)、10480A_(2)中胚轴长度差变化较大,顶土能力较强,是杂交测配的优良亲本。[结论]为保证高粱出苗率,在遗传育种过程中,要注重选育中胚轴伸长能力大、顶土能力强的亲本种质,尤其是母本的选育。

水稻中胚轴伸长性状QTL分析

【目的】水稻中胚轴伸长可以在直播方式下为种子出苗提供顶土能力,培育中胚轴长的水稻品种对水稻直播技术的推广具有重要意义。【方法】将籼稻品种贵9B和粳稻品种热研2号杂交衍生的100个RILs及籼稻品种V20B和粳稻品种CPSlo17杂交衍生的150个RILs为材料,于2020年种植于海南三亚,于2021年种植于贵州贵阳,连续2年收获种子后通过测定中胚轴长度,以2套重组自交系(recombinant inbred lines,RILs)为作图群体进行QTL定位分析。【结果】以贵9B和热研2号为亲本的RIL群体的QTL定位结果表明,种子发芽后,连续2年定位到3个QTL分别位于第8和第10染色体上,LOD值为2.51、2.63和3.42,可解释18.17%~20.68%的表型变异,等位基因均源于贵9B。以V20B和CPSlo17作为亲本构建的RIL群体2年定位到4个QTL,分别位于第2、4、7染色体上,LOD值分别为3.15、3.59和13.59,可解释7.37%~10.53%的表型变异,等位基因均来源于V20B。【结论】2套群体连续2年定位到的QTL位点均有一个位点重叠,说明该位点的QTL在2个生态环境下较稳定,对后续精细定位和克隆中胚轴伸长相关基因具有重要参考价值,为研究水稻中胚轴伸长分子机制及水稻直播育种提供了新的观点和见解。

水稻中胚轴伸长特性的遗传分析

以长中胚轴/短中胚轴杂交组合的2个亲本、F1、B1、B2和F2为试验材料,在小型人工气候箱黑暗条件下培养幼苗,对水稻中胚轴伸长特性的遗传规律进行了研究。结果表明,中胚轴伸长特性受2对隐性基因控制。F2群体中长、短中胚轴植株在节间长度、穗长、株高和剑叶长等性状上均存在较明显的差异,而中胚轴伸长特性与节间粗、剑叶宽、单株穗数、每穗总粒数、结实率、千粒重和单株产量等性状关系不明显。还对水稻中胚轴伸长特性的遗传改良以及长中胚轴材料在直播稻育种上的利用进行了探讨。

水稻出苗顶土动力源研究

在自然条件下,通过设计不同的出苗阻力(覆土深度2 cm、4 cm和6 cm)来测定中胚轴伸长潜力不同的水稻种质的出苗速度、成苗率和芽鞘节及中胚轴的长度,以分析影响水稻种子顶土出苗的动力源。结果表明,水稻种子的出苗动力主要来源于芽鞘节间和中胚轴的伸长。在不同阻力处理条件下,不同种质出苗动力差异较大,其中覆土2 cm的不同种质出苗率相差不明显,与室内发芽率结果比较接近,覆土4 cm和覆土6 cm的两个处理不同种质间出苗率差异明显,长胚轴种质出苗速度快,出苗率高,出苗率与室内发芽率相差不大;而短胚轴种质出苗速度慢,出苗率低,其中春江683的出苗率与室内发芽率相比分别下降了6.5和86.2个百分点。研究表明水稻中胚轴的伸长对覆土较深的种子顶土出苗起到关键作用,长胚轴种质顶土出苗动力来自于芽鞘节与中胚轴的共同作用,出苗动力强;而短胚轴顶土出苗动力主要来源于芽鞘节的伸长,出苗动力较弱。还对通过选育长胚轴直播稻品种解决直播稻易倒伏和出苗差等难题的可能性进行了探讨。

水稻籼粳交DH群体幼苗中胚轴长度的QTLs定位和上位性分析

应用籼粳交 IR6 4 / Azucena的 DH群体及其构建的分子标记遗传图谱 ,在遮光条件下 ,通过适温和低温逆境下发芽 ,测定中胚轴长度。采用 QTL Mapper基因定位软件检测控制中胚轴长度的加性效应 QTL s和加性×加性上位性 QTL s,在第 1、3、6、7、8、12等 6条染色体上定位了 8个控制中胚轴长度的 QTL s,其中在第 1、3、7、8染色体上定位了 4个具有加性效应的 QTL s,位于第 7染色体的 1个加性效应 QTL的增长等位基因来自于父本 Azucena,它能使中胚轴伸长 0 .2 6 cm,其贡献率达 17.5 % ,其余 3个加性效应 QTL s的增长等位基因来自于母本 IR6 4 ,能使中胚轴伸长 0 .10～ 0 .2 1cm,在第 3、7、12等3条染色体中共检测到 2对加性×加性上位性效应 ,其贡献率分别为 2 1.6 2 %和 2 .2 7% ,同时各检测到 2对加性效应×环境的互作效应和上位性与环境的互作效应。

中国北方杂草稻深覆土条件下出苗动力源分析

以盆栽的方式,采用不同的覆土深度,对中国北方杂草稻的出苗特性进行了研究,分析了杂草稻的出苗动力源。研究结果表明,在中国北方发现的部分杂草稻具有较强的中胚轴伸长特性和芽鞘节间伸长特性。在播种深度较浅时(3cm),杂草稻依赖于中胚轴的伸长顶土发芽;当覆土较深时(5cm),杂草稻中胚轴伸长特性更加明显,伸长长度加大,同时杂草稻的芽鞘节间也明显伸长,中胚轴和芽鞘节间的共同伸长为深覆土条件下杂草稻的出苗提供了动力基础。

玉米中胚轴长度与内源激素关系的研究

选用5个春播玉米品种,分别在26℃光照和黑暗条件下培养,测定中胚轴长度以及内源激素含量。结果显示,光照对玉米中胚轴长度有明显的抑制作用,是明显的光控调节途径。黑暗条件下5个品种玉米中胚轴长度随幼苗生长而伸长。中胚轴内源激素是调控其长度的重要物质。其中内源激素生长素(IAA)呈极显著正相关,认为IAA起主要促进作用。其中SN40×Mo17变化最显著,开始时IAA含量少、脱落酸(ABA)抑制中胚轴生长,但随着幼苗生长,IAA含量增多,促进其伸长。

水稻中胚轴长度QTL分析

为分析水稻中胚轴伸长与赤霉素的关系及其遗传基础,以沈农265(长中胚轴)和丽江新团黑谷(短中胚轴)的RIL群体为材料,结合其连锁图谱,对水和赤霉素溶液两种培养条件下的中胚轴长度进行QTL定位。结果表明,浓度为1.50μmolL^(-1)的赤霉素可显著促进中胚轴的伸长。两种培养条件下,共检测到控制中胚轴长度的5个QTL,分布在第1、第2、第3、第6和第11号染色体上,LOD值在3.65～15.52范围内,单个QTL对表型贡献率在7%～33%之间。其中qML3、qML6和qML11在2种处理条件下均被检测到,qML1和qML2仅在水培条件下被检测到。与其他研究比较发现,主效基因qML3可以在不同群体和不同环境下稳定表达。

杂草稻中胚轴伸长动态及其与籽粒淀粉酶活性和可溶性糖含量的关系

采用恒温培养箱,在黑暗条件下进行发芽试验,观察杂草稻中胚轴伸长的动态过程,分析杂草稻中胚轴伸长过程中籽粒淀粉酶活性和可溶性糖含量的变化及相互关系。结果表明,黑暗条件下发芽后1d到3d,杂草稻中胚轴伸长非常慢,发芽后4d到5d中胚轴伸长加快,发芽后7天中胚轴伸长逐渐停止,其最终长度显著长于辽粳294。杂草稻发芽过程中,籽粒α-淀粉酶活性、β-淀粉酶活性和可溶性糖含量均呈逐渐上升趋势。可溶性糖含量与α-淀粉酶活性、β-淀粉酶活性均呈极显著正相关。中胚轴伸长长度与籽粒α-淀粉酶活性、β-淀粉酶活性、可溶性糖含量也均呈极显著正相关。杂草稻发芽初期淀粉酶活性增强,促进了籽粒中淀粉水解为可溶性糖,为中胚轴伸长生长提供了物质和能量基础。

北方杂草稻出苗特性的研究

以43份北方杂草稻为试材,栽培稻秋光为对照,对北方杂草稻的出苗动力及其与出苗率、秧苗素质的关系进行了研究。结果表明:杂草稻的耐深播性强于秋光。在覆土3 cm条件下,杂草稻的出苗动力主要源于中胚轴的伸长。在覆土5 cm条件下,大部分杂草稻的芽鞘节间也发生伸长,与中胚轴共同成为出苗动力。中胚轴长度与出苗率及秧苗素质的相关性随覆土深度的增加而加大。杂草稻有较强的耐深播性,但不同材料间仍有较大差异。随着覆土深度的增加,中胚轴的伸长对提高出苗率及秧苗素质有显著的作用。

提高水稻直播育种水平相关理论研究进展

出苗难、除草难度大、后期倒伏是影响直播稻生产的主要因素,综述了与直播稻有关水稻中胚轴伸长特性、耐低氧发芽、抗除草剂、抗倒性的研究现状,并展望了适合于直播高产栽培是今后育种的首要目标。

赤霉素和脱落酸对谷子黄化幼苗中胚轴伸长生长的调节

对冀谷１１号及矮秆突变体矮宁黄、ＣＨ８４１１３黄化幼苗中胚轴伸长生长的研究发现，０．１μｍｏｌ／Ｌ左右的赤霉素和脱落酸可显著促进谷子中胚轴的伸长，其中ＧＡ３的效应高于ＧＡ４的，ＡＢＡ和ＧＡ之间有叠加效应，矮秆突变体对ＧＡ和ＡＢＡ的反应尤为敏感；Ｓ－３３０７和高浓度（５μｍｏｌ／Ｌ左右）的ＧＡ、ＡＢＡ抑制中胚轴的伸长，Ｓ－３３０７预处理使ＧＡ的效应延缓。