Identification of KASP markers and putative genes for pre-harvest sprouting resistance in common wheat(Triticum aestivum L.)

Common wheat(Triticum aestivum L.)is the most important crop in the world and a typical allopolyploid with a large and complex genome.Pre-harvest sprouting(PHS)leads to a significant reduction in grain quality worldwide.PHS is a complex trait with related QTL located on different chromosomes.However,the study of markers and genes related to PHS resistance is limited especially for whitegrained wheat.Four pairs of near isogenic lines(NILs)from a white-grained wheat cross of Chara
DM5637B*8 targeting a major QTL for PHS resistance(Qphs.ccsu-3A.1)on wheat chromosme 3AL were genotyped using the 90K SNP Illumina iSelect array.Ten SNPs were identified,with a 75%-100%consistency between genotype and phenotype in the resistant or susceptible isolines.The 10 SNPs were converted to cost-effective kompetitive allele-specific PCR(KASP)markers.Screening of 48 wheat cultivars with different phenotypes of PHS identified four KASP markers with 81.3%-85.4%conformity between genotype and phenotype.Further investigation revealed that the four SNPs(BS00022245_51,Kukri_c49927_151,BS00022884_51 and BS00110550_51)corresponding to the four validated KASP markers are residing in three independent genes(TraesCS3A03G1072800,TraesCS3A03G1072400,TraesCS3A03G1071800)close to each other with a distance of 4.28-4.48 Mb to the targeted QTL.These three annotated genes have potential functions related to PHS resistance.Our study revealed that combined use of NILs and the 90K SNP chip is a powerful approach for developing KASP markers and mining functional genes in wheat.The KASP markers for PHS resistance on chromosome 3AL are useful for high-throughput evaluation and marker-assisted selection,and the three identified genes could lead to a better understanding of the genetic pathways controlling PHS.

Evaluation of Pre-Harvest Sprouting(PHS)Resistance and Screening of High-Quality Varieties from Thirty-Seven Quinoa(Chenopodium quinoa Willd.)Resources in Chengdu Plain

Pre-harvest sprouting(PHS)will have a serious effect both on the yield and quality of quinoa(Chenopodium quinoaWilld.).It is crucial to select and breed quinoa varieties with PHS resistance and excellent agronomic traits for guidance production and utilization of quinoa.A comprehensive evaluation of the PHS resistance and agronomic traits of 37 species of quinoa resources was conducted in Chengdu Plain.The evaluation used various methods,including grain germination rate(GR),grain germination index(GI),total spike germination rate(SR),total grain germination index(SI),grey correlation analysis(GCA),cluster analysis and correlation analysis.Results showed significant differences in PHS resistance and agronomic traits amongst the 37 quinoa resources.CDU-23 was most resistant to PHS within 24 h,with a germination rate of 2.67%and 0%according to the GR and SR results,respectively.However,in the same time,CDU-31 showed the maximum susceptibility to PHS based on the SR of 31.07%,while CDU-34 was the most sensitive to PHS according to the GR of 100%.The comprehensive evaluation identified one and nine kinds of high resistance species for grain and whole spike germination,respectively.In both cases,the coefficients of variation(CV)for these parameters were 34.78%and 82.13%,respectively.GCA results showed that the magnitude of the association between each trait and yield in the thirty-seven quinoa resources was in the following order:thousand grain weight>seed length>seed area>seed width.Although the seed weight of CDU-18 reached 3.7010 g,the seed weight of CDU-5 was only 1.6030 g.However,the size of the seeds,their width and area did not correlate with their 1000-grain weight.There was a complex correlation between PHS resistance index and agronomic traits.Based on clustering analysis,thirty-seven quinoa resources were classified into three taxa.It was found that various taxa differed in PHS resistance and agronomic traits.Several comparisons of the aggregated data led to the selection of five varieties of quinoa,of which CDU-2 presented excellent agronomic qualities and strong PHS resistance.This study has provided a reference for breeding excellent quinoa varieties with PHS resistance.

QTL mapping revealed TaVp-1A conferred pre-harvest sprouting resistance in wheat population Yanda 1817×Beinong 6

Pre-harvest sprouting （PHS） occurs frequently in most of the wheat cultivation area worldwide, which severely reduces yield and end-use quality, resulting in substantial economic loss. In this study, quantitative trait loci （QTL） for PHS resistance were mapped using an available high-density single nucleotide polymorphism （SNP） and simple sequence repeat （SSR） genetic linkage map developed from a 269 recombinant inbred lines （RILs） population of Yanda 1817xBeinong 6. Using phenotypic data on two locations （Beijing and Shijiazhuang, China） in two years （2012 and 2013 harvesting seasons）, five QTLs, designated as QPhs.cau-3A. 1, QPhs.cau-3A.2, QPhs.cau-5B, QPhs.cau-4A, and QPhs.cau-6A, for PHS （GP） were detected by inclusive composite interval mapping （ICIM） （LOD≥2.5）. Two major QTLs, QPhs.cau-3A.2 and QPhs.cau-5B, were mapped on 3AL and 5BS chromosome arms, explaining 6.29-21.65% and 4.36-5.94% of the phenotypic variance, respectively. Precise mapping and comparative genomic analysis revealed that the TaVp-1A flanking region on 3AL is responsible for QPhs.cau-3A.2. SNP markers flanking QPhs.cau-3A.2 genomic region were developed and could be used for introgression of PHS tolerance into high yielding wheat varieties through marker-assisted selection （MAS）.

Isolation and Functional Characterization of a B3 Transcription Factor Gene <i>FUSCA3</i>Involved in Pre-Harvest Sprouting Resistance in Wheat

Pre-harvest sprouting (PHS) reduces yields and grain quality, resulting in seriously economic losses in wheat. It has been showed that PHS is significantly correlated to seed dormancy levels. FUSCA3 (FUS3) gene is considered to be the key regulator of seed dormancy. However, little information is available about the function of FUS3 gene (TaFUS3) in wheat. In this study, three homologous genes were identified in wheat grain, and their functions were investigated by gene silencing. Three full-length DNA (3477, 3534 and 3501 bp) and cDNA (1015, 1012 and 1015 bp) sequences encoding a B3 transcription factor, designated TaFUS3-3A, TaFUS3-3B and TaFUS3-3D, were first isolated from common wheat. The transcription of three TaFUS3 genes in seed development and germination process was detected. TaFUS3-3B and TaFUS3-3D had similar expression profiles, and high levels of gene transcripts were detected in seeds at 25 DAP (days after pollination) and after 24 h of imbibition. However, the transcription of TaFUS3-3A was not detected. Silencing of TaFUS3 in common wheat spikes resulted in increased seed germination and PHS. Compared with wild-type, the TaFUS3-silenced plants showed increased expression of genes related to GA biosynthesis and ABA metabolism, and decreased expression of genes associated with ABA biosynthesis. Moreover, silencing of TaFUS3 in wheat plants led to a decrease in embryo sensitivity to ABA and changed the expression of genes involved in ABA signal transduction. The results of gene silencing indicated that TaFUS3 plays a positive role in wheat seed dormancy and PHS-resistance, which might be associated with ABA, GA level and signal transduction.

QTL mapping for pre-harvest sprouting in a recombinant inbred line population of elite wheat varieties Zhongmai 578 and Jimai 22

Pre-harvest sprouting(PHS)is one of the serious global issues in wheat production.Identification of quantitative trait loci(QTL)and closely-linked markers is greatly helpful for wheat improvement.In the present study,a recombinant inbred line(RIL)population derived from the cross of Zhongmai 578(ZM578)/Jimai 22(JM22)and parents were phenotyped in five environments and genotyped by the wheat 50 K single-nucleotide polymorphism(SNP)array.Two QTL of germination index(GI),QGI.caas-3A and QGI.caas-5A,were detected,explaining 4.33%–5.58%and 4.43%–8.02%of the phenotypic variances,respectively.The resistant effect of QGI.caas-3A was contributed by JM22,whereas that of QGI.caas.5A was from ZM578.The two QTL did not correspond to any previously identified genes or genetic loci for PHSrelated traits according to their locations in the Chinese Spring reference genome,indicating that they are likely to be new loci for PHS resistance.Four kompetitive allele-specific PCR(KASP)markers K_AX-109605367and K_AX-179559687 flanking QGI.caas-3A,and K_AX-111258240 and K_AX-109402944flanking QGI.caas-5A,were developed and validated in a natural population of 100 wheat cultivars.The distribution frequency of resistance alleles at Qphs.caas-3A and Qphs.caas-5A loci were 82.7%and57.1%,respectively,in the natural population.These findings provide new QTL and tightly linked KASP markers for improvement of PHS resistance in wheat.

TaSRO1 interacts with TaVP1 to modulate seed dormancy and pre-harvest sprouting resistance in wheat

Dormancy is an adaptive trait which prevents seeds from germinating under unfavorable environmental conditions.Seeds with weak dormancy undergo pre-harvest sprouting(PHS)which decreases grain yield and quality.Understanding the genetic mechanisms that regulate seed dormancy and resistance to PHS is crucial for ensuring global food security.In this study,we illustrated the function and molecular mechanism of TaSRO1 in the regulation of seed dormancy and PHS resistance by suppressing TaVP1.The tasro1 mutants exhibited strong seed dormancy and enhanced resistance to PHS,whereas the mutants of tavp1 displayed weak dormancy.Genetic evidence has shown that TaVP1 is epistatic to TaSRO1.Biochemical evidence has shown that TaSRO1 interacts with TaVP1 and represses the transcriptional activation of the PHS resistance genes TaPHS1 and TaSdr.Furthermore,TaSRO1 undermines the synergistic activation of TaVP1 and TaABI5 in PHS resistance genes.Finally,we highlight the great potential of tasro1 alleles for breeding elite wheat cultivars that are resistant to PHS.

Breeding for pre-harvest sprouting resistance in bread wheat under rainfed conditions

Pre-harvest sprouting in wheat is the germination of seeds within the spikes when rains occur after or during grain ripening, which occurs commonly in the barani tract of Pakistan. Therefore, 10 cultivars and five advanced lines of spring bread wheat were evaluated for pre-harvest sprouting resistance. After natural rainfall,seeds were immediately collected from the wet spikes and tested for germinating ability. Three different germination tests were applied to hand-threshed seed:(1) spikes threshed on the day of sampling and germination tested immediately,(2) spikes threshed on the day of sampling and germination tested 1 week later, and(3) spikes threshed 1 week after sampling and germination test immediately after threshing. Seeds and spikes kept for 1 week were place on blotting paper at room temperature.Cultivars BARS-09, 09 FJ17, Doukkala-12, NARC-09 and Ouassou-20 exhibited higher sprouting resistance while other genotypes were susceptible to pre-harvest sprouting in each of the three tests. A diallel crossing was conducted with six susceptible and two resistant genotypes to assess the genetic behavior of pre-harvest sprouting resistance.The combining ability(CA) demonstrated a higher proportion of additive genetic effects for sprouting resistance, because of higher variance of general and specific CA for both parameters under study. Doukkala-12 and BARS-09 showed increased pre-harvest sprouting resistance in their F1 descendants.

植物生长抑制剂对藜麦农艺性状和穗发芽抗性的影响

藜麦果实无休眠或休眠期短,成熟时云南大部分地区雨季尚未结束,在收获前期遇持续阴雨天气或在潮湿环境下易发生穗发芽现象,严重影响藜麦产量和品质。为有效地控制藜麦穗发芽,以易穗发芽的WQ6为试验材料,分别在初花期(B1)、盛花期(B2)、灌浆期(B3)分别喷施脱落酸(A1)、矮壮素(A2)、多效唑(A3)3种植物生长抑制剂,以喷施清水为对照(CK),探究不同时期喷施不同植物生长抑制剂对藜麦农艺性状和穗发芽抗性的影响。通过隶属函数法综合分析穗发芽抗性,发现各处理的穗发芽隶属函数均值都小于CK,盛花期喷施矮壮素处理的隶属函数平均值最低,穗发芽抗性最强。各处理均能极显著矮化植株、影响产量,A2B1、A1B2、A2B2、A3B2、A1B3处理能够显著增加产量。综合藜麦产量、穗发芽抗性以及抑制剂成本,在盛花期喷施200mg·L^(-1)多效唑效果最好。

中国小麦抗穗发芽种质资源的挖掘与创制

根据4年的表型数据,并结合实验室开发和鉴定的13个分子标记,对我国833份小麦种质资源(主要包括278份小麦微核心种质、124份地方品种和431份现代推广品种及高代品系)穗发芽抗性进行鉴定。结果表明,13个分子标记鉴定的抗/感穗发芽等位类型间相对发芽指数(RGI)差异均达显著或极显著水平,其中TaMFT-222和TaMFT-194标记鉴定的差异最大,U值分别为14.98^(**)和11.30^(**),均达极显著水平,其优异等位类型可以降低相对发芽指数0.21~0.32。其次是Sdr2A、CNGC2AL、Vp1-b2、TaMKK3-A、PM19、CAPS-2AL、A17-19和EX06323标记,其等位类型间穗发芽抗性差异也均达极显著水平;Qsd1和Barc321标记也能显著区分穗发芽抗性。共计鉴定出63份穗发芽抗性较好的种质资源,其中达到抗的有41份,多为红皮品种和地方品种;达到中抗的有22份,白皮半冬性居多。利用分子标记辅助选择,并结合杂交聚合,创制出12份穗发芽抗性水平达到中抗和抗的种质资源,至少携带3个抗穗发芽基因/位点。该结果为抗穗发芽新品种选育提供重要遗传资源。

Sdr4 dominates pre-harvest sprouting and facilitates adaptation to local climatic condition in Asian cultivated rice

Pre-harvest sprouting(PHS),which reduces grain yield and quality,is controlled by seed dormancy genes.Because few dormancy-related genes have been cloned,the genetic basis of seed dormancy in rice(Oryza sativa L.)remains unclear.Here,we performed a genome-wide association study and linkage mapping to dissect the genetic basis of seed dormancy in rice.Our findings suggest that Seed Dormancy4(Sdr4),a central modulator of seed dormancy,integrates the abscisic acid and gibberellic acid signaling pathways at the transcriptional level.Haplotype analysis revealed that three Sdr4 alleles in rice cultivars already existed in ancestral Oryza rufipogon accessions.Furthermore,like the semi-dwarf 1(SD1)and Rc loci,Sdr4 underwent selection during the domestication and improvement of Asian cultivated rice.The distribution frequency of the Sdr4-n allele in different locations in Asia is negatively associated with local annual temperature and precipitation.Finally,we developed functional molecular markers for Sdr4,SD1,and Rc for use in molecular breeding.Our results provide clues about the molecular basis of Sdr4-regulated seed dormancy.Moreover,these findings provide guidance for utilizing the favorable alleles of Sdr4 and Rc to synergistically boost PHS resistance,yield,and quality in modern rice varieties.

山西小麦地方品种萌发期的抗旱性

利用20%PEG-6000作为渗透介质进行室内模拟干旱胁迫,测定小麦发芽率、发芽势、胚芽鞘长、主胚根长;采用模糊隶属函数与抗旱系数相结合的方法对品种萌发期的抗旱性进行综合评价,并利用灰色关联分析了各个形态指标与抗旱性的关系。结果表明,干旱胁迫下,各品种的发芽率、发芽势、胚芽鞘长、主胚根长均比对照不同程度地降低或缩短,而且不同品种间差异达显著或极显著水平;萌发期各品种抗旱性表现依次为:白和尚头>晋麦47>竹杆青>四月黄>小红麦>灯笼红>红皮冬麦>忻县冬麦>中麦9号。应用灰色关联分析的方法研究萌发期发芽势、发芽率、胚芽鞘长和主胚根长的抗旱系数4个性状指标与抗旱性之间的灰色关联度,结果表明,关联度依次为:发芽率>主根胚长>胚芽鞘长>发芽势,表明发芽率与萌发期抗旱性关系最为密切,其可作为萌发期抗旱性鉴定指标。

白皮小麦收获前穗发芽及品种抗性机制探讨

对48个白皮小麦种质及2个红皮小麦种质连续3年测定结果,品种间收获前穗发芽率、籽粒发芽率及收获后种子休眠特性差异显著。穗发芽敏感性因年份和种子发育时期而异,开花后35～40天较敏感,品种间差异大。休眠期长短与穗发芽率间呈极显著负相关(平均r^2=0.6170)。收获后的种子α-淀粉酶活性、降落值,与穗发芽率间分别呈极显著正相关(r^2=0.4609)和极显著负相关(r^2=0.3014)。种子吸水率与穗发芽率间相关显著,但程度较低。颖壳浸提液对种子发芽有明显抑制作用,以浸种24小时较显著且品种间差异大。相对而言,抗穗发芽品种的抑制作用多大于敏感品种,但有的抗性品种没有明显的颖壳抑制作用。表明,白皮小麦抗穗发芽性是复杂的,休眠特性是其主要机制,α淀粉酶活性、降落值及颖壳内含物亦有一定作用,不同品种其抗性机制有所不同。

六倍体小黑麦萌发期抗旱性分析

利用20%PEG-6000(-0.975 MPa)为渗透介质室内模拟干旱,分析六倍体小黑麦萌发期发芽率、发芽势、胚芽鞘长、根长、根数的变化,采用模糊隶属函数与抗旱系数相结合的方法对品种萌发期的抗旱性进行综合分析,并利用灰色关联分析法分析各个形态指标与抗旱性的关系,结果表明:干旱胁迫下,小黑麦各品种的发芽率、发芽势、胚芽鞘长、根长都比对照不同程度地降低或缩短,不同品种之间的差异达显著或极显著水平,但根数却增减不一,表明根数对水分胁迫的反应方向不一致;品种"Tornado"(S9)萌发期综合抗旱性强,为小麦抗旱育种提供了种质资源;发芽率与六倍体小黑麦萌发期抗旱性的关联度最大,可作为形态指标加以利用。

以活力抗旱指数作为玉米萌芽期抗旱性评价指标的初探

首次提出了活力抗旱指数,并以此为指标,在PEG人工模拟水分胁迫的条件下,对18个不同的玉米品种进行抗旱性研究。结果表明:活力抗旱指数和常用的萌发抗旱指数有很高的相关性,且能更好的反映不同品种幼苗水分胁迫下的差别,可以作为玉米萌发阶段抗旱性鉴定的筛选方法。

长江中下游麦区主要小麦品种穗发芽抗性及鉴定方法比较

为了解长江中下游麦区当前推广小麦品种抗穗发芽的特性,采用整穗发芽法、整粒发芽法和半粒发芽法鉴定了20个小麦品种的穗发芽抗性。结果表明,品种间穗发芽抗性存在显著差异,扬麦16、扬麦18、扬麦20、扬麦22、扬辐麦4号、宁麦16等13个品种穗发芽抗性较好,宁麦17、宁麦19、镇麦6号、镇麦8号、镇麦9号、扬麦13和扬麦14等6个品种穗发芽抗性中等,郑麦9023易穗发芽;20个品种的整穗发芽率和取穗当天整粒发芽率差异不显著,且两种方法的发芽率呈极显著正相关,说明整穗发芽法和整粒发芽法都可用于穗发芽抗性鉴定;整粒发芽法中取穗当天与取穗10d后籽粒发芽率较为一致,且极显著正相关,说明这一时段内都可进行抗穗发芽鉴定;取穗30d后所有红粒品种的发芽率都极显著提高,说明此时种子休眠开始解除。18个红粒小麦品种的半粒发芽率极显著高于整粒发芽率,说明种皮对穗发芽有较强的抑制作用。白粒品种郑麦9023收获30d后发芽率与收获当天穗上发芽率、籽粒发芽率,整粒法与半粒法发芽率差异均不显著,说明该品种几乎无休眠特性,且其种皮对穗发芽的抑制作用较小。

不同麦区小麦品种穗发芽抗性及其与穗部性状的相关性

为探究不同麦区小麦品种的穗发芽抗性及其与穗部性状的相关性,以黄淮冬麦区(南片和北片)、北部冬麦区及长江中下游麦区的235份小麦品种为试验材料,选用2个能够有效用于穗发芽抗性筛选的分子标记Vp1B3和WMC104,结合整穗发芽试验,对各麦区小麦品种穗发芽抗性进行鉴定,并分析穗发芽抗性与穗部及籽粒性状间的相关性。结果表明,长江中下游麦区小麦穗发芽抗性显著高于其他三个麦区,其供试小麦品种60%以上达到抗性等级,其余三个麦区中达到抗性等级的品种均不超过30%;供试品种中,扬麦10号、荆麦103、川农16等15个品种为高抗穗发芽品种,发芽率均值为4.85%,是穗发芽抗性育种中的重要抗性资源。整穗发芽率与穗部及籽粒性状相关性分析表明,穗发芽率与籽粒颜色极显著正相关,与小穗密度、千粒重、籽粒宽度等显著正相关。在育种过程中,可将籽粒颜色、小穗密度、千粒重及籽粒宽度等作为抗穗发芽小麦品种的重要参考指标。

小麦穗发芽抗性鉴定及相关分子标记的有效性验证

为筛选出抗穗发芽小麦种质及可用于种质评价和分子标记辅助育种的高效分子标记,以41份黄淮南片区试品系和309份中外小麦品种为材料,选用4个与穗发芽抗性相关的标记,即STS标记Vp1B3和MST101、STMS标记wmc104及SSR标记Xgwm155,结合整穗发芽鉴定试验,评价筛选抗穗发芽小麦种质,同时对上述4个分子标记进行有效性验证。结果表明,标记Vp1B3和wmc104可有效用于小麦穗发芽抗性筛选,而标记Xgwm155和MST101与穗发芽抗性不相关;350份供试材料中41份国家区试材料的整穗发芽率普遍偏高,均值达到43.94%;而另外309份材料中,淮麦20、轮选987、长旱58等18份材料为高抗穗发芽品种,平均整穗发芽率为2.56%,是穗发芽抗性育种中的重要抗性资源。

春小麦育种材料抗旱性和穗发芽抗性分子标记鉴定

为给春小麦生育早期抗旱和收获期抗穗发芽育种提供抗性亲本,利用与小麦抗旱性相关的分子标记TaNRX-B1a1、TaNRX-B1b1和FerA1-intrl以及与小麦穗发芽抗性相关的分子标记Vp1B3、Vp1A3和Tamyb10D对137份内蒙古春小麦育种材料进行检测。结果表明,73B609等共58份材料的抗旱性分子标记均属于抗旱基因型,占供试材料的42.34%,其单倍型组合为TaNRX-B1a/TaFer-A1a,可以用作抗旱育种的亲本材料。中国春既属于Vp-1Bb等位基因类型,同时用标记Tamyb10D检测时也属于抗穗发芽类型。辽春10号在3个分子标记的检测中均属于抗穗发芽的等位基因类型,同时又是TaNRX-B1a/TaFer-A1a单倍型,所以辽春10号在抗穗发芽和抗旱性聚合育种中可以作为亲本使用。

水稻不同品种的穗发芽及其对外源激素的反应

选择 8份早籼稻品种在开花后不同天数取穗子进行保湿培养 ,观察其抗穗发芽能力的差异。结果显示不同品种之间的抗穗发芽能力存在较大差异 ;处理以开花后 2 2d左右的穗子保湿培养 6d考查穗发芽 ,就能较好地比较出品种之间的差异。以 3种不同浓度的赤霉素 (GA3 )和脱落酸 (ABA)进行溶液培养 ,30mg/kg的赤霉素有明显促进穗发芽的效果 ,脱落酸抑制穗发芽的效果在不同品种之间表现出比较大的差异。

小麦穗发芽抗性鉴定及机制分析

采用整穗、颖壳＋整粒、整粒和半粒发芽法鉴定了105个引自国内外普通小麦品种（系）的穗发芽抗性。结果表明,不同品种间穗发芽差异性较大,穗发芽率为0.72%-100%,平均发芽率为78.09%。从中鉴定出极抗品种1份（ONSY85）,高抗品种2份（扬麦16和扬麦11）,抗性品种4份（凤麦36、绵阳45、凤麦32、扬麦158）,以上品种均为红粒。临麦15、凤麦34、小偃93166、靖麦12、周麦17、云麦29、X9610穗发芽率大于98%,穗易发芽。用K-均值聚类法分为5类。半粒发芽率与整粒发芽率,整粒发芽率与颖壳加整粒发芽率,整粒发芽率与整穗发芽率均差异显著且正相关,表明虽然种皮和颖壳水溶性物质对穗发芽有显著影响,但是籽粒的休眠性起主要作用。4种穗型的穗发芽率没有差异,表明穗型对穗发芽没有影响。