A novel transcription factor FnMYB4 regulates pigments metabolism of yellow leaf mutants in Fragaria nilgerrensis

The strawberry species Fragaria nilgerrensis Schlechtendal ex J.Gay,renowned for its distinctive white,fragrant peach-like fruits and strong disease resistance,is an exceptional research material.In a previous study,an ethyl methane sulfonate(EMS)mutant library was established for this species,resulting in various yellow leaf mutants.Leaf yellowing materials are not only the ideal materials for basic studies on photosynthesis mechanism,chloroplast development,and molecular regulation of various pigments,but also have important utilization value in ornamental plants breeding.The present study focused on four distinct yellow leaf mutants:mottled yellow leaf(MO),yellow green leaf(YG),light green leaf(LG),and buddha light leaf(BU).The results revealed that the flavonoid content and carotenoid-to-chlorophyll ratio exhibited a significant increase among these mutants,while experiencing a significant decrease in chlorophyll and carotenoid contents compared to the wild type(WT).To clarify the regulatory mechanisms and network relationships underlying these mutants,the RNA-seq and weighted gene coexpression network(WGCNA)analyses were employed.The results showed flavonoid metabolism pathway was enriched both in MO and YG mutants,while the chlorophyll biosynthesis pathway and carotenoid degradation pathway were only enriched in MO and YG mutants,respectively.Subsequently,key structural genes and transcription factors were identified on metabolic pathways of three pigments through correlation analyses and quantitative experiments.Furthermore,a R2R3-MYB transcription factor,FnMYB4,was confirmed to be positively correlated with flavonoid synthesis through transient overexpression,virus-induced gene silencing(VIGS),and RNA interference(RNAi),accompanying by reoccurrence and attenuation of mutant phenotype.Finally,dual-luciferase(LUC)and yeast one-hybrid assays confirmed the binding of FnMYB4 to the FnFLS and FnF3H promoters,indicating that FnMYB4 positively regulates flavonoid synthesis.In addition,correlation analyses suggested that FnMYB4 also might be involved in chlorophyll and carotenoid metabolisms.These findings demonstrated the pivotal regulatory role of FnMYB4 in strawberry leaf coloration.

Screening of Leaf Shape Mutants Induced by EMS and Analysis of Agronomic Traits in Azuki Bean (Vigna angularis)

[Objective] M3 progenies of Jingnong 6 variety induced by EMS chemical mutagenesis were screened and identified for obtaining valuable mutation material.[Method] Azuki bean cultivar Jingnong 6 was treated with EMS.The mutation rate,mutation types,agronomic traits and yield components of the leaf mutants were analyzed.[Result] The results showed that there is the most abundant mutational type of leaf shape and the highest mutation frequency treated with 0.9% EMS for 24 hours.Comprehensive analysis on agronom...

Characterization and Candidate Gene Analysis of the Yellow-Green Leaf Mutant ygl16 in Rice(Oryza sativa L.)

Leaf color mutants are ideal materials for studying many plant physiological and metabolic processes such as photosynthesis,photomorphogenesis,hormone physiology and disease resistance.In this study,the genetically stable yellow-green leaf mutant ygl16 was identified from mutated“Xinong 1B”.Compared with the wild type,the pigment concentration and photosynthetic capacity of the ygl16 decreased significantly.The ultrastructural observation showed that the distribution of thylakoid lamellae was irregular in ygl16 chloroplasts,and the grana and matrix lamellae were blurred and loose in varied degrees,and the chloroplast structure was disordered,while the osmiophilic corpuscles increased.The results of the genetic analysis and mapping showed that the phenotype of ygl16 was controlled by a pair of recessive nuclear gene.The gene located in the 56Kb interval between RM25654 and R3 on the long arm of chromosome 10.The sequencing results showed that the 121st base of the first intron of the candidate gene OsPORB/FGL changed from A to T in the interval.qRT-PCR results showed that the expression of chlorophyll synthase-related genes in the mutant decreased.

Quality Research on the Purple Leaf Mutant of Rice——PLM

[Objective] The aim was to carry out the quality research on a purple leaf mutant （PLM） of rice and provide the basis for applied research of purple rice.[Method] A newly discovered purple mutant of rice and its hybrid filial generations （F1 and F2） were employed as the experimental materials to determine its characteristic indexes,such as grain type,chalky grain rate,chalkiness,1 000-grain weight,brown rice percentage,protein content,amylose content,gelatinization temperature and consistency.[Result] The grain type and brown rice percentage of the parent （pro-Z） both reached standard of Ⅰ Grade,while chalky grain rate,chalkiness,amylose content and consistency did not meet the requirements of the standard.The F2 generation displayed some optimized properties,including larger grain,lower amylose content,reduced chalkiness,lower chalky grain rate and softened consistency.[Conclusion] The majority of the characteristic indexes of pro-Z did not meet the requirements of standard,but the qualities of F2 generation were all optimized to some extent.

水稻黄绿叶突变体yellow-green leaf 4的表型鉴定及候选基因定位和功能分析

[目的]本研究旨在对水稻黄绿叶突变体yellow-green leaf 4(ygl4)进行表型分析及基因定位和功能分析,探讨水稻叶绿体发育分子机制。[方法]水稻黄绿叶突变体ygl 4来自粳稻品种‘中花11’化学诱变突变体库。ygl 4与籼稻品种‘N22’杂交构建F_(2)分离群体以进行YGL 4基因定位,并利用实时定量PCR和亚细胞定位等技术对基因进行初步功能分析。[结果]相比于野生型,ygl 4在幼苗2~3叶龄出现黄绿叶症状,在6月下旬移栽大田后,黄绿叶症状加剧,甚至开始出现白化,突变性状可以一直保持到全生育期直到种子成熟。温敏性试验表明,ygl 4突变体在20℃表现出更严重黄化表型。透射电镜观察表明,ygl 4的叶片细胞结构中出现了较多的双膜囊泡结构。基因定位显示,ygl 4突变性状由1个隐性核基因LOC_Os 04g42000突变导致。该基因编码一个核黄素合成途径中的关键酶,6,7-二甲基-8-核糖醇基二氧四氢蝶啶合酶(LS),且突变体叶色表型可被外施核黄素恢复为正常表型。亚细胞定位结果显示YGL4定位于叶绿体。实时定量PCR分析表明,在突变体中,叶绿素暗反应阶段参与5-氨基乙酰丙酸(ALA)到原叶绿素酸酯合成过程的基因表达上调。[结论]水稻YGL 4编码LS基因,并通过调控植物体内核黄素合成途径影响叶色和叶绿体发育。

Identification of the genetic locus associated with the crinkled leaf phenotype in a soybean(Glycine max L.)mutant by BSA-Seq technology

The leaf is the main photosynthetic organ of plants,and it plays a significant role in the yield of crop species.Identifying the causal mutations and candidate genes that underlie leaf phenotypic variation is an important breeding target in soybean grain yield improvement.An ethyl methyl sulfonate(EMS)-induced soybean mutant DWARFCRINKLEDLEAF1(DCL1)with an aberrant crinkled leaf phenotype was identified in the background of the soybean cultivar Zhongpin 661(Zp661).We constructed an F2 segregating population from a cross between Zp661 and DCL1 in order to investigate the genomic locus associated with the crinkled leaf trait.Using bulk segregant analysis(BSA)combined with the whole-genome resequencing method,the Euclidean distance(ED)correlation algorithm detected 12 candidate genomic regions with a total length of 20.32 Mb that were linked to the target trait.Following a comparative analysis of the sequence data for the wild-type and mutant pools,only one single nucleotide mutation(C:G>T:A)located on the first exon of Glyma.19G207100 was found to be associated with the trait.Candidate gene validation based on a CAPS marker derived from the detected single-nucleotide polymorphism(SNP)indicated a nucleotide polymorphism between the two parents.Therefore,our findings reveal that Glyma.19G207100,which is renamed as GLYCINE MAX DWARF CRINKLED LEAF 1(GmDCL1),is a promising candidate gene involved in the morphogenesis of the crinkled leaf trait of the soybean mutant DCL1.This study provides a basis for the functional validation of this gene,with prospects for soybean breeding targeting grain yield enhancement.

Genetic Analysis and Molecular Mapping of Novel White Striped Leaf Mutant Gene in Rice

A new white striped leaf mutant wsll was discovered from Nipponbare mutated by ethyl methanesulfonate. The mutant showed white striped leaves at the seedling stage and the leaves gradually turned green after the tillering stage. The chlorophyll content of wsll was significantly lower than that of wild-type during the fourth leaf stage, tillering stage and booting stage. The numbers of chloroplast, grana and grana lamella were reduced and the thylakoids were degenerated in wsll compared with wild type. Genetic analysis showed that the wsll was controlled by a single recessive gene. Molecular mapping of the wsll was performed using an F2 population derived from wsll/Nanjing 11. The wsll was finally mapped on the telomere region of chromosome 9 and positioned between simple sequence repeat markers RM23742 and RM23759 which are separated by approximately 486.5 kb. The results may facilitate map-based cloning of wsll and understanding of the molecular mechanism of the regulation of leaf-color by WSL1 in rice.

Anatomical and Chemical Characteristics of a Rolling Leaf Mutant of Rice and Its Ecophysiological Properties

The anatomical and chemical characteristics of a rolling leaf mutant （rlm） of rice （Oryza sativa L.） and its ecophysiological properties in photosynthesis and apoplastic transport were investigated. Compared with the wild type （WT）, the areas of whole vascular bundles and xylem as well as the ratios of xylem area/whole vascular bundles area and xylem area/phloem area were higher in rim, whereas the area and the width of foliar bulliform cell were lower. The Fourier transform infrared （FTIR） microspectroscopy spectra of foliar cell walls differed greatly between rim and WT. The rim exhibited lower protein and polysaccharide contents of foliar cell walls. An obvious reduction of pectin content was also found in rim by biochemical measurements. Moreover, the rate of photosynthesis was depressed while the conductance of stoma and the intercellular CO2 concentration were enhanced in rim. The PTS fluorescence, which represents the ability of apoplastic transport, was 11% higher in rim than in WT. These results suggest that the changes in anatomical and chemical characteristics of foliar vascular bundles, such as the reduction of proteins, pectins, and other polysaccharides of foliar cell walls, participate in the leaf rolling mutation, and consequently lead to the reduced photosynthetic dynamics and apoplastic transport ability in the mutant.

Morphological Structure and Genetic Mapping of New Leaf-Color Mutant Gene in Rice (Oryza sativa)

Leaf-color mutations are a widely-observed class of mutations, playing an important role in the study of chlorophyll biosynthesis and plant chloroplast structure, function, genetics and development. A naturally-occurring leaf-color rice mutant, Baihuaidao 7, was analyzed. Mutant plants typically exhibited a green-white-green leaf-color progression, but this phenotype was only expressed in the presence of a stress signal induced by mechanical scarification such as transplantation. Prior to the appearance of white ~eaves, mutant plant growth, leaf color, chlorophyll content, and chloroplast ultrastructure appeared to be identical to those of the wild type. After the changeover to white leaf color, an examination of the mutated leaves revealed a decrease in total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid content, a reduction in the number of chloroplast grana lamella and grana, and a gradual degradation of the thylakoid lamellas. At maturity, the mutant plant was etiolated and dwarfed compared with wild-type plants. Genetic analysis indicated that the leaf mutant character is controlled by a recessive nuclear gene. Genetic mapping of the mutant gene was performed using an F2 population derived from a Baihuaidao 7 ~ Jiangxi 1587 cross. The mutant gene was mapped to rice chromosome 11, positioned between InDel markers L59.2-7 and L64.8-11, which are separated by approximately 740.5 kb. The mutant gene is believed to be a new leaf-color mutant gene in rice, and is tentatively designated as gwgl.

Heredity and gene mapping of a novel white stripe leaf mutant in wheat

Spotted leaf(spl)mutant is a type of leaf lesion mimic mutants in plants.We obtained some lesion mimic mutants from ethyl methane sulfonate(EMS)-mutagenized wheat(Triticum aestivum L.)cultivar Guomai 301(wild type,WT),and one of them was named as white stripe leaf(wsl)mutant because of the white stripes on its leaves.Here we report the heredity and gene mapping of this novel wheat mutant wsl.There are many small scattered white stripes on the leaves of wsl throughout its whole growth period.As the plants grew,the white stripes became more severe and the necrotic area expanded.The mutant wsl grew only weakly before the jointing stage and gradually recovered after jointing.The length and width of the flag leaf,spike number per plant and thousand-grain weight of wsl were significantly lower than those of the WT.Genetic analysis indicated that the trait of white stripe leaf was controlled by a recessive gene locus,named as wsl,which was mapped on the short arm of chromosome 6 B by SSR marker assay.Four SSR markers in the F2 population of wsl×CS were linked to wsl in the order of Xgpw1079–Xwmc104–Xgwm508-wsl–Xgpw7651 at 7.1,5.2,8.7,and 4.4 c M,respectively and three SSR markers in the F2 population of wsl×Jimai 22 were linked to wsl in the order of Xgwm508–Xwmc494–Xgwm518-wsl at 3.5,1.6 and 8.2 c M,respectively.In comparison to the reference genome sequence of Chinese Spring(CS),wsl is located in a 91-Mb region from 88 Mb(Xgwm518)to 179 Mb(Xgpw7651)on chromosome 6 BS.Mutant wsl is a novel germplasm for studying the molecular mechanism of wheat leaf development.

Identification and Genetic Analysis of a Novel Rice Spotted-Leaf Mutant with Broad-Spectrum Resistance to Xanthomonas oryzae pv. oryzae

A spotted-leaf mutant of rice HM143 was isolated from an EMS-induced IR64 mutant bank. Brown lesions randomly distributed on leaf blades were observed about 3 wk after sowing. The symptom lasted for the whole plant growth duration. Histochemical analysis indicated that cell death occurred in and around the site of necrotic lesions accompanied with accumulation of hydrogen hyperoxide. Agronomic traits were largely similar to the wild type IR64 except seed setting rate and 1 000-grain weight which were significantly decreased in the mutant. Disease resistance of the mutant to multiple races of Xanthomonas oryzae pv. oryzae was significantly enhanced. Genetic analysis showed that the mutation was controlled by a single recessive gene, tentatively termed splHM143. In addition, using molecular markers and 1023 mutant type individuals from an F2 segregating population derived from the cross HM143/R9308, the spotted-leaf gene was finally delimited to an interval of 149 kb between markers XX25 and ID40 on the long arm of chromosome 4. splHM143 is likely a novel rice spotted-leaf gene since no other similar genes have been identified near the chromosomal region.

Genetic Analysis and Mapping of a Thermo-sensitive White Stripe-Leaf Mutant at Seedling Stage in Rice(Oryza sativa)

A thermo-sensitive white stripe-leaf mutant （tws） was selected from the M2 progeny of a japonica variety, Jiahua 1, treated by ^60 Co γ-radiation. In comparison with the wild type parent, the mutant displayed a phenotype of white stripe on the 3rd and 4th leaves, but began to turn normal green on the 5th leaf when grown at low temperatures （20℃ and 24℃）. Furthermore, the content of total chlorophyll showed an obvious decrease in the leaves with white stripe. These results suggest that the expression of the mutant trait was thermo-sensitive and correlated with the leaf age of seedlings. The genetic analysis indicated that the mutant trait was controlled by a single recessive nuclear gene, designated as tws. In addition, by using SSR markers and an F2 segregating population derived from the cross between the tws mutant and 9311, tws was mapped between the markers MM3907 and MM3928 with a physical distance of 86 kb on dce chromosome 4.

Resistance to Bacterial Leaf Blight in a Somaclonal Rice Mutant HX-3 at Cellular Level

The interaction between rice host and its pathogen Xanthomonas oryzae pv. oryzae (Xoo) at cellular level was studied by using a resistant somaclonal mutant HX-3 and its susceptable donor Minghui 63. After inoculation with Xoo strain Zhe 173 (Chinese pathotype Ⅳ), the activity of superoxide dismutase (SOD) and peroxidase (POD) in the callus of Minghui 63 was increased dramatically, and the active oxygen(O2 ) was produced at a higher rate; Meanwhile, the callus grew slowly with the reduction of protein content Compared to the activity of SOD and POD, the production rate of Oa and the fresh weight in HX-3 callus varied little after the inoculation It could be proposed that there were great differences between the resistance of HX-3 and Mighui 63 at cellular level. There was no difference detected concerning resistance to bacterial leaf blight in HX-3 between the plant and the callus.

Quantitative Proteomics Analysis Identifies the Potential Mechanism Underlying Yellow-Green Leave Mutant in Wheat

Enhancing photosynthesis efficiency is considered as one of the most crucial targets during wheat breeding.However,the molecular basis underlying high photosynthesis efficiency is not well understood up to now.In this study,we investigated the protein expression profile of wheat Jimai5265yg mutant,which is a yellow-green mutant with chlorophylls b deficiency but high photosynthesis efficiency.Though TMT-labeling quantitative proteomics analysis,a total of 72 differential expressed proteins(DEPs)were obtained between the mutant and wild type(WT).GO analysis found that they significantly enriched in thylakoid membrane,pigment binding,magnesium chelatase activity and response to light intensity.KEGG analysis showed that they involved in photosynthesis-antenna protein as well as porphyrin and chlorophyll metabolism.Finally,118 RNA editing events were found between mutant and WT genotype.The A to C editing in the 3-UTR of TraesCS6D02G401500 lead to its high expression in mutant through removing the inhibition of tae-miR9781,which might have vital role in regulating the yellow-green mutant.This study provided some useful clues about the molecular basis of Jimai5265yg mutant as well as chlorophylls metabolism in wheat.

Characterization and Genetic Analysis of Rumpled and Twisted Leaf Mutant(rtl1) in Rice

A rumpled and twisted leaf 1(rtl1) mutant was generated from a japonica cultivar Nipponbare by ethyl methanesulfonate treatment,which was characterized as rumpled and twisted leaf at the seedling stage.The F2 populations were constructed by crossing with indica cultivars TN1 and Zhefu 802,respectively.Genetic analysis demonstrated that the phenotype was controlled by a single recessive nuclear gene.The closely linked simple sequence repeat(SSR) marker RM1155 was obtained from bulked segregant analysis.Subsequently,sequence tagged site(STS) markers were developed using the published rice genome sequence.Finally,RTL1 was located between an STS marker T1591 and an SSR marker RM1359,at the distances of 0.48 cM and 0.96 cM,respectively.These results will facilitate the cloning of the target gene in further studies.

Analysis of variation in temperature-responsive leaf color mutant lines induced from Gamma irradiation in rice(Oryza sativa L.)

Eight lines of temperature-responsive leaf colormutants induced by applying 300 Gy Gamma-ray irradiation to Thermo-sensitive genic malesterile line 2177s,were obtained through con-tinuous selection in seven generations..Theleaves of these lines started to become greenafter the fourth leaf extension,and except

一个水稻“斑马叶”叶色突变体基因zebra leaf2(zl2)的图位克隆

从粳稻品种Asominori组培后代中获得一个稳定遗传的黄绿相间叶色突变体(zebra leaf 2,zl2)。该突变体在苗期表现为黄绿相间的斑马状,分蘖后期斑马叶性状逐渐减弱,到抽穗期叶片逐渐变为淡黄色。与野生型相比,zl2在3叶期、分蘖盛期、抽穗期及成熟期叶片的叶绿素、类胡萝卜素含量显著降低,成熟后其结实率、千粒重、株高也显著下降。电镜观察结果显示,苗期zl2叶片黄色部分叶肉细胞中叶绿体显微结构发生了明显的异常,而绿色部分与野生型基本一致。遗传分析结果表明,zl2突变性状受一对隐性核基因控制。从zl2与籼稻品种南京11衍生的F2群体中挑选1607株表现为突变性状的分离单株,最终将该突变基因定位于第11染色体约164.3kb的区域内。基因预测表明该区域内存在13个ORFs,其中ORF12编码一个类胡萝卜素异构酶,序列分析表明突变体中的该基因第10个内含子与第11外显子的交界处碱基A突变为T,导致cDNA发生错误剪切,缺失4个碱基,产生移码突变,并于第395个氨基酸处提前终止。RT-PCR分析表明,相对野生型在突变体中ZL2的表达量显著下降,同时叶色相关基因PORA、RbcL、RbcS、Cab1、Cab2、psaA、psbA、OsDVR表达量也显著下降,而HEMA1、YGL1、V1、V2、SPP、OsPPR的表达量显著上升。结果表明ZL2在水稻叶绿素合成及叶绿体发育中起着重要作用。

水稻窄叶突变体Narrow leaf11(nal11)的基因定位

【目的】定位和分析水稻窄叶突变体基因,为水稻叶片发育调控及株型育种提供参考依据。【方法】用甲基磺酸乙酯(EMS)诱导泸恢17,获得稳定的窄叶突变体(Narrow leaf 11,nal11),调查其与野生型泸恢17抽穗期功能叶的长和宽、分蘖数及成熟期株高。对nal11和绵恢727正反交获得的F2代进行遗传分析及基因定位。【结果】nal11抽穗期剑叶、倒2叶和倒3叶的宽度与野生型泸恢17存在显著(P<0.05,下同)或极显著(P<0.01,下同)差异,分别为野生型泸恢17的60.7%、57.9%和75.8%,但长度无显著差异(P>0.05);nal11株高为野生型泸恢17的90.3%,存在显著差异;nal11分蘖数极显著增加,为野生型的150.0%。nal11和绵恢727正反交后,F1代均表现正常叶宽,F2代叶宽发生性状分离,正常叶宽与窄叶植株数比例经χ2检验均符合3∶1,表明nal11是受核单基因控制的隐性突变。利用SSR标记将nal11定位在水稻第4号染色体RM7290和RM16720标记之间约322 kb范围内,其与2个标记的遗传距离均为0.8 c M,覆盖了32.236 kb的物理区域,在定位区域内有5个注释基因,即Os04g26834、Os04g26850、Os04g26870、Os04g26880和Os04g26841,其序列与前人克隆的窄叶基因无重复。【结论】获得一个新的水稻窄叶突变体(nal11),其窄叶性状由1个隐性核基因控制,定位于水稻第4号染色体RM7290和RM16720标记之间,对功能叶、株高和分蘖数的表型有明显影响。

Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis

Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana. However, the leaf development that is specific to plants has been little studied. With the aim of contributing to the genetic dissection of leaf development, we have performed a large-scare screening for mutants with abnormal leaves. Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethylmethae sulfonate (EMS) mutagenesis, four independent mutant lines have been identified and studied genetically. Phenotypes of these mutant lines represent the defects of four novel nuclear genes designated LL1 (LOTUS LEAF 1), LL2 (LOTUS LEAF 2), URO (UPRIGHT ROSETTE), and EIL (ENVIRONT CONDITION INDUCED LESION). The phenotypic analysis indicates that these genes play important roles during leaf development. FOr the further genetic analysis of these genes and the map-based cloning of LL1 and LL2, we have mapped these genes to chromosome regions with an efficient and rapid mapping method.

Identification and Fine Mapping of a Gene Related to Pale Green Leaf Phenotype near the Centromere Region in Rice(Oryza sativa)

A thermo-insensitive pale green leaf mutant （pgl2） was isolated from T-DNA inserted transgenic lines of rice （Oryza sativa L. subsp, japonica cv. Nipponbare）. Genetic analysis indicated that the phenotype was caused by a recessive mutation in a single nuclear-encoded gene. To map the PGL2gene, an F2 population was constructed by crossing the mutant with Longtefu （Oryza sativa L. subsp, indica）. The PGL2 locus was roughly linked to SSR marker RM331 on chromosome 8. To finely map the gene, 14 new InDel markers were developed around the marker, and PGL2 was further mapped to a 2.37 Mb centromeric region. Analysis on chlorophyll contents of leaves showed that there was no obvious difference between the mutant and the wild type in total chlorophyll （Chl） content, while the ratio of Chl a / Chl b in the mutant was only about 1, which was distinctly lower than that in the wild type, suggesting that the PGL2 gene was related to the conversion between Chl a and Chl b. Moreover, the method of primer design around the centromeric region was discussed, which would provide insight into fine mapping of the functional genes in plant centromeres.