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.

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。

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.

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.

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.

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.

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.

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.

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

一株拟南芥宽叶形突变体atscamp的分离鉴定

叶片是主要的光合作用器官,选育利于光合作用的叶片形态已成为重要的育种目标。atscamp是从拟南芥(Arabidopsis thaliana)突变体库(约6000株系)中筛选获得的1株叶片宽大突变体。Tail-PCR分析该突变体为AT1G11180位点的插入,该基因位点编码1个分泌载体膜蛋白(SCAMP)。RT-PCR检测显示,该基因转录表达水平基本为零。进一步研究发现,该突变体叶片的宽度和叶面积极显著大于野生型植株(P<0.01),但是冠幅基本保持不变;同时atscamp突变体叶绿素含量增加,叶绿素最大荧光、PSⅡ潜在光化学效率显著增加(P<0.05);相应地,突变体植株蒸腾系数(Tr)、净光合速率(Pn)和叶片水分利用效率(WUE)显著增加(P<0.05)。拟南芥AT1G11180基因的时空特异性表达分析显示,该基因仅在叶片中高表达,在其他器官中表达量很低;且随着植物发育成熟,该基因表达量逐渐增加。研究结果表明AtSCAMP基因在叶形发育中发挥着重要作用。

谷子黄绿叶突变体ygl7的精细定位及候选基因分析

叶色突变体是研究C4光合途径及叶绿素代谢机制的理想材料。为了研究谷子黄绿叶突变的分子机制,为黄绿叶基因的功能研究及叶绿素代谢的分子机制解析奠定基础,在长农35号甲基磺酸乙酯(EMS)突变体库中鉴定到1份可稳定遗传的谷子黄绿叶突变体ygl7,并对突变体及野生型进行农艺性状调查、光合色素含量及光合参数测定、叶绿体超微结构观察;同时对突变体叶色进行遗传分析,采用BSA法进行基因初定位,利用F_(2)群体进行精细定位,并根据功能注释结合RNA-Seq预测候选基因。采用qRT-PCR进行表达模式分析,采用酵母双杂交试验进行蛋白互作验证。结果表明,与野生型相比,ygl7苗期、拔节期叶片呈明显黄绿色,抽穗期逐步转为浅绿色,ygl7整个生育期叶绿素含量及光合速率都显著低于野生型,且叶绿体结构出现异常。遗传分析表明,ygl7黄绿叶表型由1对隐性单基因控制,基因精细定位将黄绿叶基因定位于第Ⅶ染色体434.9 kb区间内,候选基因分析预测编码原卟啉Ⅸ镁鳌合酶Ⅰ亚基的Seita.7G290300为调控黄绿叶的候选基因。qRT-PCR结果显示,Seita.7G290300在叶片中高表达,且在突变体中的表达量低于野生型;叶绿素合成途径(CHLD、CHLI)和光系统(LHCB1、LHCB6)相关基因在突变体中均下调表达。酵母双杂交试验表明,SiYGL7与MORF_(2)存在互作。

大豆类病变皱叶突变体NT301遗传分析和2对基因定位

通过研究类病变突变体,挖掘抗病基因,利用分子设计育种的方法快速培育优良抗病大豆新品种,可减轻化学农药对环境的污染,降低病害的抗药性。本研究以^(60)Coγ诱变获得的类病变皱叶突变体NT301为父本,分别与W82、KF1和KF35进行杂交,构建了F_(2)和F_(2:3)分离群体,通过SSR标记和SNP分析,将目标基因1(rl1)缩小到18号染色体937 kb区间内,包含66个候选基因,将目标基因2(rl2)缩小到8号染色体130 kb区间内,包含15个候选基因。接着,本研究利用基因芯片技术对近等基因系进行了基因表达谱研究,得到了差异表达基因参与的KEGG调控通路。另外对8号染色体的15个候选基因进行半定量与荧光定量RT-PCR表达分析发现,只有基因Glyma.08G332500在突变体NT301与野生型中的差异达到了4倍,推测Glyma.08G332500基因是突变体NT301的候选基因。

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。

绿豆窄叶突变体vrnl11的精细定位

绿豆叶形突变体和叶形调控基因的鉴定,可为品种叶形的遗传改良提供种质资源,也有利于解析叶片发育的遗传调控机理。从皖科绿3号EMS诱变突变体库中筛选到窄叶突变体vrnl11,利用vrnl11/皖科绿3号和vrnl11/中绿1号的杂交后代进行遗传分析,用χ^(2)检验确定F_(2)群体中不同表型植株的分离模式。以vrnl11和中绿1号及中绿5号构建的2个F_(2)群体为定位群体,利用BSA测序技术和图位克隆的方法完成vrnl11的精细定位。表型鉴定结果表明,vrnl11叶片宽和叶面积较野生型皖科绿3号分别减少25.7%和21.7%。遗传分析表明,vrnl11的窄叶表型受单隐性核基因调控。BSA测序分析将突变位点定位在第11染色体上15.0 Mb至末端4.7 Mb的区间内。利用新开发的多态性分子标记将vrnl11定位在标记nl-61和nl-46之间186.5 kb的区间内,包含9个预测基因。这些研究结果为克隆vrnl11和解析绿豆叶片生长发育的分子调控机理提供理论依据。

一份新的玉米永久性失绿突变体chs10的鉴定及基因克隆

【目的】利用60Co-γ射线处理自交系齐319获得了一份新的玉米叶色突变体,对该突变体进行遗传分析、基因定位与克隆,并对候选基因的功能进行初步分析。【方法】以该突变体为亲本,构建遗传分析群体和基因定位群体;通过图位克隆技术获得关键候选基因;利用生物信息学方法分析关键候选基因的结构和进化关系;通过qRT-PCR技术检测候选基因在不同组织中的表达差异;同时运用烟草瞬时表达技术对候选基因进行亚细胞定位表达分析。【结果】鉴定了一份玉米永久性失绿突变体chs10(Permanent chlorosis 10),chs10自V2时期开始,叶片从幼苗基部到顶部逐渐由绿转黄,后期的生长发育过程中不再复绿,并且,整个植株包括叶鞘、叶环、茎秆、苞叶和雄穗也均为黄色。该突变体受一对隐性核基因控制,可稳定遗传,植株生长发育正常,可正常授粉结实。突变基因被定位于玉米第10染色体长臂标记SNP-2和SNP-3之间约0.17 Mb范围内,确定了关键候选基因Zm00001d025860,qRT-PCR结果显示Zm00001d025860在玉米根、茎、叶和叶鞘中均有表达,但在叶片中高表达,亚细胞定位结果显示目标蛋白被定位在细胞膜和叶绿体中。利用STRING预测发现Zm00001d025860与卟啉结合蛋白GUN4基因互作,GUN4与叶绿素合成中的镁离子螯合酶(MgCh)具有反馈调节作用。【结论】Zm00001d025860基因的突变导致突变体chs10叶色的改变,并且Zm00001d025860可能通过参与叶绿素合成途径来调控叶色变化。突变体chs10的发现一方面丰富了玉米叶色突变体研究的基因资源,同时为解析镁离子螯合酶在叶绿素合成途径中的作用机理奠定了材料基础。

西瓜黄化斑点叶片的生理特性与遗传倾向

【目的】探究西瓜黄化斑点叶片的生理特性与遗传倾向,为该材料在实际应用及后续基因定位与克隆提供参考依据。【方法】以黄化斑点叶西瓜TNY1201和普通西瓜1182为材料,对其叶片表型、解剖结构以及光合生理特性进行对比分析,同时建立六世代群体进行遗传倾向研究。【结果】TNY1201从第一片真叶开始就具有黄化斑点性状,与普通西瓜叶片相比,具有面积大、密度小的气孔,叶片上下表皮细胞形状不规则,栅栏组织和海绵组织排列松散,叶片紧密度小,海绵组织所占体积较大;TNY1201净光合速率与叶绿素含量均显著低于1182,气孔导度、胞间CO_(2)浓度显著高于1182;将TNY1201与1182进行正反交与回交,遗传倾向表现为F2中叶片有斑与无斑的分离比为3:1,回交BC1P1叶片有斑与无斑分离比为1∶1。【结论】TNY1201叶片叶绿素含量、净光合速率均显著低于普通西瓜叶片。TNY1201叶片的黄化斑点由一对显性核基因控制。