Molecular Markers and Agronomic Traits of a New Kind of Genic Male Sterile Material Mian 7AB-4-2 in Brassica napus L.

[ Objective] The study was to investigate the agronomic traits and breeding characteristics of genic male sterile material Mian 7AB-4- 2 in Brassica napus. [ Method] The differences in agronomic traits and polymorphisms in SSR markers, between the genic male sterile material Mian 7AB-4-2 in Brassica napus and its sisterly line Mian 7AB-4-1 were investigated by hybridization and molecular identification; and the percentage of sterile individuals of Mian 7AB-4-2 and of the hybrids with its sisterly line Mian 7AB-4-1 from test cross and back cross were also studled. [ Result] Mian 7AB-4-2 was not significantly different in agronomic traits from its sisterly line Mian 7AB-4-1 at 0.05 probability level. The per- centages of sterile individuals in the pollinated fertile Mian 7AB-4-2 plants were over 60%, and that in its sisterly line Mian 7AB-4-1 was about 25%. In test crosses with other nine sterile lines, Mian 7AB-4-1 kept the percentage of sterile individuals of sterile lines over 90%, and the percentage of sterile individuals from back cross over 80%. With regard to molecular markers, Mian 7AB-4-2 and its sisterly line Mian 7AB-4-1 were different in the band number from SSR primers a2 and E10. [ Conclusion] The results indicate that Mian 7AB-4-2 is helpful for rapeseed breeding, quickening the application of new materials in field breeding.

Establishment on Isolated Microspore Culture Optimized System for Restorer of New Cytoplasmic Male Sterile (NER) of Brassica napus L.

[Objective]The aim was to establish optimized system for NER isolated microspore culture of Brassica napus L.. [Method]Twenty varieties of NER of Brassica napus were grown under uncontrolled temperature and light conditions,and their isolated microspore from anthers were used as explants in vitro culture. The influencing factors of microspore culture were preliminarily studied. [Result]The difference of genotypes was important influencing factors to embryoid yield. The embryoid yield increased by supplementing with 6-BA and NAA,culturing in solid-liquid double layer medium with activated charcoal; The difference was not significant of embryoid yield between culturing in medium supplemented with colchicines and the CK. The rates of cotyledonous embryoids directly developed into normal plantlets increased through enriching with 0.1-0.2 mg/L NAA and being treated on slim illumination two days before being inducted into normal plantlets. [Conclusion]The technical system of microspore culture of restorer of new cytoplasmic male sterile （NER） was established,which and lays a foundation for accelerating genotype purification of NER introgressive line.

Identification of a Cytoplasmic Male Sterile Line NEA in Brassica napus L.and Its Genetic Studies

Male sterile NEA plants were identified in progenies of the radiated 92P x Aggregate-silique in Brassica napus L. in 1993. Their progeny plants from test crossing and open pollination were 100% male sterile. The double-low male sterile lines JL-4 and JL-18 were bred through successive backcrossing of the double -low variety No.4 and No. 18 in Brassica napus L.to NEA. Restorer lines 6720 and 6722 with significant heterosis in F1 were developed. The results from investigating the restoring-ntaintaining relationship and inheritance of the restorer gene indicated that JL-4 and JL-18 were different from both PolCMS and Shan 2A type, and their restorer gene was controlled by a pair of dominant genie genes.

Differentially expressed miRNAs in anthers may contribute to the fertility of a novel Brassica napus genic male sterile line CN12A

In Brassica napus L.(rapeseed),complete genic male sterility(GMS)plays an important role in the utilization of heterosis.Although microRNAs(miRNAs)play essential regulatory roles during bud development,knowledge of how GMS is regulated by miRNAs in rapeseed is rather limited.In this study,we obtained a novel recessive GMS system,CN12AB.The sterile line CN12A has defects in tapetal diferentiation and degradation.llumina sequencing was employed to examine the expression of miRNAs in the buds of CN12A and the fertile line CN12B.We identifed 85 known miRNAs and 120 novel miRNAs that were expressed during rapeseed anther development.When comparing the expression levels of miRNAs between CN12A and CN12B,19 and 18 known miRNAs were found to be difrentially expressed in 0.5--1.0 mm buds and in 2.5--3.0 mm buds,respectively.Among these,the expression levels of 14 miRNAs were higher and the levels of 23 miRNAs were lower in CN12A compared with CN12B.The predicted target genes of these iferentially expressed miRNAS encode protein kinases,F-box domain-containing proteins,MADS-box family proteins,SBP-box gene family members,HD ZIP proteins,floral homeotic protein APETAL A 2(AP2)。and nuclear factor Y,subunit A.These targets have previously been reported to be involved in pollen development and male sterility,suggesting that miRNAs might act as regulators of GMS in rapeseed anthers.Furthermore,RT-qPCR data suggest that one of the dffrentially expressed miRNAs,bna-miR159,plays a role in tapetal differentiation by regulating the expression of transcription factor BnMYB101 and participates in tapetal degradation and infuences callose degradation by manipulating the expression of BnA6.These findings contibute to our understanding of the roles of miRNAs during anther development and the occurrence of GMS in rapeseed.

Analysis on Combining Ability for Characters of Male Sterile Lines in Rapeseed(Brassica napus L.)

The male sterile line is very important in the hybrid breeding program of rapeseed. This study was conducted to evaluate the combining ability of many characters of male sterile lines in Brassica napus L. Ten recessive genetic male sterile （RGMS） lines were used as parents to produce 45 single cross hybrids by using a half diallel cross method. These 45 crosses and their 10 parents were evaluated at Guiyang during 2007-2008. The results showed that both general combining ability （GCA） and specific combiing ability （SCA） effects were important for all characters, but additive gene effects were more predominant than non-additive gene effects. Qianyou 8AB and You 2894AB gave respective highly significant GCA effects of 230.94 and 127.65 kg-hm-2 for seed yield. Lines You 2894AB, QH303-4AB, You 157AB and You 2341AB gave highly significant GCA effects for oil content of 0.99, 1.62, 1.20 and 1.53%, respectively. The crosses among lines Qianyou 3AxQianyou 8B, Qianyou 8AxYou 2894B, You 2894AxQianyou 6B, Qianyou 8AxQH303-4B and Qianyou 8Ax Qianyou 6B gave high SCA effects of 616.29, 398.71,356.48, 394.24 and 303.79 kg hm-2 for seed yield, respectively. All these crosses also gave high seed yield indicating that these crosses could be used in the breeding program.

Correlation Analysis on Male Sterile Lines Characters in Rapeseed(Brassica napus L.)

[Objective] This study was conducted to evaluate the associations of many characters of male sterile lines in Brassica napus L.[Method]10 recessive genetic male sterile（RGMS）lines were used as parents to cross in a half diallel cross method to produce 45 single cross hybrids.These 45 crosses and their 10 parents were evaluated at Guiyang during 2007-2008.[Result]The positive correlations of seed yield with plant height,pods per plant,days to flowering and days to maturity were significant.Their respective correlation coefficients（r）were 0.644,0.583,0.281 and 0.341.Highly significant and negative correlation was observed between seeds per pod and seed size（-0.533）.Direct path coefficients were observed for pods per plant（0.556 2）,seeds per pod（0.407 4）,1 000-seed weight（0.481 3）,and plant height（0.401 7）to seed yield.[Conclusion]Pods per plant,seeds per pod,1 000-seed weight and plant height were positive correlated with seed yield and showed high direct contributions to yield.Other characters including branches per plant,days to flowering,days to maturity and oil content had low contributions to seed yield.

The unstability of cytoplasmic male sterility of Brassica napus L.

The unstability of cytoplasmic male sterilities (CMS) of Brassica napus Lwas stud-ied from 1989 to 1992,and the results indicated: 1. The occurrence of trace-pollen plants In sterilematerials was caused by nuclear polygenes of the maintainers; 2. Progenies of partial sterility typeshowed segregation of sterile, partially sterile (with trace pollen )and fertile plants; 3. High andlow temperature CMS lines were crossed and its progenies manifested complex segregation; 4.There was a tendency to increase the fertility with successive selfing of the high temperatureCMS line.

Influential Factors of Restorer of New Cytoplasmic Male Sterile(NER) on Anther Culture

Some influential factors of anther culture were studied preliminarily by conducting anther culture of the restorers of new cytoplasmic male sterile （NER）. Several results were obtain from this experiment and they were listed as follow：① MS cultrure medium with such hormones as 2,4-D 2 mg/L,6-BA 0.5 mg/L, NAA 0.5 mg/L was the best suitable for callus induction of NER. ②The difference of induction rate was significantly different between different plant age groups. From the 110th day to 141th day,the induction rate was increased with the increase of age and the difference of induction rate reached 0.01 significant difference level. The induction rate reached the highest value in the 141th day then it declined gradually. ③The combined use of 2, 4-D and 6-BA with proper increase of 2,4-D was good for inducing callus. ④The green plantlet induction rate of NER was increased when the concentration of 6-BA increased from 2 mg/L to 4 mg/L. Adding ZT from 0.5 mg/L to 2 mg/L. 6-BA would led 2.47% increase of green plantlet olantlet induction rate.

Tissue-specific Expression of Acetolactate Synthase (ALS), Male Sterility-inducing Effect of Tribenuron-methyl and Its Effect on ALS Activity in Brassica napus L.

[Objectives]This study was conducted to provide a basis for the rapid identification of the drug spraying effect in early stage and the molecular mechanism of chemical hybridizing in Brassica napus L.[Methods]Quantitative RT-PCR analysis showed that ALS was constitutively expressed in various tissues of 096030,including flower buds,four floral organs (calyxes,petals,stamens and pistils),roots,stems and leaves.ALS was prominently expressed in leaves and was expressed weakly in the petals and stamens.The male sterility-inducing effects of tribenuron-methyl on such two Brassica napus L.varieties as Ningyou18 and 096030 were investigated.[Results]Plants were twice sprayed with 0.2 μg/ml tribenuron-methyl on leaves.The results showed that 8-10 ml of tribenuron- methyl was applied per plant for the first time at bolting stage with 1-2 mm flower buds on 15-20 cm inflorescence,and the second spray was performed with 8-10 ml of tribenuron-methyl per plant 10 d later.The results showed that the percentage of the full sterile plants reached 100%,which lasted for the whole flowering period,and the relative seed setting rate was only about 4%.Thus,this method could fullfill the requirement of hybrid seed production in field.The in-vivo enzyme activity of acetolactate synthase (ALS) was assayed using 2 mm buds collected 3 d after spray.The results showed that 0.2 μg/ml tribenuron-methyl inhibited ALS activity.The ALS activity of Ningyou 18 (CK) and Ningyou 18 (0.2 μg/ml) was 3.20 and 1.30 μmol/(mg·h),respectively,and the ALS activity of 096030 (CK) and 096030 (0.2 μg/ml) was 3.37 and 1.25 μmol/(mg·h),respectively.The relative enzyme activity of ALS in Ningyou18 and 096030 was 40.63% and 37.23%,respectively,both of which decreased significantly.[Conclusions]These results showed that the change of ALS activity may be used as an index for quickly identifying and predicting the chemical hybridizing effect of tribenuron-methyl.

Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was discussed.

Differential Expression Analysis of Genie Male Sterility A/B Lines in Chinese Cabbage-Pak-Choi (Brassica campestris ssp. chinensis Makino)

To determine differential expression of genie male sterility A/B lines in Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis Makino var. communis Tsen et Lee), we used the RNA fingerprinting technique, cDNA-AFLP analysis, in different developmental stages and different tissues. While no obvious differential expressions were observed in rosette leaves, florescence leaves, and scapes, some differential expressions were found in alabstrums of A/B lines and among leaves, scapes and alabstrums. We analyzed the al-abstrums collected in different developmental stages with 10 primer combinations. We got a unique band between middle size alabstrums and large alabstrums in B line in one of the ten pair primers, and in another one pair, one band reflecting a higher gene-expression level in A line than that in B line was obtained. No unique bands were found with the other primer combinations. The bands reflecting different gene-expression level were confirmed by Northern hybridization. The results indicated that cDNA-AFLP was a suitable tool for studying differential expression of genie male sterility in plants. SDS-polyacrylamide gel electrophoresis patterns of soluble proteins further verified the difference in A/B lines.

甘蓝型油菜(B.napus L.)细胞质雄性不育系MICMS的选育

本研究选用“陆奥”油菜为母本,“五十铃”油菜为父本进行品种间杂交,以父本连续回交,育成了新的质核互作结构的甘蓝型油菜雄性不育系 MICMS。该不育系花器官形态和花药解剖结构的变异现象与已知的油菜细胞质雄性不育系相似。通过不同品种测交,已鉴定出对 MICMS 具有较好恢、保能力的品种。

甘蓝型油菜(Brassica napus L.)隐性核不育两型系22118AB的基因型分析及利用途径探讨

甘蓝型油菜（BrassicanapusL．）核不育系22118A的育性是由两对隐性基因所控制．不育株的基因型为双隐性纯合体aabb（为了便于分析．作者把这两对基因分别用a和b表示，下同）．两对基因中只要有一个为显性．植株全部表现为可育；只要有一对为显性纯合体AA或BB的材料都可作为恢复系，核不育两型系22118AB中的可有株基因型为Aabb或aaBb，采用兄妹交配aabb×Aabb或aabb×aaBb，其后代育性分离稳定为1：1．在杂种优势利用中可以采用二系法制种．用两型系中的可育株作父本进行测文，有望找到核质工作的保持系，从而实现三系配套，生产杂交种子。

甘蓝型油菜(Brassica napus L.)隐性核不育花叶两型系的选育

用隐性核不育两型系内不育株和花叶自交系杂交 ,F1全部表现为雄性可育 ,叶型为半花叶。F2 群体出现分离 ,花叶、半花叶、圆叶三种类型植株的分离比例符合 1∶2∶1,可育植株与不育植株的分离比例符合 15∶1。在F2 群体中选花叶可育株自交 ,同时与花叶不育株兄妹交 ,F3群体内所有单株均为花叶。这些可育株自交育性分离比例为 3∶1、相应兄妹交育性分离比例为

甘蓝型油菜(Brassicanapus L.)无蜡粉隐性核不育两型系的选育

用隐性核不育两型系内不育株和无蜡粉自交系杂交 ,F1代全部表现为雄性可育 ,且茎叶表面被有蜡粉 ;F2 代群体出现有蜡粉可育、无蜡粉可育和有蜡粉不育三种类型的植株 ;在F2代群体中选无蜡粉可育株自交 ,F3代群体内所有单株都无蜡粉 ,并出现 15∶1、3∶1和 10 0 %可育等三种育性表现 ;从育性分离比例为 3∶1的二个F3代群体内选株自交和兄妹交 ,一部分F4代群体的育性表现为 10 0 %可育 ,相应的F3代兄妹交群体的育性也表现为 10 0 %可育 ;另一部分F4 代群体的育性表现为 3∶1,相应的F3代兄妹交群体的育性表现为 1∶1。这些可育株自交育性分离比例为 3∶1、相应兄妹交育性分离比例为

甘蓝型(Brassica napus L.)显性核不育双低油菜杂交新品种核杂3号的选育

以甘蓝型油菜显性核不育杂合型不育株204A与恢复系83248为材料,通过杂交、自交、兄妹交和测交等育种方法,成功地转育成双低纯合两型系48AB。然后在显性核不育三系技术基础上,用纯合两型系48AB与临保系C1按核不育二系法生产全不育系(48A×C1)CA,再用该系与恢复系RH-5杂交选育成显性核不育双低油菜杂交品种核杂3号。该杂交品种不仅产量优势显著,而且双低品质优良。

甘蓝型油菜(Brassica napus L.)核质互作型雄性不育系3213A的遗传性

以新发现的天然不育株 32 13A为材料 ,采用正反交、自交和测交等方法 ,分析研究其后代的育性分离表现。结果表明 ,32 13A的育性不但受一对细胞核基因控制 ,还受细胞质属性影响 ,属核质互作型雄性不育。不育株花粉虽然微量 ,但能被稳定、彻底恢复可育。 32 13A的遗传性表明 ,该不育性可用于三系配套 ,生产杂交种。

油菜(Brassica napus L.)胞质不育杂种及其三系苔段离体培养分化出苗能力比较

在B_5培养基上,进行胞质不育杂种及三系苔段的离体培养,结果发现杂种苔段的分化出苗率(72.2%)、正常苗块率(26.7%)及每块苗数(4.2株)均优于三系,且玻璃化苗和分化不良苗率较低.

甘蓝型油菜BnPHL12基因克隆及功能分析

【目的】在甘蓝型油菜中克隆拟南芥MYB-CC家族转录因子AtPHL12的同源基因BnPHL12并进行生物信息学、组织表达模式、亚细胞定位及转录活性研究,为BnPHL12的基因功能研究奠定基础。【方法】利用序列比对方法(BLAST)在甘蓝型油菜泛基因组数据库BnIR中鉴定AtPHL12的同源基因BnPHL12,在“Westar”品系中克隆AtPHL12的同源基因,进行生物信息学及进化分析。利用qRT-PCR及GUS染色方法检测AtPHL12同源基因在不同组织中的表达水平,利用原生质体转化的方法检测BnPHL12的亚细胞定位及转录活性。利用农杆菌侵染介导的转化方法,创建BnA01PHL12过量表达转化植株,并进行BnA01PHL12功能分析。【结果】在“Weatar”材料中克隆到4个AtPHL12的同源基因,命名为BnA01PHL12(705 bp),BnC01PHL12(705 bp),BnA05PHL12(705 bp)和BnC05PHL12(696 bp),分别编码234、234、234、231个氨基酸,与AtPHL12的序列相似性大于70%,均含有保守的MYB DNA结合结构域和Coiled-Coiled结构域。BnA01PHL12与BnC01PHL12分别来源于白菜的BraA01t04110Z、甘蓝的BolC01g050170.2J。BnPHL12的同源基因在根、茎、叶、花蕾中均有表达,在根和叶的表达水平更高;BnA01PHL12与BnC01PHL12在花药发育的早期和晚期均有较高的表达水平。BnA01PHL12定位在细胞核中并具有转录激活活性。在甘蓝型油菜和拟南芥中,利用绒毡层特异表达基因BnA9启动子驱动BnA01PHL12表达,导致转基因材料雄性不育。【结论】在甘蓝型油菜中,AtPHL12的4个同源基因具有保守的MYB和CC结构域,在营养器官和生殖器官中均有表达。通过在绒毡层细胞中过量表达BnA01PHL12,创制了新的雄性不育材料,为研究花药发育提供良好基础。

甘蓝型油菜三隐性核不育材料L7AB及临保系的选育

用从贵州省油料研究所选育的高不育株率材料中发现的异型不育株作母本,自育另一黄籽双低高不育株率材料2822B作父本杂交,其后代经多年测交、自交选育、鉴定和恢复系的筛选,已成功选育了稳定的双低三基因隐性核不育系、保持系和优良恢复系,从而实现了三系配套。