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×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-maintaining 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 genic genes.

Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice

The DNA fragments about 1 600 bp were amplified using random amplified polymorphism DNA (RAPD) primer OPA12 with the templates of mitochondrial DNA of Zhenshan 97A and Zhenshan 97B,and were sequenced.The nucleotide sequences and lengths of the fragments from Zhenshan 97A and Zhenshan 97B showed no difference.The precise length of the fragment was 1 588 bp.Sequence characterized amplification region (SCAR) primers were then developed to discriminate the cytoplasmic male sterile (CMS) lines and their maintainer lines.A specific 1 588 bp fragment could be amplified with SCAR primers,CHI19F2/CHI19R2 and CHI20F3/CHI23R3,in the mitochondrial DNA of Zhenshan 97A,but not Zhenshan 97B.Furthermore,the specific fragment could be also amplified from the total DNA from green leaf tissues of Zhenshan 97A with SCAR primers,but not Zhenshan 97B.With the corresponding primers,the specific fragment could also be amplified from the total DNA of green leaves of other two CMS lines with wild abortive type cytoplasm (CMS-WA),namely Zhenpin A and Tianfeng A,but not in their maintainer lines.Moreover,using total DNA as template,each of the four pairs of SCAR primers could also be used to amplify the 1 588 bp fragment in CMS-ID (Indonesia paddy type) line Ⅱ-32A,but not in II-32B,and the specific fragment was amplified from the DNA of both F1 and F2 seedlings of Shanyou 63.The results of detecting the genetic purity of a man-made mixture of the seeds of Zhenshan 97A using CHI20F3/CHI23R3 were completely consistent with the phenotypes.Taken together,these results indicated that the specific 1 588 bp-fragment amplified by CHI20F3/CHI23R3 was the unique amplification products of CMS mitochondrial DNA,and could be used to distinguish CMS-WA and CMS-ID lines from their corresponding maintainer lines at the seedling stage.

Differences in Nuclear DNA Between Male-Sterile and Male-Fertile Lines of Sorghum bicolor

Cytoplasmic male sterility (cms) is determined by nuclear-cytoplasmic interactions. Up tonow, most studies are focused on the comparison of cytoplasmic DNAs of male-sterile lines and male-fertilelines, and analysis of nuclear DNA has not been documented yet. In order to find out the possible difference innuclear genome of male-sterile line A1 Tx623 and corresponding male-fertile line Tx623 of sorghum, randomamplified polymorphic DNA (RAPD) approach was used to analyze their cytoplasmic and nuclear genomes.Total DNAs of them were amplified at first to screen primers, which were able to generate reproducible bandsspecific to male-sterile line or male-fertile line. Then the selected primers were used to amplify their mitochon-drial DNA (mtDNA) and chloroplast DNA (cpDNA). The origins of all the polymorphic fragments were ana-lyzed. After ruling out those amplified from cytoplasmic DNA, seventeen polymorphic fragments were deter-mined to be amplified from nuclear DNA. These fragments originated from nuclear DNA indicate that diffe-rences in sequence exist between the nuclear DNA of male-sterile line and male-fertile line of sorghum, whichdo not agree with the traditional standpoint that they have identical nucleus.

New Temperature Sensitive Genic Male Sterile Lines with Better Outcrossing Ability for Production of Two-Line Hybrid Rice

An investigation was carried out with three newly developed temperature sensitive genic male sterile(TGMS) lines for their floral traits, seed production potential and outcrossing ability in ten cross combinations. In the TGMS lines, fertile pollens had an average diameter of 0.89 mm while the sterile pollens was with 0.02 mm diameter.TS-29-150 GY produced the biggest fertile pollens with 0.92 mm and other two lines produced relatively smaller pollens with 0.91 and 0.85 mm. Pollen fertility during the fertility reversion period was an average of 60.7%. TS-29-150 GY had the maximum of 66.9% spikelet fertility whereas other two lines(TNAU18S and TNAU60S) had relatively lower spikelet fertility of 27.8% and 26.7%, respectively.Average of 17.00 g of seed yield was obtained in the TGMS lines during the fertility reversion period. TS-29-150 GY had the highest value of 21.20 g of seed yield while TNAU18 S and TNAU60 S produced 16.6 g and13.2 g of seed yield, respectively. The low seed production ability of these three TGMS lines was attributed only to the environmental conditions prevailing during the period. All three TGMS lines had considerable outcrossing potential of 41.2%, 24.6% and 25.0%, respectively. The cross combinations viz.TNAU18S/IET21508(36 g/plant), TNAU18S/IET21044(13 g/plant), TNAU18S/IET21009(26.5 g/plant),TNAU60S/CB-09-106(26.2 g/plant), TNAU60S/IET21009(14 g/plant) and TS29-150-GY/DRR 3306(39.2g/plant) showed perfect synchronization with acceptable hybrid seed yield, indicating suitability of TGMS system under Indian condition. Based on the outcrossing related traits viz. panicle exertion, angle of glume opening, stigma length and pollen size, TNAU18 S was identified as the best, followed by TS-29-150 GY.

Comparisons on Genetic Diversity among the Isonuclear-Alloplasmic Male Sterile Lines and Their Maintainer Lines in Rice

Four sets of rice isonuclear-alloplasmic lines including 16 male sterile lines and their maintainer lines were analyzed by using 91 pairs of SSR primers to study the genetic diversity of nuclear genome and their relative relationships. A total of 169 alleles were detected in the 16 lines, with a frequency of polymorphic loci of 53.85% and an average number of alleles per locus of 1.8, and the average gene diversity was 0.228. Four sets of the isonuclear-alloplasmic male sterile lines shared 146 identical alleles, corresponding to 86.39% of the total alleles; meanwhile, there are 23 different alleles among the tested materials, being 13.61% of the total alleles. On average, 78.70% identical alleles and 21.30% different alleles of the total alleles were detected between the isonuclear-alloplasmic male sterile lines and their maintainer lines. There were 53.85% identical alleles and 46.15% different alleles of the total alleles among the homozygous allonucleus male sterile lines. The fingerprints were established for some male sterile lines and maintainer lines. All the materials tested were divided into three groups at the 0.2 genetic distance based on the cluster analysis. Eight lines of Huanong A and Huayu A (including Huanong B and Huayu B) were in the first group, four lines of Kezhen A (including Kezhen B) in the second group, and four lines of Zhenshan 97A (including Zhenshan 97B) in the third group. For the isonuclear-alloplasmic male sterile lines, the similarity coefficient between Y (Yegong) type and WA (wild abortive) type or between CW (Raoping wild rice) and WA type reached 87-98%.

Improving Blast Resistance of a Thermo-Sensitive Genic Male Sterile Rice Line GD-8S by Molecular Marker-Assisted Selection

The broad-spectrum blast resistance gene Pi-1, from donor line BL122, was introduced into a thermo-sensitive genic male sterile rice line GD-8S, which possessed good grain quality but high susceptibility to rice blast, by using backcross breeding and molecular marker-assisted selection. Five elite improved male sterile lines, RGD8S-1, RGD8S-2, RGD8S-3, RGD8S-4 and RGD8S-5, were selected based on the results of molecular marker analysis, spikelet sterility, recovery rate of genetic background and agronomic traits. Thirty-three representative blast isolates collected from Guangdong Province, China were used to inoculate the improved lines and the original line GD-8S artificially. The resistance frequencies of the improved lines ranged from 76.47% to 100%, much higher than that of the original line GD-8S (9.09%). On the agronomic characters, there were no significant differences between the improved lines and GD-8S except for flag leaf length and panicle number per plant. The improved lines could be used for breeding hybrid rice with high blast resistance.

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 3A×Qianyou 8B,Qianyou 8A×You 2894B,You 2894A×Qianyou 6B,Qianyou 8A×QH303-4B and Qianyou 8A× 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.

Verification of Glyphosate Resistance, Lepidopteran Resistance and Wide Compatibility of Male Sterile Line E1C4008S in Rice

Weeds and insect pests are two important biotic stresses resulting in yield loss in rice, and wide compatibility is the essential characteristic of breeding inter-subspecific hybrid rice. This study focused on glyphosate resistance, lepidopteran resistance and wide compatibility as well as identification of molecular and some agronomic characteristics of transgenic male sterile line E1 C4008 S. The results indicated that glyphosate resistance gene Epsps^# and lepidopteran resistance gene Cry1 ca^# were transferred into japonica wide compatibility male sterile line 4008 S by Agrobacterium-mediated method, and four independent transformation events named E1 C4008 S-1, E1 C4008 S-2, E1 C4008 S-3 and E1 C4008 S-4 were obtained, in which E1 C4008 S-3 and E1 C4008 S-4 were of single copy insertion. The EPSPS protein contents of E1 C4008 S-3 and E1 C4008 S-4 in different organs were significantly different both in descending order of leaf > stem > root, and ranged from 300.58 to 1410.69 μg/g at the tillering stage. The glyphosate tolerable concentration(dosage) of E1 C4008 S-3 and E1 C4008 S-4 reached at least 16 g/L(54.42 kg/hm^2), and the seeds of E1 C4008 S-4 can germinate normally on the medium containing 1 g/L glyphosate. The CRY1 C protein contents of E1 C4008 S-3 and E1 C4008 S-4 in different organs were significantly different both in descending order of leaf > stem > root, and ranged from 0.62 to 2.43 μg/g at the tillering stage. The larvae mortalities of rice leaf rollers fed on leaves of E1 C4008 S-3 and E1 C4008 S-4 for 5 d were 95.35% and 97.77%, respectively, while the average mortalities of silkworms fed with protein extracts from leaves of E1 C4008 S-3 and E1 C4008 S-4 reached 94.55% and 83.64%, respectively. The results suggested that wide compatibility and evaluated agronomic traits of E1 C4008 S-4 were not significantly changed by insertion of the exogenous genes. Overall, a novel male sterile germplasm with glyphosate resistance, lepidopteran resistance and wide compatibility was verified to be developed in rice.

Inheritance of the Male Sterility in a New Photo/Thermo-Sensitive Genie Male Sterile Line B06S of Rice

The major mate sterile genes in a new photo/thermo-sensitive genic male sterile (PTGMS) line B06S of rice were analyzed by the manipulation of mixture distribution theory. The results indicated that a pair of major male sterile nuclear genes with large effects were responsible for controlling the male sterility of B06S.

Genetic Mechanism of Dominant Earliness in Kefeng A,a New Rice Cytoplasmic Male Sterile Line

Kefeng A is an early maturing indica cytoplasmic male sterile (CMS) line of rice. Combinations derived from Kefeng A and late maturing indica restorer lines showed dominant earliness to various extents. To understand the genetic basis of dominant earliness, the genotype of photoperiod-sensitive genes in Kefeng A was analyzed using a complete set of heading time near isogenic lines (NILs) EG0 to EG7, ER, LR, T65, T65Eb, T65Ebm, T65m, NIL(Hd1) and NIL(Hd4). Results indicated that Kefeng A contained two dominant photoperiod-sensitive alleles E1 and Se-1u on E1 and Se-1 loci, respectively, and the genotype of photoperiod-sensitivity genes for heading time in Kefeng A was E1E1e2e2E3E3Se-1uSe-1uEf-1Ef-1. Based on the detected heading time genotype, in combination with the heading time of Kefeng A and the early maturing phenomenon in its derived F1 hybrids, it is speculated that Kefeng A might carry a dominant inhibitor gene Su-E1 for the dominant photoperiod-sensitive gene E1, and a recessive inhibitor gene i-Se-1 for another dominant photoperiod-sensitive gene Se-1. The reason why F1 hybrids from Kefeng A exhibited early maturing was hereby analyzed and the breeding value of dominant earliness related genes in Kefeng A was discussed.

Influence of Genetic Drift of Restoring Gene (Rf) on Seed Purity of Yuetai A, a Honglian-Type Cytoplasmic Male Sterile Line in Hybrid Rice

The seed samples of Yuetai A, a Honglian (HL) type cytoplasmic male sterile (CMS) line in hybrid rice were investigated to assess the seed purity and to analyze the cause of off-type plants by imitating the biological contaminant to Yuetai A in Nanjing, Jiangsu Province and Lingshui, Hainan Province during 2004–2006. The seed impurity of Yuetai A mainly resulted from the genetic drift of restoring fertility gene (Rf) after biological contamination but not from its sterility unstability. All of the ten maintainer lines, five restorer lines and three thermo-sensitive genic male sterile lines used in the study could pollinate Yuetai A and Yuetai B to produce F1 plants, directly or indirectly resulting in Rf-gene drifting into Yuetai A and generating ‘iso-cytoplasm restoring-generations’. Furthermore, high outcrossing rate and similar heading date of Yuetai A with many varieties used in rice production might easily result in the biological contamination. After removing all plants with Rf-gene mixed in Yuetai A and preventing Rf-gene drifting into Yuetai A, the seed purity of Yuetai A and Yuetai B had been raised to 100%.

Maxie A- a novel cytoplasm male sterile line

The pollen abortion of Maxie A and Zhengshan97 A belong to stainless type with KI-I2 solu-

Breeding double low super hybrids with dominant genic male sterility three lines in Brassica napus

A double low homozygous two-type sterile line ZY-2AB, its near-isogenic temporary maintainer line ZY-4, and the full sterile line ZY-3 derived from the cross of ZY-2A with ZY-4, were developed to breed three-line dominant GMS hybrid in Brassica napus. Three years data showed that the ratios of sterile and fertile plants fit the expected 1:1 ratio in ZY-2AB, and the 1:0 ratio in ZY-3, respectively. Some double low super hybrid combinations were screened out when the full sterile line ZY-3 was used as female parent, among which the combination 01Z24 increased 36.88 percent more yield than control and the combination 98Z131 has been passed through the Sichuan Provincial Regional Test and registered in Sichuan Province. Two years data showed that the agronomic performance of the two-line hybrids derived from crosses of ZY-2A with restorer lines versus that of the three-line hybrids produced by ZY-3 was not different for any of the nine most important agronomic characteristics, revealing that near-isogenic line of homozygous two-type line was used as temporary maintainer could effectively avoid the disadvantage of three-way cross hybrid in breeding three-line dominant GMS hybrid.

Development of Insect-Resistant Hybrid Rice by Introgressing the Bt Gene from Bt Rice Huahui 1 into Ⅱ-32A/B,a Widely Used Cytogenic Male Sterile System

Huahui 1 is an elite transgenic male sterile restorer line of wild rice abortive-type that expresses a Bacillus thuringiensis(Bt) δ-endotoxin and provides effective and economic control of lepidopteran insects. To exploit Huahui 1 to develop a new Bt rice, the insertion site of the Bt gene was determined by thermal asymmetric interlaced PCR(TAIL-PCR). Bt was located in the promoter region of LOC.Os10g10360, approximately 5.35 Mb from the telomere of the short arm of chromosome 10. For the first time, a Bt cytoplasmic male sterile(CMS) system was developed by introgressing Bt from Huahui 1. The recipient CMS system used consisted of Indonesia paddy rice-type II-32B(maintainer line) and II-32A(male sterile line). Marker-assisted selection was used to increase selection efficiency in the backcrossing program. In BC5F1, the Bt plant 85015-8 was selected for further analyses, as it had the highest SSR marker homozygosity. In addition, the linkage drag of the foreign Bt gene in 85015-8 was minimized to 8.01-11.46 Mb. The foreign Bt gene was then delivered from 85015-8 into II-32 A. The resultant Bt II-32 A and Bt II-32 B lines were both resistant to lepidopteran in field trials, and agronomic traits were not disturbed. The maintainability of II-32 B, and the male sterility and general combining ability of II-32 A, were not affected by the Bt introgression. This study demonstrates a simple and fast approach to develop Bt hybrid rice.

Factors Influencing the Outcrossed Seed-Setting Obstacles of Photo-thermo Sensitive Genie Male Sterile Rice

Using photo-thermo sensitive genic male rice (PTGMS) Pei' ai 64S, W7415S, W6154S, N26S, Annong S, Nongken 58S,7001S and 5088S as female parents and conventional indica lines 8258 and U89 as male parents, the factors affecting outcrossed seed-setting were analyzed. The PTGMS had obstacles in outcrossed seed setting influenced by inheritance and environment at varying degrees. Environmental temperature was regarded as the main factor that resulted in the outcrossed seed-setting obstacles.The sensitive stage was at the early stage of grain filling for outcrossed seed setting. There existed remarkable differences at the sensitivity stage, the duration of sensitive period, the sensitive level and the effective level of outcrossed seed-setting obstacles caused by environmental temperature among different PTGMS lines. Therefore, attention should be paid to outcrossed seed-setting obstacles in selection and utilization of PTGMS lines.

小麦光温敏核雄性不育系BS366和BS1086杂种一代结实率研究

为了解小麦光温敏核雄性不育系BS366和BS1086的育性恢复状况,评估恢复系人工定向改良对其恢复能力的效果,在不育生态区对以55份定向改良的恢复系和33份非改良恢复系(常规小麦)为父本配制的杂种一代的结实率进行了分析。结果表明,BS366和BS1086杂种一代结实率变化范围为15.42%~140.34%和20.28%~119.07%(国际法),平均结实率分别为73.02%和72.21%,二者差异不显著,说明2份母本的育性恢复能力差异不显著。BS366和BS1086杂种一代结实率主要分布于40%~110%和60%~100%间,BS1086杂种一代结实率分布更为集中。BS366和BS1086与改良父本的杂种一代平均结实率分别为84.45%和78.97%,与非改良父本的杂种一代平均结实率分别为53.97%和60.95%,改良父本的平均恢复力高于非改良父本,表明定向改良有利于提高恢复系的恢复力。BS366和BS1086与相同父本的杂种一代结实率差异在-41.66%~61.93%之间,表明相同父本对不同母本的育性恢复力存在差异。BS366和BS1086与相同改良父本的杂种一代中有61.82%的结实率差异分布于-10%~20%之间,与相同非改良父本的杂种一代中有51.52%的结实率差异在-30%~0%之间,表明同一改良父本对不同母本的育性恢复力差异小于非改良父本。BS366与14份父本、BS1086与7份父本的杂种一代结实率高于对照,其中父本14YH261、14YH551、SD036与2份母本杂种一代的结实率均高于对照,说明具有可用恢复力的父本不仅数量极少,而且对不同母本的恢复力存在较大差异。杂种一代主茎穗的平均结实率极显著大于分蘖穗。因此,建议对不同母本分别开展强恢复力的恢复系筛选,并以中选强恢复系为亲本进行定向改良,有效增加杂种一代结实率达对照水平的组合数量,为获得更多强优势杂交组合奠定基础。

适合机械化移栽结球甘蓝新品种‘沪甘2150’的选育

‘沪甘2150’是由甘蓝胞质雄性不育系CMS轮01-181为母本和结球甘蓝自交系ZS008-183为父本选配而成的宜机化结球甘蓝新品种,从定植到开始采收约为70 d。该品种生长势强,中抗黑腐病;叶球翠绿,蜡粉少,球形圆正,结球紧实。苗期株型直立性好,适合机械化移栽。叶球质量为1.03 kg,每667 m^(2)平均产量为4004.06 kg,较对照品种‘绿宝石’增产12.08%。

从一道高考题谈利用雄性不育系生产杂交种子

以一道利用雄性不育系生产杂交种子的高考题引发的思考为切入点,阐明利用雄性不育系批量生产杂交种子的技术原理,提出利用转基因技术生产杂交种的技术要点仍是选育不育系、保持系和恢复系并年年留种,为生物学教学提供有价值的参考。

Difference and Inheritance of the Sterility Maintenance of CMS in Indica Rice

The inheritance of the sterility maintenance ability was studied for three types of CMS lines (Zhenshan97A, XieqingzaoA and Ⅱ-32A) and several lines in indica rice. The result showed that the abilities to keep the male sterility of the maintainer lines strengthened obviously with the progressive backcrossing. The critical stage to maintain the sterility of CMS lines happened usually in BC5-6 generation. The selection of the recurrent parent gave an obvious influence on the pollen fertility distribution of the later generations. The author has discussed ways of improving of the maintainer lines.

Thoughts and Practice on Some Problems about Research and Application of Two-Line Hybrid Rice

The main problems about research and application of two-line hybrid rice were reviewed, including the confusing nomenclature and male sterile lines classification, the unclear characteristics of photoperiod and temperature responses and the unsuitable site selection for male sterile line and hybrid rice seed production. In order to efficiently and accurately use dual-purpose genic male sterile lines, four types, including PTGMS (photo-thermo-sensitive genic male sterile rice), TGMS (thermo-sensitive genic male sterile rice), reverse PTGMS and reverse TGMS, were proposed. A new idea for explaining the mechanism of sterility in dual-purpose hybrid rice was proposed. The transition from sterile to fertile was involved in the cooperative regulation of major-effect sterile genes and photoperiod and/or temperature sensitive ones. The minor-effect genes with accumulative effect on sterility were important factors that affected the critical temperature of sterility transfer. In order to make better use of dual-purpose lines, the characterization of responses to photoperiod and temperature of PTGMS should be made and the identification method for the characterization of photoperiod and temperature responses of PTGMS should also be put forward. The optimal ecological site for seed production could be determined according to the historical climate data and the requirements for the meteorological conditions during the different periods of seed production.