Genetic analysis and fine mapping of tms9, a novel thermosensitive genic male‐sterile gene in rice (Oryza sativa L.)

Two‐line hybrid rice as a novel hybrid breeding method has huge potential for yield increasing and utilization of intersubspecific heterosis, and it is of major significance for the food security of rice‐consuming populations. Zhu1S is a thermosensitive genic male‐sterile line of rice with low critical temperature and excellent combining ability, which has been widely exploited as a female parent in Chinese two‐line hybrid rice breeding. Here, genetic mapping in F₂ populations was used to show that its male sterility is inherited as a single recessive gene and that responsible gene (termed tms9) lies on the short arm of chromosome 2. A high‐resolution linkage analysis which was based on the Zhu1S/R173 F₂ population found that the thermosensitive genic male‐sterile gene tms9 of Zhu1S was fine mapped between insertion–deletion (Indel) markers Indel 37 and Indel 57, and the genetic distance from the tms9 to the two markers was 0.12 and 0.31 cM, respectively. The physical distance between the two markers was about 107.2 kb. Sequence annotation databases showed that the two Indel markers (Indel 37 and Indel 57) were located on two BAC clones (B1307A11 and P0027A02). There are sixteen open reading frames (ORF) present in this region. The results of this study are of great significance for further cloning tms9 and molecular marker–assisted selection.     

Improving male fertility restoration of common wheat for Triticum timopheevii cytoplasm

The fertile pure line R3‐37 of common wheat with cytoplasm of Triticum timopheevii Zhuk. is an R‐line (restorer) that can restore the male fertility of A‐lines (male sterile lines) with T. timopheevii cytoplasm. In breeding hybrid wheat, the hybrid of the cross R3‐37/ Baimian3 was found to be completely male sterile, indicating that Baimian3 has some genes that are epistatic to the Rf genes in R3‐37. In order to elucidate the essence of this phenomenon, the male fertilities of the hybrids of 27 crosses including R3‐37 and/or Baimian3 were studied. The results show that inheritance of male fertility of the hybrid R3‐37/Baimian3 involves interactions among Rf alleles, male fertility‐inhibiting genes and genetic background. Although more than 70 different kinds of male sterile cytoplasm to common wheat have been discovered, the systems of hybrid wheat production based on male sterile cytoplasm are all the A‐line/R‐line type and all have similar problems of hybrid fertility restoration. This study confirmed that there is a new model (A‐line/R*‐line//R‐line) for producing hybrid wheat with high fertility restoration. In the new model, the completely male sterile hybrids of A‐line/R*‐line can act as common A‐line.     

Mapping of male sterility gene ms10 in chilli pepper (Capsicum annuum L.)

Most of the hybrid seed in chilli are produced manually, but the use of male sterility (MS) can reduce the cost of hybrid seed production. MS‐12, a nuclear male‐sterile (NMS) line developed at Punjab Agricultural University, Ludhiana (India), has been utilized to develop commercial F₁ hybrids. A recessive gene, designated as ms10, governs MS in MS‐12. Due to recessive gene control, development of new NMS lines incorporating ms10 gene is tedious and time‐consuming. We identified SSR markers AVRDC‐PP12 and AVRDC_MD997* linked to the ms10 gene. A total of 558 primer pairs were screened following bulked segregant analysis (BSA). Linkage analysis in 210 F₂ plants indicated that the two SSR markers were linked to the ms10 gene and the marker AVRDC‐PP12 was closest to the gene at 7.2 cM distance. The marker was mapped to chromosome 1 at genome position 175 694 513 to 175 694 644. Until more closely linked markers are developed, the marker AVRDC‐PP12 would facilitate transfer of ms10 gene through marker‐assisted selection (MAS). Fine mapping would lead to cloning of the ms10 gene.     

Identification of AFLP markers linked to one recessive genic male sterility gene in oilseed rape, Brassica napus

The commercial utilization of heterosis in seed yield by means of hybrid varieties is of great importance for increasing oilseed rape production in China. This requires a functional system for the production of hybrid seed. The Brassica napus oilseed rape line 9012AB is a recessive epistatic genic male sterility (GMS) two‐type line, in which the sterility is controlled by two pairs of recessive duplicate sterile genes (ms1 and ms2) interacting with one pair of a recessive epistatic inhibitor gene (rf). Homozygosity at the rf locus (rfrf) inhibits the expression of the recessive male sterility trait in homozygous ms1ms1ms2ms2 plants. This study was conducted to identify molecular markers for one of the male fertility/sterility loci in the B. napus male sterility line 9012AB. Sterile bulk (BS) and fertile bulk (BF) DNA samples prepared from male sterile and male fertile plants of the homozygous two‐type line 9012AB were subjected to amplified fragment length polymorphic (AFLP) analysis. A total of 256 primer combinations were used and seven markers tightly linked to one recessive genic male sterile gene (ms) were identified. Among them, six fragments co‐segregated with the target gene in the tested population, and the other one had a genetic distance of 4.3 cM. The markers identified in this study will greatly enhance the utilization of recessive GMS for the production of hybrid seed in B. napus oilseed rape in China.     

Development of a genetic marker linked to a new thermo-sensitive male sterile gene in rice (<Emphasis Type="Italic">Oryza sativa L</Emphasis>.)

Application of the thermo-sensitive genic male sterile (TGMS) system has a great potential to increase the efficiency of hybrid rice breeding. An indica rice TGMS mutant, 0A15-1, was crossed with a fertile indica line Guisi-8 to map the gene responsible to the TGMS. A RAPD (random amplified polymorphic DNA) maker, S187-770, linked to the TGMS gene at a distance of 1.3 cM in coupling phase was identified. The S187-770 was then cloned and sequenced to develop a dominant SCAR (sequence characterized amplified region) marker. Homology search against rice genome DNA sequence database indicated that S187-770 located on the short arm of chromosome 3 and close to centromere as a single copy sequence. This SCAR marker can be used in the marker-assisted transfer of this gene to different genetic background. As no other TGMS gene has been mapped on rice chromosome 3, the gene from 0A15-1 is a new TGMS gene and tentatively designated tms6(t).     

Development of thermo-sensitive cytoplasmic male sterile (TCMS) lines of wheat characterized by complete male sterility at lower-temperatures and partially restored fertility at higher-temperatures

A ‘‘two-line hybrid system’’ was developed using thermo-sensitive cytoplasmic male sterility (TCMS) from Aegilops kotschyi and is proposed as a means for seed multiplication and production of hybrid wheat. The TCMS line is crossed to a pollinator line during the normal wheat-growing season for production of hybrid wheat seeds, whereas the TCMS line is maintained through self-pollination in an environment with high temperatures. The male sterile wheat line KTP3314A, containing Ae. kotschyi cytoplasm and the short arm of chromosome 1B from Triticum spelta, was studied in different temperature regimes. KTP3314A was completely male sterile under normal wheat growing temperatures (<18 °C). Sterility of KTP3314A was partially restored when plants were grown at temperatures above 20 °C during growth stages 45–52. Male fertility was not correlated with day-length. A series of TCMS lines derived from KTP3314A showed similar environmental responses and performed well in hybrid wheat production at lower temperatures. Fertility of these TCMS lines at higher temperatures makes it possible to produce large quantities of male sterile parental seeds. Based on these findings, application of the TCMS-dependent two-line system in hybrid wheat breeding appears to have a promising future for hybrid wheat production.     

Identification of AFLP markers linked to the epistatic suppressor gene of a recessive genic male sterility in rapeseed and conversion to SCAR markers

A recessive epistatic genic male sterility (REGMS) two‐type line, 9012AB, has been used for rapeseed hybrid seed production in China. The male sterility of 9012AB is controlled by two recessive duplicate sterile genes (ms1 and ms2) interacting with one recessive epistatic suppressor gene (esp). Homozygosity at the esp locus (espesp) suppresses the expression of the recessive male sterility trait in homozygous ms1ms1ms2 ms2 plants. In this study, we used a combination of bulked segregant analyses and amplified fragment length polymorphism (AFLP) to identify markers linked to the suppressor gene in a BC₁ population. From the survey of 1024 AFLP primer combinations, eight markers tightly linked to the target gene were identified. The two closest markers flanking both sides of Esp, P9M5₃₇₀ and S16M14₇₈₀, had a genetic distance of 1.4 cM and 2.1 cM, respectively. The AFLP fragment from P4M8₁₉₀, which co‐segregated with the target gene was converted into a sequence characterized amplified region marker. The availability of linked molecular markers will facilitate the utilization of REGMS in hybrid breeding in Brassica napus.     

Molecular mapping of a dominant genic male sterility gene Ms in rapeseed (Brassica napus)

Rs1046AB is a genic male sterile two‐type line in rapeseed that has great potential for hybrid seed production. The sterility of this line is conditioned by the interaction of two genes, i.e. the dominant genic male sterility gene (Ms) and the suppressor gene (Rf). The present study was undertaken to identify DNA markers for the Ms locus in a BC₁ population developed from a cross between a male‐sterile plant in Rs1046AB and the fertile canola‐type cultivar ‘Samourai’. Bulked segregant analysis was performed using the amplified fragment length polymorphism (AFLP) methodology. From the survey of 480 AFLP primer combinations, five AFLP markers (P10M13₃₅₀, P13M8₄₀₀, P6M6₄₁₀, E7M1₂₃₀ and E3M15₁₀₀) tightly linked to the target gene were identified. Two of them, E3M15₁₀₀ and P6M6₄₁₀, located the closest, at either side of Ms at a distance of 3.7 and 5.9 cM, respectively. The Ms locus was subsequently mapped on linkage group LG10 in the map developed in this laboratory, adding two additional markers weakly linked to it. This suite of markers will be valuable in designing a marker‐assisted genic male sterility three‐line breeding programme.     

Introgression of Xa4, Xa7 and Xa21 for resistance to bacterial blight in thermosensitive genetic male sterile rice (Oryza sativa L.) for the development of two-line hybrids

Bacterial blight (BB) of rice caused by X. oryzae pv. oryzae is a major production constraint in commercial hybrid rice production in the Philippines because most of the parental lines used in hybrid production do not carry resistance genes against the pathogen. In this study, three bacterial blight resistance genes, Xa4, Xa7 and Xa21, were introgressed to a temperature-sensitive genetic male sterile (TGMS1) line. A three-way cross of AR32-19-3-3/TGMS1//IRBB4/7 (PR36944) was made to produce 1,364 F₂ plants carrying various combinations of Xa4, Xa7 and Xa21. Individual plants were characterized for reaction to bacterial blight PXO61 (race 1), PXO86 (race 2), PXO99 (race 6) and pollen sterility. Of 144 F₂ plants demonstrating resistance against PXO61, PXO86 and PXO99, 22 exhibited highly resistant phenotypes with mean lesion lengths ranging from 0.37–2.97 cm. Analysis of disease reaction identified 20 potential TGMS F₂ plants containing Xa4, Xa7 and Xa21 while 78 plants with Xa4 + Xa7. Phenotypic and polymerase chain reaction (PCR) analyses confirmed PR36944-450, PR36944-473 and PR36944-700 as homozygous for Xa7 and Xa21 and highly resistant to all three Xoo races. Fertility of PR36944-450 and PR36944-700 was restored at permissive temperature in a growth chamber. BB-resistant TGMS lines should facilitate breeding two-line hybrids in the tropics.     

AFLP and SCAR markers linked to the suppressor gene (Rf) of a dominant genetic male sterility in rapeseed (Brassica napus L.)

Rs1046AB is a line which is true breeding for a dominant genetic male sterility gene (Ms) but which is a mixture of male fertile and sterile individuals (a two-type line) because it is segregating for a dominant suppressor gene (Rf). This system provides a promising alternative to the CMS system for hybrid breeding in Brassica napus. In order to identify molecular markers linked to the rf gene, a near-isogenic line (NIL) population from the cross between a sterile individual (MsMsrfrf) and a fertile individual (MsMsRfrf) in Rs1046AB was subjected to amplified fragment length polymorphism (AFLP) analysis, with a combination of comparing near isogenic lines (NILs) and bulked segregant analysis (BSA). From 2,816 pairs of AFLP primers, six fragments showing polymorphism between the fertile and sterile bulks as well as the individuals of the bulks were identified. Linkage analysis indicated that the six AFLP markers are tightly linked to the Rf gene and all are distributed on the same side. The minimum genetic distance between the Rf gene and a marker was 0.7 cM. Since the AFLP markers are not suitable for large-scale application in MAS (marker-assisted selection), our objective was to develop a fast, cheap and reliable PCR-based assay. Consequently, three of the four closest AFLP markers were converted directly to sequence characterized amplified region (SCAR) markers. For the other marker a corresponding SCAR marker was successfully obtained after isolating the adjacent sequences by PCR Walking. The available SCAR markers of the Rf gene will greatly facilitate future breeding programs using dominant GMS to produce hybrid varieties.     

YM型小麦温敏雄性不育系不育基因的QTL定位

YM型小麦温敏雄性不育系的不育基因被定位在1Bs染色体片段上, 但已发现的相邻分子标记与该基因的遗传距离较大, 达10 cM以上。为寻找与该基因连锁更紧密的分子标记, 以YM型温敏雄性不育系ATM3314与恢复系中国春杂交的F₂代200株为作图群体, 从1Bs的22个SSR引物中筛选出5个在亲本和F₂代中分离的SSR引物, 构建了1个包含5个标记的1Bs局部遗传连锁图谱。结合F₂代个体的育性调查, 采用复合区间作图法在YM型温敏雄性不育系的1Bs染色体上检测到不育基因的1个主效QTLrfv1-1和1个微效QTLrfv1-2。rfv1-1位于SSR标记Xgwm18和Xwmc406之间, 与两标记的遗传距离分别为6.0 cM和4.6 cM, LOD值为8.80, 加性效应23.87, 显性效应10.44, 可解释表型变异的23.91%; rfv1-2位于Xwmc406和Xbarc8之间, 与两标记的遗传距离分别为4.0 cM和3.4 cM, LOD值为3.10, 加性效应17.59, 显性效应5.99, 可解释表型变异的7.78%。本研究初步定位了YM型小麦温敏雄性不育系1Bs染色体片段上不育基因的QTL, 为进一步准确定位该基因奠定了基础。     

Genetic and cytological analysis of a new spontaneous male sterility in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

A male sterile plant appeared in the radish breeding program at the Hubei Academy of Agricultural Sciences, Hubei, China. In its progeny, a two-type (half of plants male sterile, the other half male fertile) line 01GAB was established. An F₂ population of 260 plants from a cross of male-sterile 01GAB and a male fertile line 9802H segregated for male fertility in a 3:1 ratio indicating that fertility was restored by a single dominant gene, here designated RsMs. A PCR-based DNA marker specific to the male fertility Rfob gene in 9802H was absent in 01GAB. Linkage analysis placed the RsMs locus 10.7 cM away from the Rfo locus. In an F₂ population of hybrids between 01GAB and male fertile 9802B, a co-dominant DNA marker for the RSultr3.2A (a radish sulfate transporter gene) locus was linked to the RsMs locus at 1.5 cM suggesting that fertility restoration in 01GAB was located in the region with known male sterility restorers in radish. However, no maintainer for the 01GAB source of male sterility has been identified so far. Cytological observations have shown that the abnormalities in male sterile anthers first appeared in tapetum at the tetrad stage, followed by a hypertrophy of the tapetal cells at the vacuolate microspore period. These results suggest that male sterility in 01GAB is likely to be genetic in nature, or it may represent a new type of the cytoplasmic male sterility.     

Molecular markers linked to <Emphasis Type="Italic">Bn</Emphasis>;<Emphasis Type="Italic">rf</Emphasis>: a recessive epistatic inhibitor gene of recessive genic male sterility in <Emphasis Type="Italic">Brassica napus </Emphasis>L.

7–7365AB is a recessive genic male sterile (RGMS) two-type line, which can be applied in a three-line system with the interim-maintainer, 7–7365C. Fertility of this system is controlled by two duplicate dominant epistatic genes (Bn;Ms3 and Bn;Ms4) and one recessive epistatic inhibitor gene (Bn;rf). Therefore an individual with the genotype of Bn;ms3ms3ms4ms4Rf_ exhibits male sterility, whereas, plant with Bn;ms3ms3ms4ms4rfrf shows fertility because homozygosity at the Bn;rf locus (Bn;rfrf) can inhibit the expression of two recessive male sterile genes in homozygous Bn;ms3ms3ms4ms4 plant. A cross of 7–7365A (Bn;ms3ms3ms4ms4RfRf) and 7–7365C (Bn;ms3ms3ms4ms4rfrf) can generate a complete male sterile population served as a mother line with restorer in alternative strips for the multiplication of hybrid seeds. In the present study, molecular mapping of the Bn;Rf gene was performed in a BC₁ population from the cross between 7–7365A and 7–7365C. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) technique was used to identify molecular markers linked to the gene of interest. From a survey of 768 primer combinations, seven AFLP markers were identified. The closest marker, XM5, was co-segregated with the Bn;Rf locus and successfully converted into a sequence characterized amplified region (SCAR) marker, designated as XSC5. Two flanking markers, XM3 and XM2, were 0.6 cM and 2.6 cM away from the target gene, respectively. XM1 was subsequently mapped on linkage group N7 using a doubled-haploid (DH) mapping population derived from the cross Tapidor × Ningyou7, available at IMSORB, UK. To further confirm the location of the Bn;Rf gene, additional simple sequence repeat (SSR) markers in linkage group N7 from the reference maps were screened in the BC₁ population. Two SSR markers, CB10594 and BRMS018, showed polymorphisms in our mapping population. The molecular markers found in the present study will facilitate the selection of interim-maintainer.     

Development of dominant nuclear male-sterile lines with a blue seed marker in durum and common wheat

In order to develop genie male‐sterile lines with a blue seed marker, male‐sterile plants, controlled by a dominant nuclear gene Ms2, were used as female parents against a 4E disomic addition line ‘Xiaoyan Lanli’(2n= 44, AABBDD+4EII) as the male parent to produce monosomic addition lines with blue seed. Male‐sterile plants from the monosomic addition lines were pollinated with durum wheat for several generations and in 1989 a male‐sterile line with the blue grain gene and the male‐sterile gene Ms2 on the same additional chromosome was detected and named line 89‐2343. Using this line, the blue seed marker was successfully added to a short male‐sterile line containing Ms2 and Rht10. The segregation ratios of male sterility and seed colour as well as the chromosome figurations of different plants indicated that the blue grain genes, Ms2 and Rht10 were located on the same additional chromosome. Cytological analysis showed that the blue marker male‐sterile lines in durum wheat and common wheat were monosomic with an additional chromosome 4E. The inheritance ratio for blue seed male‐sterile plants and white seed male‐fertile plants was 19.7% and 80.3%, respectively, in common wheat. The potential for using blue marker sterile lines in population improvement and hybrid production is discussed.     

A new male sterile mutant LZ in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Genetic male sterility (GMS) genes in wheat (Triticum aestivum L.) can be used for commercial hybrid seed production. A new wheat GMS mutant, LZ, was successfully used in the 4E-ms system for producing hybrid wheat, a new approach of producing hybrid seed based on GMS. Our objective was to analyse the genetic mechanism of male sterility and locate the GMS gene in mutant LZ to a chromosome. We firstly crossed male sterile line 257A (2n = 42) derived from mutant LZ to Chinese Spring and several other cultivars for determining the self-fertility of the F₁ hybrids and the segregation ratios of male-sterile and fertile plants in the F₂ and BC₁ generations. Secondly, we conducted nullisomic analysis by crossing male sterile plants of line 257A to 21 self-fertile nullisomic lines as male to test the F₁ fertilities and to locate the GMS gene in mutant LZ to a chromosome. Thirdly, we conducted an allelism test with Cornerstone, which has ms1c located on chromosome 4BS. All F₁s were male fertile and the segregation ratio of male-sterile: fertile plants in all BC₁ and F₂ populations fitted 1:1 and 1:3 ratios, respectively. The male sterility was stably inherited, and was not affected by environmental factors in two different locations or by the cytoplasm of wheat cultivars in four reciprocal cross combinations. The results of nullisomic analysis indicated the gene was on chromosome 4B. The allelism test showed that the mutant LZ was allelic to ms1c. We concluded that the mutant LZ has common wheat cytoplasm and carries a stably inherited monogenic recessive gene named ms1g.     

水稻温敏核雄性不育系培矮64S广亲和位点S-5的SSR紧密连锁标记

广亲和(WC)是水稻(OryzasativaL.)亚种间杂种优势利用的重要遗传工具。本研究对我国南方大面积推广应用的亚种间两系杂交稻亲本培矮64S的WC基因S-5n进行分子标记定位。从培矮64S//T8/秋光三交F1中选出由263个高育和低育极端类型组成的标记群体,选用来源于CornellSSR连锁图的20个标记和根据GenBank数据库公布的序列合成的9个SSR标记,通过BSA(分离群体分析)法对标记群体进行分析,该S-5位点精确定位于第6染色体上,距SSR标记GXR6和RM276的距离只有0.2cM。培矮64S的S-5n对育性贡献可使小穗结实率从平均43.5%提高到77.5%,贡献率达34.0%,表明它是一个很强的控制广亲和特性的主效基因。本研究所获得的这些紧密连锁标记对分子标记辅助选择培育广亲和水稻品种和基因克隆具有重要的利用价值。     

Development of a cytoplasmic male‐sterile line NJCMS4A for hybrid soybean production

Cytoplasmic male‐sterile (CMS) lines are being used to produce hybrid seeds. Thus far, four CMS sources in soybean [Glycine max (L.) Merr.] have been reported in China. However, they are not sufficient or efficient in meeting the requirements of commercial soybean hybrid seed production. In this study, 33 varieties were tested for CMS using 45 crosses among 37 landraces and 17 annual wild soybean accessions (Glycine soja Sieb. et Zucc.). The cross of N23661 × N23658 showed partial to complete male sterility in backcross generations, while the corresponding reciprocal cross showed normal male fertility. Thus, the cytoplasm of N23661 is male‐sterile, the continuously backcrossed line is a male‐sterile line (designated NJCMS4A), and N23658 is its maintainer (designated NJCM4B). The male fertility of NJCMS4A was restored by another accession, Nansheng9403. Accordingly, NJCMS4A along with its maintainer and restorer composes a complete set of three lines for producing hybrid soybean. Using mitochondrial markers and sequence analyses, NJCMS4A is a CMS line with its cytoplasm not identical to the four previously reported CMS sources in soybean.     

Mapping a gene conferring resistance to <Emphasis Type="Italic">Wheat yellow mosaic virus</Emphasis> in European winter wheat cultivar ‘Ibis’ (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Wheat yellow mosaic, caused by Wheat yellow mosaic virus (WYMV), is one of the most devastating soil-borne diseases of winter wheat (Triticum aestivum L.) in Japan. Yellow-striped leaves and stunted spring growth, symptomatic of WYMV infection, result in severe yield loss. A new putative WYMV resistance gene in the European wheat cultivar ‘Ibis’ was mapped in the cluster of microsatellite markers including Xcfd16, Xwmc41, Xcfd168 and Xwmc181 on the long arm of chromosome 2D at the distances of 2.0 cM, 4.0 cM, 7.1 cM and 12.4 cM, respectively. WYMV-resistant cultivars contained a common haplotype of the four markers, whereas moderately susceptible and susceptible cultivars did not. These results should be useful in marker-assisted selection for WYMV resistance in wheat.     

Localization of Genes in Rye that Restore Male Fertility to Hexaploid Wheat with timopheevi Cytoplasm

Four sets of wheat-rye addition lines were screened to localize genes in rye that restore male fertility to hexaploid wheat with timopheevi cytoplasm. One gene, designated Rfc3, was physically located in the distal 40 % of the long arm of chromosome 6R. No allelic variation at Rfc3 was found; normal male fertility was consistently observed in all F₁ hybrid combinations tested. A second gene, designated Rfc4, was located on the long arm of chromosome 4R. Variation between chromosomes 4R in the level of restoration was observed; fertility in hybrids ranged from 0 % to about 50 % of normal. Attempts to genetically map Rfc4 were inconclusive but suggested it was located 16.1 cM from the telomere of the long arm and at least 8.0 cM from the centromere. These restorers, particularly Rfc3, may have potential in hybrid wheat breeding programs and can be manipulated for production of male sterile triticale lines.     

Factors responsible for levels of male sterility in photoperiod-sensitive cytoplasmic male sterile (PCMS) wheat lines

A `two-line system' using photoperiod-sensitivecytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm has been proposed as a newmeans of producing hybrid wheat. The PCMS line ismaintained by self-pollination under short-dayconditions (14.5 h light period), and F<sub>1</sub> seedscan be produced by outcrossing of the PCMS line witha pollinator under long-day conditions (15 h lightperiod). As the levels of male sterility in PCMSlines under the long-day conditions is a crucialfactor in determining hybrid purity of the F<sub>1</sub>seeds, a study was conducted into the effect ofseeding rate on male sterility in PCMS lines. Threedifferent density levels were tested using analloplasmic line of Japanese wheat cultivar `Norin 26'which exhibits PCMS. Levels of male sterility of thePCMS line increased at sparse planting, because tiller(ear) number per plant increased at low seedingdensity and late-appearing ears tended to exhibithigher levels of male sterility than early-appearingears. On the other hand, male sterility levels of thePCMS lines depended on genotype, e.g., the PCMS`Fujimikomugi' was completely male sterile, whereasthe PCMS `Norin 26' showed partial male sterility. APCMS line showing complete male sterility, such as thePCMS `Fujimikomugi', should produce F<sub>1</sub> seeds withhigh purity. However, the PCMS `Fujimikomugi' showeda lower female fertility. For practical use, it isnecessary to produce PCMS lines having high malesterility with high female fertility under long-dayconditions.