Conversion of the genic male sterility (GMS) system of bell pepper (Capsicum annuum L.) to cytoplasmic male sterility (CMS)

Non‐pungent bell pepper (Capsicum annuum L.) lacks the cytoplasmic male sterility (CMS) nuclear restorer allele, Rf, and CMS cannot be employed in its F₁ hybrid seed production. To demonstrate that the genic male sterility (GMS) system in non‐pungent bell pepper can be converted to the CMS male sterility system, the conversion of GMS to CMS for non‐pungent bell pepper line GC3 was conducted by introgression of S‐type cytoplasm and the Rf allele from tropical pungent donors. After morphological traits were evaluated, two lines from BC₁F₁ containing S‐type cytoplasm and four lines from BC₂F₂ containing Rf allele, phenotypically similar to GC3, were obtained and could be employed as CMS male sterile lines and restorer lines for non‐pungent bell pepper. Four molecular markers potentially linked to traits of interest were also evaluated in BC₁F₁ and BC₁F₂ populations. This is the first time that GMS has been successfully converted to CMS in bell pepper, a significant contribution for bell pepper hybrid seed production.     

Modified complementation test of male sterility mutants in pepper (Capsicum annuum L.): preliminary study to convert male sterility system from GMS to CMS

The male sterility system in hybrid seed production can eliminate the cost of emasculation and ensure seed hybridity through avoidance of self pollination. GMS and CMS are two types of male sterility system that currently employed in pepper breeding. Conversion from GMS to CMS will increase the male sterility proportion of female parent from 50 to 100%. In this study, segregation analysis of four male sterile mutants consisting of one CMS mutant (CA1) and three GMS mutants (GA1, GA3 and GA4) showed that each had single recessive gene inheritance. A modified complementation test was performed by replacing male sterile mutants with their maintainer line as male parent. The nuclear restorer gene for CMS was independent of all nuclear restorer genes for GMS and all nuclear restorer genes for GMS were independent each other. Further observation on CMS and GMS male sterility loci revealed that GA1 and GA3 had mutated in both nuclear restorer genes for CMS and GMS, while CA1 and GA4 each carried mutation in single male sterility system of nuclear restorer gene for CMS and GMS, respectively. Conversion from GMS to CMS in the case of lines carried mutations in both sterility systems required only S-type cytoplasm donor, while lines carried mutation in single nuclear restorer gene for GMS required not only S-type cytoplasm but also rf allele donors. The important finding is the broader function of maintainer line in certain male sterility system that can be used as a maintainer or restorer line for other male sterility systems. We also confirmed that line CC1 is the general restorer for both CMS and GMS systems.     

Development of cytoplasmic-genic male sterility in safflower

An interspecific cross was made between Carthamaus oxyacantha and the cultivated species C. tinctorius to develop a cytoplasmic‐genic male sterility (CMS) system in safflower. C. oxyacantha was the donor of sterile cytoplasm. The 3: 1 segregation pattern observed in BC₁F₂ suggested single gene control with dominance of male‐fertility over male‐sterility. The information obtained from crossing male sterile X male fertile plants in BC₁F₃ and BC₁F₄ generations showed statistically significant single gene (1: 1) segregation for male sterility vs. male fertility. The results demonstrated that C. tinctorius possesses a nuclear fertility restorer gene and that a single dominant allele restored fertility (Rf) in progeny carrying CMS cytoplasm of C. oxyacantha. Male sterility occurred with the homozygous recessive condition (rfrf) in a sterile C. oxyacantha cytoplasm background and not in the normal cytoplasm of C. tinctorius. The genetic background of different restorer lines of C. tinctorius having normal cytoplasm did not effect fertility restoration. The absence of male sterile plants in C. tinctorius populations ruled out the possibility of genetic male sterility. Normal meiosis in F₁ and BC₁F₂ ruled out a cytogenetic basis for the occurrence of male sterility.     

A unique introgression from Moricandia arvensis confers male fertility upon two different cytoplasmic male-sterile lines of Brassica juncea

A Brassica juncea line carrying an introgression from Moricandia arvensis restored male fertility to two cytoplasmic male‐sterile (CMS) B. juncea lines carrying either M. arvensis or Diplotaxis catholica cytoplasm. Genetics of fertility restoration was studied in the F₁, F₂, F₃ and backcross generations of the cross between CMS and fertility‐restorer lines. No male‐sterile plants were found in F1‐F₃ generations of the cross between CMS [M. arvensis] B. juncea and the restorer. However, a 1: 1 segregation for male sterility and fertility was observed when the F1 was pollinated with non‐restorer pollen from a euplasmic line. These results clearly show that restoration is mono‐genic and gametophytic. In CMS lines carrying D. catholica cytoplasm, the restorer conferred male fertility to the F1 and showed 3: 1 and 1: 1 segregations for male fertility and sterility in F₂ and BC1 generations, respectively, indicating a monogenic, sporophytic mode of fertility restoration. The results were also supported by pollen stainability in the F1 which was about 65% in M. arvensis‐based CMS and >90% in D. catholica‐based CMS. The above results are discussed in the light of previous molecular studies which showed association between CMS and atpA in both systems.     

Genetic linkage between male sterility and non‐spiny trait in safflower (Carthamus tinctorius L.)

Genetic male sterility (GMS) exists naturally in safflower (Carthamus tinctorius L.). In the existing safflower GMS lines, sterile and fertile plants are distinguishable at flowering. This causes delay in fertile plants rouging and reduction in hybrid purity. In this investigation, a cross between a spiny GMS parent 13‐137 and a spiny non‐GMS parent ‘A1’ was effected. One sib cross, SC‐67, producing non‐parental‐type non‐spiny sterile and spiny fertile plants in F₃ was advanced to F₉ through sib crossing between non‐spiny sterile and spiny fertile plants. Mendelian digenic segregation was not observed for non‐spiny trait and male sterility. The results revealed strong linkage between these traits. The linkage was confirmed in F₂ generations of crosses between a non‐spiny marker‐linked GMS line (MGMS) and five elite lines. Male sterility–linked non‐spiny trait could distinguish sterile and fertile plants at elongation stage. The MGMS would be useful in production of pure F₁ hybrid seed and development of elite populations.     

Moricandia arvensis cytoplasm based system of cytoplasmic male sterility in Brassica juncea: Reappraisal of fertility restoration and agronomic potential

Cytoplasmic male sterility (CMS) system based on the cytoplasm from Moricandia arvensis (mori) was investigated for fertility restoration and agronomic potential. Fertility restorer gene for mori CMS was introgressed from cytoplasm donor species as all the evaluated Brassica juncea genotypes (155) acted as sterility maintainers. The allosyndetic pairing between M^a and the A/B genome chromosomes in the monosomic addition plants (2n= 18II + 1M^a) facilitated the gene introgression. Partial fertility restoration (43–52% pollen grain stainability) in F₁ hybrids and absence of segregation for male sterility in F₂ progenies suggested gametophytic control of fertility restoration. The pollen fertility in the F₁ hybrids was, however, sufficient to ensure complete seed set upon bag selfing. Introgression from M. arvensis also helped in correction of chlorosis associated with mori cytoplasm in CMS and fertile alloplasmic B. juncea plants. Yield evaluation of thirty F₁ hybrids having the same nuclear genotype but varied male sterilizing cytoplasms (mori, oxy, lyr, refined ogu), in comparison to respective euplasmic hand bred control hybrids, allowed an estimate of yield penalty associated with different CMS systems. It ranged from 1.8% to 61.6%. Hybrids based on cytoplasmically refined ogu were most productive followed by those based on cytoplasmically refined mori CMS. The male sterility systems emanating from somatic hybridization were found superior than those developed from sexual hybridization.     

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.     

Tournefortii male sterility system in Brassica napus. Identification, expression and genetic characterization of male fertility restorers

A germplasm collection of 152 diverse rapeseed accessions from Canada, China, France, India, Poland and South Korea was assayed for identifying new fertility restorers and sterility maintainers for a Tournefortii (tour) cytoplasmic male sterility (CMS) system in rape‐seed. Only 16 (10.5%) genotypes showed complete fertility restoration following hybridization with tour CMS line NE 409A. Notable among these were GSL 8851, GSL 8953, Mokpo # 9, Mali, Buk‐wuk‐13, Kuju‐27 and Mokpo # 84. As many as 78 (51.3%) genotypes were perfect maintainers of sterility, the remaining 58 (38.2%) genotypes were classified as partial maintainers. To study the inheritance of fertility restoration, 20 CMS (tour) rapeseed lines were crossed with the four best fertility restorers, namely GSL 8851, GSL 8953, Kuju‐27 and Mokpo # 9, to obtain F₂ and test cross populations. Segregation data indicated that fertility restoration for tour CMS was governed by two genes, of which, one is stronger than the other (χ²_12:3:1). Differences in gene interactions were also observed (χ²_9:3:4) which could be explained on the basis of influence of female parent genotypes/or modified expression of the restorer gene(s) in different genetic backgrounds. Tests of allelism indicated that the restorer genes present in the four restorers evaluated were allelic.     

Establishment of a random-mating population of 'Polima' CMS restorers in Brassica napus by use of dominant genie male sterility

Five restorers of ‘polima’ cytoplasmic male sterility (pol CMS) cannot restore the fertility in dominant genie male sterility (DGMS). A dominant male sterility gene from both, a DGMS line Rs l046AB and DGMS hybrid ‘Zhongza No. 3’, was successfully introduced into Polima cytoplasm. A random-mating population of pol CMS restorers was established by using many double-low pol CMS restorers as pollinators to cross continuously to the DGMS plants which had Polima cytoplasm.     

Cytological observation of anther structure and genetic investigation of a new type of cytoplasmic male sterile 0A193‐CMS in Brassica rapa L.

Cytoplasmic male sterility (CMS), a maternally transmitted failure in pollen formation, is an effective pollination control system in hybrid rapeseed (Brassica napus) breeding. However, CMS is not widely used in the related oilseed species Brassica rapa. In the past years, several male sterile plants have been isolated from the B. rapa landrace ‘0A193’, collected in Shaanxi, China, in 2011. It is noteworthy that the fertility expression of 0A193‐CMS was affected by temperature. In contrast to pol CMS, fertility tests with 18 B. rapa and 9 B. napus accessions suggest that a different system of maintaining and restoring is responsible for the observed phenotype. Further on, genetic investigation evidenced that fertility of 0A193‐CMS is controlled by both cytoplasmic and one pair of nuclear recessive genes. Interestingly, plants of the 0A193‐CMS type possess a highly specific fragment of the mitochondrial gene orf222, a crucial regulator of male sterility in nap CMS. Our study broadens the CMS resources in B. rapa and provides a highly applicable alternative to pol CMS and ogu CMS for hybrid breeding production.     

Maternal inheritance of male sterility in the progeny of a natural hybrid between Cajanus lineatus and C. cajan

Adoption of pigeonpea hybrids in central and southern India is showing high impact with on‐farm yield advantages of >30%. The hybrid pigeonpea technology, the first in any legume crop, is based on a cytoplasmic‐nuclear male‐sterility (CMS) system. For a long‐term sustainability of hybrid programme, it is imperative that both nuclear diversity and cytoplasmic diversity are maintained among hybrid parents. In this context, a continuous search for new CMS‐inducing cytoplasms is necessary. This paper reports detection of maternal inheritance of male sterility in the progeny derived from a natural hybrid between a wild relative [Cajanus lineatus (W. & A.) Maesen comb. nov.] of pigeonpea and an unknown pigeonpea [Cajanus cajan (L.) Millsp.] genotype. In the present study, the male sterility was maintained up to BC₇F₁ generation by an advanced breeding pigeonpea line ICPL 99044. This male sterility inducing cytoplasm of C. lineatus was tagged as A₆. In future, this CMS genetic stock can be used to develop a range of new pigeonpea hybrids with high yield and adaptation.     

Discovery of new male-sterile cytoplasm sources and development of a new cytoplasmic-nuclear male-sterile line NJCMS3A in soybean

Most of the cytoplasmic-nuclear male-sterile (CMS) lines of soybean were developed only from a limited cytoplasm sources and performed not as good as required in hybrid seed production, therefore, to explore new male-sterile cytoplasm sources should be one of the effective ways to improve the pollination and hybridization for a better pod-set in utilization of heterosis of soybeans. In the present study, total 80 crosses between 70 cultivated and annual wild soybean accessions and three maintainers (N2899, N21249, and N23998) of NJCMS1A were made for detecting potential new sources with male-sterile cytoplasm. The results showed that in addition to the crosses with N8855.1 (the cytoplasm donor parent of NJCMS1A) and its derived line NG99-893 as cytoplasm parent, there appeared three crosses, including N21566 × N21249 and N23168 × N21249, with male-sterile plants in their progenies. According to the male fertility performance of backcrosses and reciprocal crosses with the tester N21249, the landrace N21566 and annual wild soybean accession N23168 were further confirmed to have male-sterile cytoplasm. Accordingly, it was understood that the source with male-sterile cytoplasm in soybean gene pool might be not occasional. The results also showed that the genetic system of male sterility of the newly found cytoplasm source N21566 was different from the old cytoplasm source N8855.1, while N23168 was to be further studied. Based on the above results, the derived male-sterile plants from [(N21566 × N21249) F₁ × N21249] BC₁F₁ were back-crossed with the recurrent parent N21249 for five successive times, and a new CMS line and its maintainer line, designated as NJCMS3A and NJCMS3B, respectively, were obtained. NJCMS3A had normal female fertility and stable male sterility. Its microspore abortion was mainly at middle uninucleate stage, earlier than that of NJCMS1A and NJCMS2A. The male fertility of F₁s between NJCMS3A and 20 pollen parents showed that 7 accessions could restore its male fertility and other 13 could maintain its male sterility. The male sterility of NJCMS3A and its restoration were controlled by one pair of gametophyte male-sterile gene according to male fertility segregation of crosses between NJCMS3A and three restorers. The nuclear gene(s) of male sterility in NJCMS3A appeared different from the previously reported CMS lines, NJCMS1A and NJCMS2A. The development of NJCMS3A demonstrated the feasibility to discover new CMS system through choosing maintainers with suitable nuclear background.     

A simple method to control the seed purity of japonica hybrid rice varieties using PCR-based markers

Cytoplasmic male sterility (CMS) by the cms‐bo cytoplasm and its restoration by the nuclear restorer gene, Rf‐1, are used for seed production of japonica hybrid rice varieties. To produce pure hybrid seeds, a prerequisite is to properly manage the seed purity of parental lines, especially CMS lines. In this study, three dominant polymerase chain reaction (PCR)‐based markers (M1, M2 and M3) were developed to detect mutual contamination in seed batches of CMS lines, maintainer lines, restorer lines and hybrids. M1 detected the mitochondrial sequence that was present in the cytoplasm of common japonica varieties and absent in the cms‐bo cytoplasm. M2 and M3 detected the chromosomal sequence related to the Rf‐1 allele in restorer lines and the rf‐1 allele in common japonica varieties, respectively. By the strategic use of these markers, japonica hybrids and their parental lines could be efficiently distinguished from each other. Furthermore, sensitivity tests for the three markers with a series of crude DNA samples prepared from polished grains demonstrated that these markers could detect one contaminating grain among 500 or 1000 grains. Therefore, the bulk PCR analyses with the markers developed here probably make it possible to control the seed purity of japonica hybrids properly by selecting appropriate seed batches of their parental lines quickly and efficiently.     

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.     

Identification and transfer of a new cytoplasmic male sterility source from Oryza perennis into indica rice (O. sativa)

Summary Most of the commercial hybrids of indica rice are based on wild abortive (WA) source of cytoplasmic-genetic male sterility (CMS). Such cytoplasmic uniformity may lead to genetic vulnerability to disease and insect pests. To overcome this problem, diversification of CMS sources is essential. Crosses of 46 accessions of O. perennis and two accessions of O. rufipogon as female parents were made with two restorers (IR54, IR64) of WA cytosterility. Sterile hybrids were backcrossed with the respective recurrent parents. Of all the backcross derivatives, one line having the cytoplasm of O. perennis Acc 104823 and the nuclear background of IR64 was found to be stable for male sterility. The newly developed CMS line has been designated as IR66707A. This line is completely sterile (0% seed set) under selfed conditions. Crosses of IR66707A with 10 restorers of WA cytoplasm showed almost complete (93–100%) pollen sterility, indicating that the male sterility source of IR66707A is different from WA sterility. Southern hybridization of IR66707A, O. perennis (cytoplasmic donor), IR66707B (maintainer) and V20A (WA cytoplasm) using mitochondrial DNA specific probes (5 endonucleases × 8 probes) showed identical banding patterns between IR66707A and O. perennis. However, in more than half of the combinations, different banding patterns were observed between IR66707A and IR66707B and between IR66707A and V20A. The results suggest that IR66707A has the same cytoplasm as the donor (O. perennis), and CMS may not be caused by any major rearrangement or modification of mtDNA. The new CMS source identified will be useful in cytoplasmic diversification in hybrid rice breeding.     

Mitochondrial genome variation in Allium ampeloprasum and its wild relatives

Limitation in mitochondrial genome diversity of leek, revealed by restriction fragment length polymorphism (RFLP) analyses with mitochondrial gene probes, prevent a cytoplasmic male sterility (CMS) system in elite populations. However, mitochondrial genome diversity was detected in Allium ampeloprasum L. wild accession and landraces, as well as in pearl onion. Within this plant material, nine mitotypes were distinguished and could be used in order to broaden the genetic basis of leek. A chimeric mitochondrial gene configuration is usable as a marker for the sterility inducing cytoplasms (S₁) in chives (Allium schoenoprasum L.) and in onion (Allium cepa L.) for (S) and (T) cytoplasm. This chimeric mitochondrial gene configuration is also present in the subgenus Allium, revealed by polymerase chain reaction (PCR). However, only a faint amplicon was observed in a few accessions investigated herein, suggesting that this fragment might be present to a lesser level in mitochondrial DNA, as a sublimon.     

胞质雄性不育和核雄性不育辣椒内源激素含量的变化

采用酶联免疫吸附法(ELISA)测定和比较分析了CMS和GMS辣椒不育系(株)和相应保持系(可育株)花蕾和叶片中的内源IAA,Z ZR,GA3,ABA含量及比值的变化。结果表明,CMS雄性不育系和保持系花蕾中IAA,Z ZR,GA3和ABA含量的变化趋势与GMS不育株和可育株间变化趋势基本相同,即不育系(株)IAA,Z ZR,GA3和ABA含量低于保持系(可育株);叶片中Z ZR,GA3和ABA含量变化趋势GMS和CMS也基本一致,即不育系(株)中的含量高于保持系(可育株);花蕾中IAA/ABA,(Z ZR)/ABA,GA3/ABA激素比例的变化GMS与CMS也相同,即不育系(株)比值小于保持系(可育株)。说明虽然GMS和CMS辣椒控制雄性不育的基因不同,但在调节雄性不育时激素的变化规律大多相同,辣椒雄性不育在生理生化机理上可能存在一些相同的特点。     

Identification of a Stable Maintainer Line for 'Polima' Cytoplasmic Male Sterility in Rapeseed (B. napus L.)

‘Polima’ cytoplasmic male sterility (CMS) was transferred to three different genotypes of B. napus i.e. GSL-1, ISN706 and HNS-8 by repeated backcrossing and was found to be completely stable in the nuclear background of synthetic B. napus genotype, ISN706, derived from a cross B. campestris ssp. oleifera var. brown sarson ×B. oleracea var. botrytis cv. Tusa Katki'. The BC₅ and BC₆ generation lines of ISN706, GSL-1 and HNS-8 with ‘Polima’ CMS were grown under a range of temperature and photo-period conditions to test their stability. No breakdown in sterility was observed in ‘Polima’ ISN706 and, therefore, this genotype can be used as a maintainer for hybrid seed production.     

Detection and characterization of two new CMS systems in faba bean (Vicia faba) *

Wide crosses were made to identify new cytoplasmic male sterility (CMS) systems in faba beans, based on the interaction of cytoplasm with restorer and maintainer alleles. A total of 330 F₁ hybrids were produced in both reciprocal forms. Male sterile segregates were observed in one reciprocal version in the F₂ generation of six crosses. Two of these crosses with female parents originating from Afghanistan and Egypt expressed stable male sterility in subsequent backcross generations. Based on the female parents of the two crosses, these two CMS systems were designated CMS 199 and CMS 297. CMS 199 was more stable than CMS 297 during backcross generations and across different environments. Maintainer and restorer lines for both CMS systems were identified. Lower expression of male sterility occurred in CMS 297 in the greenhouse during the winter generations than in isolation cages during the summer generations, which may be utilized to maintain male sterile lines by selfing. Regarding practical applications, the CMS 199 shows great promise for hybrid breeding in faba beans.     

油菜野芥NSa细胞质雄性不育系的特异性分子鉴定

常规细胞质类型鉴定方法花费时间长、工作量大、鉴定效率低、应用性较差。基于基因组重复序列的PCR技术(rep-PCR)在重复序列丰富的线粒体和原核生物的基因组鉴定中效果较好, 本研究对8份油菜资源的细胞质进行分子鉴定,结果表明rep-PCR可以明确区分甘蓝型油菜中常见的6种雄性不育细胞质。同时对甘蓝型油菜和野芥(Sinapis arvensis)体细胞杂交创建的、育性较为稳定的NSa野芥雄性不育胞质进行了分子特异性鉴定,获得了两条NSa不育胞质的特征DNA条带。但波里马和萝卜质不育细胞质的特异性引物在NSa中不能扩增出特异性片段,说明NSa不属于这两种细胞质类型。本研究结果为该不育胞质系统的利用和知识产权保护提供了理论依据和技术支撑。