Identification of two SCAR markers co-segregated with the dominant Ms and recessive ms alleles in onion (Allium cepa L.)

Cytoplasmic genetic male-sterility is used to produce hybrid onion (Allium cepa L.) seeds worldwide. In this paper, we present the results of research aimed toward identifying PCR-based markers linked to the Ms locus through amplified fragment length polymorphism (AFLP). After screening 512 AFLP primer combinations, only one AFLP fragment was identified as being flanking linked to the dominant Ms allele. Subsequently, the AFLP marker was converted into a sequence-characterized amplified region (SCAR) marker, designated as DNF-566, co-segregated with the dominant Ms allele in first backcross (BC₁) segregated populations. Furthermore, we designed another molecular marker (RNS-357) co-segregated with the ms allele to identify different genotypes (i.e., MsMs, Msms, or msms). Both markers could be used for evaluating onion lines with different genetic backgrounds (including male-sterile lines, maintainer lines, male-fertile lines, and commercial based F₁ hybrid cultivars). The results of this study indicate that maintainer plants could be directly selected by using these 2 SCAR markers in the onion breeding process, and this may contribute significantly toward breeding onion F₁ hybrid cultivars.     

Limitations of PCR‐based molecular markers to identify male‐sterile and maintainer plants from Indian onion (Allium cepa L.) populations

The main aim of this study was to validate PCR markers for determining cytoplasm and genotypes at the Ms locus in short‐day onion. Three cytoplasmic (OSN, MKFR and accD) and four nuclear (OPT, jnurf13, AcSKP1 and AcPMS1) markers were employed. Sel. 121‐1 had 100% S cytoplasm, whereas Sel. 121‐2, ‘Pusa Red’ and ‘Pusa Madhavi’ had 88%, 33% and 17% S cytoplasm, respectively. ‘Early Grano’ and ‘Pusa Riddhi’ did not possess S cytoplasm. Observations in 33 commercial varieties revealed two with sterile (S) cytoplasm. Nuclear markers were not found in linkage disequilibrium with the Ms locus, and the constitution of Ms alleles by OPT was different from other three markers, which were in conformity with each other. The other three markers predicted that most of the plants should be homozygous recessive. Anther colour also did not confirm the sterility status. It can be concluded that accD may be used for cytoplasm determination based on the ease of its use. For the Ms locus tagging, more markers are needed to be evaluated to isolate maintainer lines from open‐pollinated populations.     

甘蓝型油菜隐性核不育系20118A的育性遗传及分子标记辅助选择

以20118A不育系和临保系与22个油菜品种(系)杂交测交后代为材料, 采用经典遗传学和分子标记辅助选择方法, 验证该不育系统遗传控制体系及等位基因分布频率; 探索利用连锁共显性标记筛选两型系和临保系基因型的高效性和准确率。研究表明, 6个品种(系)与20118A测交产生的F₂世代, 所有组合可育株∶不育株均符合3∶1或13∶3分离规律, 而与20118A-TAM杂交产生的6个F₂中1个呈13∶3分离, 其余均为全可育, 育性符合1对隐性不育基因和1对隐性上位抑制基因互作控制的遗传模式; 进而采取反向验证方法, 从1 059个F₂分离单株中, 用新发展的Bnms₃/Bnrf连锁共显性标记跟踪选择, 直接获得临保系(ms₃ms₃rfrf)、纯合不育株(ms₃ms₃RfRf)和两型系可育株(Ms₃ms₃RfRf) 70、69和135株, 经测交或互交验证, 准确率均达95%以上; 根据20个测交品种的后代分离, BnRf位点上只出现Rf和rf两个等位基因, 推测第3个等位基因存在的频率很低, 基本可以根据两基因各2等位基因互作原理开展分子标记辅助育种。     

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.     

水稻新质源(CMS-FA)雄性不育恢复基因的遗传研究

发掘水稻新型雄性不育细胞质源CMS-FA,育成系列优质米不育系和系列新质源恢复系,组配成强优势杂交稻组合的基础上研究新质源雄性不育恢复系的恢复基因遗传。采用新质源(CMS-FA)不育系金农1A与恢复系金恢3号杂交获得杂交F₁代种子,种植F₁代,收获自交F₂代种子。用F₁分别与不育系或保持系回交,获得(不育系//不育系/恢复系和不育系/恢复系//保持系)2个测交群体。同时种植P₁、P₂、F₁、F₂、B₁F₁和B₂F₁等群体,考察花粉染色率、套袋结实率和自然结实率,卡平方测验遗传分离适合度。结果表明,不育系与恢复系杂交F₁代正常可育,育性恢复(可育)基因为显性遗传。F₂代分离出可育︰不育适合3︰1,育性恢复(可育)基因为1对显性基因控制。B₁F₁和B₂F₁代2个测交群体的可育︰不育都适合1︰1分离规律,验证了F₂代育性恢复(可育)单基因的遗传模式。暂时确定新质源(CMS-FA)核质互作三系的基因型为不育系S(SS)、保持系F(SS)和恢复系S(FF)。     

Construction of high-resolution linkage map of the Ms locus, a restorer-of-fertility gene in onion (Allium cepa L.)

For the purpose of developing closely-linked molecular markers to the Ms locus, a restorer-of-fertility gene in onions (Allium cepa L.), bulked segregant analysis and randomly amplified polymorphic DNA (RAPD) analyses were utilized. Five RAPD markers polymorphic between male-fertile and male-sterile bulks were identified. These RAPD markers were converted into a simple PCR marker or cleaved amplified polymorphic sequence (CAPS) markers after sequencing the RAPD products and obtaining flanking sequences of the RAPD markers by genome walking. A linkage map was constructed with the Ms locus and flanking markers using a F₂ population. There was no recombinant between the Ms locus and two CAPS markers, jnurf05 and jnurf17. To increase resolution among these closely linked molecular markers and the Ms locus, a total of 1,346 F_2:3 and 2,927 F_2:4 plants were analyzed with two flanking markers for detection of recombinants. Segregation of male-fertility phenotypes in large-sized populations confirmed allelic segregation distortion in favor of the recessive Ms allele. Analysis of the recombinants with closely linked markers revealed only two recombinants between the Ms locus and the jnurf05 markers among 4,273 segregating plants, showing very tight linkage between the two loci. However, linkage disequilibrium between the two loci was not too strong among the breeding lines. Despite weak linkage disequilibrium, these tightly linked markers are useful in accurate marker-assisted selection of the Ms alleles and ultimate isolation of the Ms gene by map-based cloning approach.     

Inheritance of very high linoleic acid content and its relationship with nuclear male sterility in safflower

Safflower (Carthamus tinctorius L.) possesses the highest amount of linoleic acid among the 10 major vegetable oil crops of the world. Very high linoleic acid content is controlled by recessive alleles at a single locus Li. However, deviated segregations from the expected monogenic inheritance have been observed in crosses involving nuclear male‐sterile (NMS) lines. The present research was undertaken to study the inheritance of very high linoleic acid content in safflower and its relationship with nuclear male sterility. F₁, F₂, F₃, BC₁F₁ and BC₁F₂ seed generations were evaluated in a cross between CR‐142 (a line with very high linoleic acid content, 88%) and CL1 (an NMS line with wild‐type linoleic acid content, 74%). The genetics of linoleic acid content in male‐sterile plants was determined by testcrossing with CR‐142. The results confirmed monogenic inheritance. The analysis of the F₃ and BC₁F₂ to CL1 seed generations demonstrated a repulsion‐phase linkage between Li and Ms loci, the latter conferring the NMS trait. The recombination rate between Li and Ms was estimated to be 0.09.     

Development of simple PCR-based markers linked to the <Emphasis Type="Italic">Ms</Emphasis> locus,a restorer-of-fertility gene in onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.)

In order to implement reliable marker-assisted selection systems for the restorer-of-fertility locus (Ms) in onions (Allium cepa L.), simple PCR-based codominant markers linked to the Ms locus were developed. Based on the EST probe sequences of previously reported RFLP markers, full-length genomic sequences of the gene encoding putative oligopeptide transporter (OPT) was obtained by RACE. The first intron contained two 108 and 439-bp indel polymorphisms between the two Ms allele-linked OPT alleles. A simple PCR marker for OPT was developed by designing a primer pair on the flanking regions of the 108-bp indel which is created by two tandem repeats. The second simple PCR marker was developed from the EST probe encoding photosystem I subunit O (PsaO). Two 14 and 39-bp tandem repeats were identified from the 5′ upstream sequences of the PsaO-coding gene, which were isolated by genome walking. Three different compositions of these tandem repeats were identified from diverse onion germplasm. A primer set binding to the flanking sequence of these polymorphic repeats was used to amplify three different marker haplotypes. The OPT marker was tightly linked to the Ms locus at a distance of 1.5 cM, but the analysis of the linkage relationship showed little linkage disequilibrium between the marker and the Ms locus. Even so, these simple PCR markers are valuable tools for the marker-assisted selection of segregating individuals in onion F₁ hybrid breeding programs.     

Development of a cytoplasmic-nuclear male-sterile line of soybean

The F₁ of N8855 x N2899, a cross between two cultivars of G. max (L.) Merr., was found to be male-sterile. After it was back-crossed with the recurrent parent N2899 for four successive generations, a cytoplasmic-nuclear male-sterile line and its maintainer line, designated as NJCMS1A and NJCMS1B, respectively, were obtained. NJCMS1A was stable in male sterility with 98.29% plants being male-sterile at 0% pollen germination, while its female fertility was normal. The pollen abortion of NJCMS1A occurred at the binucleate stage. Then the vacuolation and disintegration of cytoplasm became further pronounced in NJCMS1A pollen. In addition, the abnormal phenomenon of 2 ∼ 3 styles surrounded by 20 ∼ 30 stamens was found in a few flowers of some male-sterile plants at BC₁F₁ and BC₂F₁. This revised version was published online in August 2006 with corrections to the Cover Date.     

大豆质核互作雄性不育系NJCMS3A双亲雄性育性基因的SSR标记

利用大豆质核互作雄性不育系NJMCS3A的质、核供体亲本N21566和N21249构建F₂和BC₁F₁育性分离群体进行雄性育性的遗传分析与基因定位。结果表明, F₁正反交可育,F₂和BC₁F₁的可育株与不育株分离比例经χ²测验分别符合3∶1和1∶1,表明NJCMS3A供体亲本雄性育性由一对基因控制,可育等位基因为显性。该基因可能是NJCMS3A的一个恢复基因。选用793对SSR引物对F₂和BC₁F₁群体分别进行育性基因定位,发现该育性基因位于O连锁群上,在Satt331和Satt477标记之间,与Satt331、CSSR133和Satt477标记距离的次序一致,分别为8.1~10.4 cM、11.4~16.4 cM、13.3~19.2 cM。     

Digenic nature of male sterility in pepper (Capsicum annuum L.)

Summary A cross was made between two nearly isogenic lines differing for male sterility genes, viz. ms₁ms₁Ms₂Ms₂ s Ms₁Ms₁Ms₂ms₂. F₁ plants yielded F₂ populations which segregated either in 3:1 or 9:7 ratios of fertile vs male sterile individuals. Test crosses between male sterile and male fertile sibs in the 9:7 segregating populations provided a few lines in which most of the progenies were male sterile. A 3:1 ratio model of male steriles vs fertiles is suggested and the value of the system is discussed.Contribution A.R.O. Agricultural Research Organization, The Volcani Center, Bet Dagan 50 250, Israel No. 3703-E, 1992 series.     

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.     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. IV. Ogura-type cytoplasm found in the wild radish, Raphanus raphanistrum

The orf138 gene, which is specific to Ogura male-sterile cytoplasm, was analysed in mitochondrial DNA (mtDNA) of the wild radish, Raphanus raphanistrum, by polymerase chain reaction (PCR), Southern hybridization and sequencing. The effect of R. raphanistrum cytoplasm on the expression of male sterility was also examined in progeny with R. sativus. A PCR-aided assay and Southern hybridization revealed that three out of six strains analysed included plants with orf138. The sequence of wild type orf138 was same as that of Ogura, except for one or two nucleotide substitutions. Southern hybridization showed a novel mtDNA configuration in R. raphanistrum, in addition to the normal and Ogura types identical to those in R. sativus. Among interspecific hybrids, all the F₁ had normal pollen fertility. In the F₂ progeny between female wild plants having orf138 and the maintainer of Ogura male sterility, male-sterile plants were segregated, fitting the ratio of 3 fertile: 1 sterile plant. R. raphanistrum has cytoplasm that induces male sterility in radishes, and contains a dominant fertility restorer gene.     

大葱雄性不育分子标记辅助选择的研究

大葱雄性不育在杂种优势利用中具有重要价值,传统方法选育不育系效率低,分子标记辅助育种可提高育种效率。本研究试图建立大葱雄性不育辅助育种的技术体系,加快大葱不育系、保持系的育种进程。利用RAPD技术分析了多态性片段S132800、S38960、S72300、S731100、S2002400与大葱育性的连锁关系,重组率分别为0、7．5％、0、4．2％、0。其中S132800、S2002400能在多数品种中区分N、S胞质,且重组率接近0,用于胞质鉴定具有很高的准确率,因而具有较高的利用价值。以S132800、S2002400为探针对不育系和保持系mtDNA的酶切片段进行了Southern杂交分析,结果表明它们在N、S胞质中存在多态性。预示着它们可能是与大葱CMS相关的片段。鉴于RAPD标记的应用有一定局限性,进一步对特异片段S132800、S2002400进行了克隆、测序和SCAR标记转化,其中S132800成功转化为SCAR标记,而S2002400转化后多态性消失。为进一步降低成本,简化了SCAR扩增产物的检测技术,初步建立了大葱不育系、保持系分子标记辅助选择的技术体系。研究表明,SCAR产物加入EB直接检测存在一定误差,而通过快速电泳可以快速、准确地鉴定单株的胞质类型。RAPD标记和SCAR标记在育种中应用有望提高大葱不育系、保持系的育种效率。     

Development of CGMS system in ridge gourd [<Emphasis Type="Italic">Luffa acutangula</Emphasis> (Roxb.) L.] for production of F<Subscript>1</Subscript> hybrids

Cytoplasmic male sterile system in ridge gourd has been converted to cytoplasmic genetic male sterile (CGMS) system through the development of analogues of male sterile (MS) line, maintainer line and fertility restorer line. These lines were developed by crossing the MS mutant, regenerated through in vitro culture, with monoecious pollen parents Deepthi, Haritham, LA 101, CO 2, IC 92761 and IC 92685. All hybrids and the BC₁ generation developed by crossing with the recurring pollen parents Deepthi, Haritham and LA 101 were male sterile. Male sterile BC₁ plants have been advanced to BC₆ generation and the parental line LA 101 was proved to be a successful maintainer line, producing male sterile progeny in successive back cross generations. Analogue of cytoplasmic male sterile line, MS LA 101, was developed through back crossing and on crossing with fertility restorer lines Arka Sumeet and LA 102, this line excelled as female parent, resulting heterotic combinations. Mitochondrial marker rpS14 and SCAR Tm-53 were identified to yield male sterility specific markers whereas SSR marker 18956 has generated the male fertility specific marker. These primers are recommended for marker assisted selection of ridge gourd, for utilizing male sterility for hybrid seed production and for developing A, B and C lines in CGMS system.     

Marker-assisted breeding for rf1, a nuclear gene controlling A1 CMS in sorghum (Sorghum bicolor L. Moench)

A study on marker-assisted selection (MAS) for the rf1 gene, which controls pollen sterility in the sorghum A1 cytoplasm, was conducted on the offspring population of two crosses between a maintainer line, BTx-622, and two sweet sorghum lines, BJ-299 and Lunen-2, to test the effectiveness of the MAS method and develop maintainer lines with sweet and juicy stalks and corresponding cytoplasmic-nuclear male sterility (CMS) lines. The simple sequence repeat marker Xtxp18 exhibited a high accuracy (95.098 %) for selecting recessive homozygotes for the rf1 gene. The segregation ratio matched the expected ratio calculated according to the reported genetic distance in the F₂ population of the two crosses used. Finally, four excellent maintainer lines/CMS line pairs (F₅/BC₃) with high stalk juice and stalk juice sugar contents were developed. The MAS method based on Xtxp18 for the sorghum rf1 gene could be used for hybrid breeding programs at a low cost in the future.     

Identification of Maintainer Genes in Brassica napus L. for the Male-Sterility-Inducing Cytoplasm of Diplotaxis muralis L.

Male fertility of F¹ interspecific hybrid plants derived from crosses between cytoplasmic male-sterile Brassica campestris in Diplotaxis muralis cytoplasm and 147 B. napus cultivars was Investigated. F¹, plants obtained, from crosses with the B. napus cultivars‘Mangum’and‘Hinchu’were male-sterile while F¹ plants derived from all other crosses were male-fertile. This indicated that these two cultivars carried maintainer genes far the male-sterility-inducing cytoplasm of D. muralis. Sterility was stable In plants derived from backcrosses of male-sterile F; plants with‘Mangun and‘Hinchu’but the seed set of backcross plants was low. With restorer genes readily available in B. napus, these findings could lead to the development of a new cytoplasmic male sterility system for the breeding of B. napus hybrid cultivars.     

洋葱雄性不育恢复位点Ms座位的SSR标记开发及其应用

本研究旨在开发稳定可靠的洋葱育性位点分子标记,并将其应用于育种实践,筛选育种系,从而缩短育种周期,节省育种成本,加速洋葱杂交种的培育进程。以洋葱育性恢复Ms座位的AFLP序列为基础,采用序列比对分析、PCR检测和表型分析等方法,开发、鉴定、应用了WH-SSR-1分子标记。结果表明:WH-SSR-1标记与Ms位点紧密连锁,Ms位点基因型为纯合显性MsMs时,有1条102 bp的扩增带;纯合隐性msms时,有1条99 bp的扩增带;杂合Msms时,有102 bp和99 bp两条扩增带。32份洋葱材料的验证结果证实了Ms座位的标记类型与基因型完全相符。随后从4个OP群体中直接获得了2个配套的不育系与保持系,‘吊玉’和‘天正红玉’因未检测到保持株或不育株,无法简单实现配套。WH-SSR-1标记与Ms位点紧密连锁,能够将其用于育种系筛选的育种实践,从OP群体中直接选择保持株和不育株,同时实现两系配套。     

Assessment of purity of rice CMS lines using cpDNA marker

AFLP technique was used to analyse the polymorphism between rice cytoplasmic male sterility (CMS) line Jin2A and its maintainer Jin2B. A stable differential band was discovered, and sequence analysis showed that Jin2A contained a more tandem repeat of 6 base pairs (AGAAAA) than Jin2B. Further studies confirmed that the diversity came from cpDNA and occurred at three kinds of abortive cytoplasmic genotypes. Accordingly, specific primers were designed and utilized to assess the purity of rice CMS lines during multiplication with pollen fertility and seed setting rates of bagged panicles as control. The result indicated that this cpDNA locus could be utilized to precisely distinguish maintainer plants from rice CMS lines. PCR analysis was consistent with that from Grow-out test in CMS line seed purity assessment during multiplication, despite it was helpless in distinguishing F₁ hybrids from CMS lines due to similar cytoplasms. Because of fewer hybrid and more maintainer off-plants, this cpDNA locus was still appropriate for seed purity assessment of rice CMS line during multiplication. This is first report that a marker on cpDNA could be utilized to assess the genetic purity of rice CMS lines with three abortive cytoplasmic genotypes.     

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.