N2O induces mitotic polyploidization in anther somatic cells and restores fertility in sterile interspecific hybrid lilies

Fertile plants undergoing male gametogenesis can be treated with nitrous oxide (N₂O) gas to obtain 2n male gametes. N₂O treatment is also expected to restore the fertility of interspecific hybrids through meiotic restitution or mitotic amphidiploidization. However, this technique has few applications to date, and it is un-known how N₂O treatment restores fertility in sterile hybrids. To establish optimal N₂O treatment conditions and determine its cytological mechanism of action, we treated various sized floral buds with N₂O gas at different anther developmental stages from fertile and sterile hybrid lilies. N₂O treatment using the optimal 1–4 mm floral buds induced mitotic polyploidization of male archesporial cells to produce 2n pollen in fertile hybrid lilies. In sterile hybrid lilies, N₂O treatment doubled the chromosome number in male archesporial cells followed by homologous chromosome pairing and normal meiosis in pollen mother cells (PMC), resulting in restoration of pollen fertility. Backcrossing the resultant fertile pollen to Lilium × formolongi produced many triploid BC₁ plants. Thus N₂O treatment at the archesporial cell proliferating stage effectively overcame pollen sterility in hybrid lilies, resulting in fertile, 2n pollen grains that could produce progeny. The procedure presented here will promote interspecific or interploidy hybridization of lilies.     

Mapping three new interspecific hybrid sterile loci between Oryza sativa and O. glaberrima

Hybrid sterility hinders the transfer of useful traits between Oryza sativa and O. glaberrima. In order to further understand the nature of interspecific hybrid sterility between these two species, a strategy of multi-donors was used to elucidate the range of interspecific hybrid sterility in this study. Fifty-nine accessions of O. glaberrima were used as female parents for hybridization with japonica cultivar Dianjingyou 1, after several backcrossings using Dianjingyou 1 as the recurrent parent and 135 BC₆F₁ sterile plants were selected for genotyping and deducing hybrid sterility QTLs. BC₆F₁ plants containing heterozygous target markers were selected and used to raise BC₇F₁ mapping populations for QTL confirmation and as a result, one locus for gamete elimination on chromosome 1 and two loci for pollen sterility on chromosome 4 and 12, which were distinguished from previous reports, were confirmed and designated as S37(t), S38(t) and S39(t), respectively. These results will be valuable for understanding the range of interspecific hybrid sterility, cloning these genes and improving rice breeding through gene introgression.     

Directional transfer of a multiple-allele male sterile line in Brassica campestris L. ssp. chinensis (L.) Makino var. rosularis Tsen et Lee

To produce hybrid seeds of Wutacai (Brassica campestris L. ssp. chinensis (L.) Makino var. rosularis Tsen et Lee), a “directional transfer program” was designed to breed the multiple-allele male sterile line of Wutacai. A multiple-allele male sterile line of Naibaicai (Brassica campestris L. ssp. chinensis L., S01) was used as the male sterile resource, and an inbred line of Wutacai (WT01) was used as the target line. Recurrent backcrossing was employed to transfer the male sterility and other botanical traits simultaneously, while the genotype was identified through test crossing. The male sterility was successfully transferred from S01 to WT01. A new male sterile line, GMS-3, with similar botanical traits to WT01, was bred. Four hybrid combinations were generated with GMS-3 as the female parent. One hybrid (C1) that contained the most desirable traits was developed from the new male sterile line.     

高粱A3细胞质雄性不育发生的细胞学观察及分析

以不育系A₃晋粱5号和相应保持系B₃晋粱5号为材料，对A₃不育系花药发育及雄配子形成过程进行了细胞学观察和分析。结果表明，与A₁、A₂型CMS不同，A₃ CMS孢母细胞减数分裂正常，从四分体形成到幼龄花粉粒发育阶段都未观察到不同于B₃晋粱5号的异常现象。A₃晋粱5号花药经石蜡包埋切片观察，在造孢细胞期、减数分裂前间期及减数分裂期，均未观察到异常现象，不育系花粉母细胞能完成正常的减数分裂过程，四分体正常游离，绒毡层发育正常。但在成熟的花药中，观察到所有的花粉粒皱缩、凹陷、变形。在开花前约1周内的花粉粒成熟期，不育系花粉粒蔗糖—淀粉代谢途径受阻，花粉粒壁上无淀粉粒沉积。A₃ CMS小孢子败育发生在小孢子形成晚期的花粉粒成熟期。     

Genetic mechanisms of postzygotic reproductive isolation: An epistatic network in rice

Products of interspecific crosses often show abnormal phenotypes such as sterility, weakness and inviability. These phenomena play an important role in speciation as mechanisms of postzygotic reproductive isolation (RI). During the past two decades, genetics studies in rice have characterized a number of gene loci responsible for postzygotic RI. I have identified 10 loci including three sets of epistatic networks in a single inter-subspecific cross (Oryza sativa ssp. indica × japonica). These results suggest that RI genes cause developmental dysfunction of vegetative and/or reproductive organs through a variety of molecular pathways. The latest molecular studies demonstrated that hybrid incompatibility is mainly due to deleterious interactions caused by species-specific mutations of two or more genes, mediated by proteins acting within the same molecular pathway. Because genetic interactions provide a perspective on gene function, epistatic networks are a key to the understanding of the molecular basis of postzygotic RI. In this review, I focus on recent progress in postzygotic RI studies in rice and discuss the evolutionary significance as well as implications for improving rice productivity.     

Plasmons and male sterility types in Solanum verrucosum and its interspecific hybrid derivatives

Summary Solanum verrucosum (2n=24) is easily crossable with many diploid species and haploids, but usually as a female partner only. All interspecific hybrids studied appeared male sterile due to interaction of S. verrucosum plasmons with genes from male parents. Seven male sterility types have been discovered in the investigations made on 21 S. verrucosum introductions and their interspecific hybrid derivatives. These are briefly discussed. S. verrucosum proves to possess at least four plasmons denoted as [Tr^s], [Ps^s], [Sv^s] and [ns^s]. Each produces a diagnostic type of male sterility in the presence of the specific plasmon-sensitive genes which are widely spread. The role of hybrid sterility as a barrier to gene exchange between populations is discussed.     

Molecular Mapping of a Pollen Killer Gene S29(t) in Oryza Glaberrima and Co-Linear Analysis with S22 in O. Glumaepatula

Two species in genus Oryza, O. glaberrima and O. glumaepatula, are valuable and potential sources of useful genes of interest for rice improvement. However, the hybrid sterility between O. sativa and these two species is a main reproduction barrier when transferring the favorable traits/genes to mbox{O. sativa.} To overcome it, the nature of hybrid sterility should be understood further. The objective in the report is to map a new hybrid sterility gene as a Mendelian factor from O. glaberrima and analyze the co-linear of hybrid sterility S loci mbox{between} mbox{O. glaberrima} and mbox{O. glumaepatula} via comparative mapping approach. A BC₂F₂ population, derived from a single semi-sterility plant of BC₂F₁ of WAB56-104/ WAB450-11-1-2-P41-HB (WAB450-6) //WAB56-104///WAB56-104 was employed to map this pollen killer in O. glaberrima since WAB450-6 is a progeny of interspecific hybrid between O. sativa and O. glaberrima. A new pollen killer locus, S29(t) in O. glaberrima, was identified and mapped to interval between SSR marker RM7033 (1.1 cM) and RM7562 (1.3 cM) on rice chromosome 2. Comparative mapping indicated that S29(t) closely corresponded to S22 which is also a pollen killer gene in O. glumaepatula and is tightly linked with RFLP marker S910 on the short arm of rice chromosome 2. The good co-linear between S29(t) and S22 implied that there might exist common (orthologous) hybrid sterility loci controlled the reproduction barrier among AA genome species of genus Oryza, which will contribute significantly to our understanding of speciation and operation of hybrid sterility between O. sativa and its AA genome relatives.     

Cytogenetic analysis of cytoplasmic male sterility in wheat line KTP116A and molecular mapping of two thermo-sensitive restoration genes

The cytoplasmic male sterile (CMS) wheat (Triticum aestivum L.) line KTP116A developed at Northwest A&F University, Yangling, China, was sterile at temperatures below 18 °C and fertile at temperatures above 20 °C during Zadok’s growth stages 45–52. The possibility of a two-line system has a promising future for hybrid wheat production. The present study describes morphological differences in pollen abortion behavior at different temperatures, and the genetics of the thermo-sensitive restorer gene(s) in line KTP116A. Cytological observations showed that abnormalities in development of male sterile anthers first appeared at the bi-nucleate stage. Only a few pollen grains go through the second mitosis to produce two sperm cells; most of those became abnormal pollen grains and shell structures without protoplast, confirming that the conversion from sterility to fertility in the CMS line was accompanied by changes in morphology and cytology. A BC₁ population of 198 plants from a cross of male-sterile KTP116A and male fertile F₁ TP116B/WM5-5 was developed to study the genetic control of thermo-sensitive sterility. Chi squared tests on data from back-crossed populations revealed that two recessive genes, designated rfv ₁ ^sp and rfv ₂ , were responsible for sterility of line KTP116A. Sixteen of 712 SSR markers were polymorphic between the parents and bulks. Four SSR markers, viz. Xgwm11, Xgwm18, Xgwm413 and Xbarc137, were linked to thermo-sensitive gene rfv ₁ ^sp on chromosome 1BS of T. spelta, and another four markers were linked to rfv ₂ located on chromosome 2A. This thermo-sensitive male sterile line can be used for production of experimental hybrids in order to test levels of heterosis.     

Genetical and morphological characterization of cold tolerance at fertilization stage in rice

Cold temperature during the reproductive phase leads to seed sterility, which reduces yield and decreases the grain quality of rice. The fertilization stage, ranging from pollen maturation to the completion of fertilization, is sensitive to unsuitable temperature. Improving cold tolerance at the fertilization stage (CTF) is an important objective of rice breeding program in cold temperature areas. In this study, we characterized fertilization behavior under cold temperature to define the phenotype of CTF and identified quantitative trait loci (QTLs) for CTF. A wide variation in CTF levels has been identified among local cultivars in Hokkaido, which is one of the most northern regions for rice cultivation in the world. Clear varietal differences in pollen germination, and pollen tube elongation due to cold temperature have been observed. These differences may confer a degree of CTF among this population. We conducted QTL analysis for CTF using 120 backcrossed inbred lines derived from a cross between Eikei88223 (vigorous CTF) and Suisei (very weak CTF). Three QTLs for CTF were identified. A clear effect by QTL, qCTF7, for increasing the level of CTF was validated using advanced progeny. These results will facilitate marker-assist selection for desirable QTLs for CTF in rice breeding program.     

Direct genotyping of single pollen grains of a self-compatible mutant of Japanese pear (Pyrus pyrifolia) revealed inheritance of a duplicated chromosomal segment containing a second S-haplotype

Radiation mutant 415-1, which is the first known diploid pollen-part self-compatible mutant of pears (Pyrus spp.), has a decreased ability to produce pollen. To determine whether the self-compatibility trait is associated with this defect, we directly analyzed the genotypes of individual pollen grains by using polymerase chain reaction amplification of DNA from single pollen grains. We isolated single pollen grains from 415-1 and succeeded in genotyping the S-RNase gene and three simple sequence repeat (SSR) markers in linkage group 17. Out of 173 individual pollen grains, 28 (16 %) were S-heteroallelic. These pollen grains had two alleles each of the S-RNase gene and of two linked SSR loci, all on a duplicated chromosomal segment, but only one allele of a non-duplicated locus farther away on the same chromosome. The segregation ratio of each marker in the pollen from 415-1 was approximately the same as that observed in outcross progeny. This suggests that the decrease in frequency of pollen with the duplicated S-haplotype occurred during meiosis or pollen formation, but that the probability of fertilization by S-heteroallelic pollen is equal to that of single-allelic pollen. However, the partial sterility in 415-1 can also be attributed to one or more unidentified lethal mutations unlinked to the duplicated segment encompassing the S-haplotype. Single-pollen genotyping can be used in a variety of applications in genetic research because in cases where all pollen genotypes are proportionately represented in the progeny, segregation ratios can be obtained without producing the next generation.     

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)。     

从携带S5n基因的水稻种质中发掘San、Sbn和Scn基因

花粉不育是籼粳杂种F₁优势利用的主要障碍之一。包括S_a、S_b和S_c等至少6个基因座位内的等位基因互作会引起花粉不育,这些座位上的中性等位基因可以克服不育性。所以,发掘和利用中性等位基因具有重要意义。本文用携带S₅ⁿ的水稻种质,分别与台中65及其携带花粉不育基因的一套近等基因系杂交,组配具有单个座位互作和多个座位同时互作的杂种F₁,首先通过观察杂种F₁的花粉育性并比较相应杂种F₁育性的差异,初步判断是否具有中性等位基因,然后,采用与S_a、S_b和S_c座位紧密连锁的分子标记对F₂植株基因型的分离进行检测,并分析其分离比例的符合度,确定存在中性等位基因的真实性。结果发现在所鉴定的6份材料中有2份(灰背子和Madhukar)同时携带S_aⁿ和S_bⁿ,3份(饭毫皮、秕五升和粤泰B)携带S_bⁿ,1份(Jackson)携带S_cⁿ。这些材料同时携带可克服杂种F₁胚囊不育和花粉不育的基因,是克服籼粳杂种F₁不育性的重要基因来源。     

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.     

Identification of four genes for stable hybrid sterility and an epistatic QTL from a cross between Oryza sativa and Oryza glaberrima

To further understand the nature of hybrid sterility between Oryza sativa and Oryza glaberrima, quantitative trait loci (QTL) controlling hybrid sterility between the two cultivated rice species were detected in BC₁F₁ and advanced backcross populations. A genetic map was constructed using the BC₁F₁ population derived from a cross between WAB450-16, an O. sativa cultivar, and CG14, an O. glaberrima cultivar. Seven main-effect QTLs for pollen and spikelet sterility were detected in the BC₁F₁. Forty-four sterility NILs (BC₆F₁) were developed via successive backcrosses using pollen sterility plants as female and WAB450-16 as the recurrent parent. Seven NILs, in which the target QTL regions were heterozygous while the other QTL regions as well as most of the reminder of the genome were homozygous for the WAB450-16 allele, were selected as the QTL identification materials. BC₇F₁ for the seven NILs showed a continuous variation in pollen and spikelet fertility. The four identified pollen sterility QTLs were located one each on chromosomes 1, 3, 7 and 7. Pollen sterility loci qSS-3 and qSS-7a were on chromosomes 3 and 7, respectively, which coincides with the previously identified S19, and S20, while loci qSS-1 and qSS-7b on chromosomes 1 and 7L appear distinct from all previously reported loci. An epistatic interaction controlling the hybrid sterility was detected between qSS-1 and qSS-7a.     

Cajanus platycarpus (Benth.) Maesen as the donor of new pigeonpea cytoplasmic male sterile (CMS) system

Cajanus platycarpus, a distantly related wild species, was successfully crossed with cultivated pigeonpea using embryo rescue and tissue culture techniques. Advance generation lines showed a range of desirable characters including cytoplasmic male sterility. A range of pigeonpea cultivars restored fertility and was maintained by a few lines including cultivar ICPL 85010. Clasmogamous flowers were observed in the male sterile lines. In such flowers anthers did not form di-adlephous bundle. Cytological analysis revealed that meiosis proceeded normally till the tetrad stage in those anthers with pollen grains. After which many of the pollen grains turned sterile. In the anthers with pollen grains, dehiscence was not observed, thus creating functional sterility. In many other anthers, pollen mother cells (PMCs) were not formed at all, giving rise to sepalous anthers. In conclusion two mechanisms of male sterility existed, one was premeiotic, where PMCs did not form and in the second, although PMCs gave rise to pollen grains, they were either partially or totally sterile accompanied by non-dehiscence of anther wall.     

Development of photoperiod- and thermo-sensitive male sterility rice expressing transgene Bacillus thuringiensis

Stem borers and leaffolders are the main pests that cause severe damage in rice (Oryza sativa L.) production worldwide. We developed the first photoperiod- and thermo-sensitive male sterility (PTSMS) rice 208S with the cry1Ab/1Ac Bacillus thuringiensis (Bt) gene, through sexual crossing with Huahui 1 (elite line with the cry1Ab/1Ac gene). The novel 208S and its hybrids presented high and stable resistance to stem borers and leaffolders, and the content of Cry1Ab/1Ac protein in chlorophyllous tissues achieved the identical level as donor and showed little accumulation in non-chlorophyllous tissue. No dominant dosage effect in the Bt gene was observed in 208S and its derived hybrids. An analysis of fertility transition traits indicated that 208S was completely sterile under long day length/high temperature, but partially fertile under short day length/low temperature. With fine grain quality and favorable combining ability, 208S had no observed negative effects on fertility and agronomic traits from Bt (cry1Ab/1Ac). Additionally, 208S as a male sterile line showed no fertility decrease caused by Bt transgenic process, as it is the case in Huahui 1. Thus, 208S has great application value in two-line hybrid production for insect resistance, and can also be used as a bridge material in rice Bt transgenic breeding.     

豫粳6号B与TR2604间杂种花粉不育基因的定位

阐明BT型杂交粳稻组合间育性差异的遗传基础有助于三系法杂交粳稻组合的选育。根据TR2604与豫粳6号A(B)、9201A(B)后代的花粉育性及小穗育性,明确了豫粳6号A(B)/TR2604 F₁不育由双亲间特异性不亲和造成。遗传分析表明豫粳6号A(B)与TR2604 F₁花粉不育受单基因S38(t)控制。以352株豫粳6号A/TR2604//TR2604、豫粳6号B/TR2604//豫粳6号B等群体中单株为定位群体,将S38(t)定位于第7染色体上标记RM18和RM234之间,与两标记遗传距离分别为0.43 cM和0.14 cM,两标记间物理距离为180 kb,相关结果为S38(t)图位克隆工作奠定了基础。     

Developing rice lines possessing neutral alleles at sterility loci to improve the width of compatibility

To improve the width of compatibility for overcoming various sterilities in inter‐subspecific hybrid rice, some elite lines combining several sterility‐neutral genes were developed and the effects on mitigating various hybrid sterilities were tested. From Akihikari// IR36/Dular, neutral genes at ga11 and six sterility loci, S5, S7, S8, S9, S15 and S16, were combined and elite lines were obtained in their successive progeny. Four of the lines tested were confirmed to combine the neutral alleles S5‐n, S7‐n, S8‐n, S9‐n, S15‐n and S16‐n at the sterility loci and, among them, two harboured an additional gamete abortion‐neutral allele, ga11‐n. F₁s, which used the lines and various testers as parents, mitigated the spikelet sterilities by six sterility loci and gamete abortion by a gametophyte gene, ga11. These lines could be selectively used as parents or donors to increase the width of compatibility of rice varieties for improving fertility in inter‐subspecific hybrid rice breeding.     

Production and characterization of an alloplasmic and monosomic addition line of Brassica rapa carrying the cytoplasm and one chromosome of Moricandia arvensis

Intergeneric hybridization was performed between Moricandia arvensis and four inbred lines of Brassica rapa following embryo rescue. Three F₁ hybrid plants were developed from three cross combinations of M. arvensis × B. rapa, and amphidiploids were synthesized by colchicine treatment. Six BC₁ plants were generated from a single cross combination of amphidipolid × B. rapa ‘Ko1-303’ through embryo rescue. One BC₂ and three BC₃ plants were obtained from successive backcrossing with B. rapa ‘Ko1-303’ employing embryo rescue. Alloplasmic and monosomic addition lines of B. rapa (Allo-MALs, 2n = 21) were obtained from backcrossed progeny of three BC₃ plants (2n = 21, 22 and 23) without embryo rescue. An alloplasmic line of B. rapa (2n = 20) degenerated before floliation on 1/2 MS medium due to severe chlorosis. Allo-MALs of B. rapa (2n = 21) showed stable male sterility without any abnormal traits in vegetative growth and female fertility. Molecular analyses revealed that the same chromosome and cytoplasm of M. arvensis had been added to each Allo-MAL of B. rapa. This Allo-MAL of B. rapa may be useful material for producing cytoplasmic male sterile lines of B. rapa.     

温光敏核不育小麦C412S的育性转换及其APRT基因的表达

以选育的小麦(Triticum aestivum L.)温光敏核不育系C412S为试材,以C412S回交转育时的受体亲本C412为常规品系对照,通过分期播种试验研究了其育性转换特性。用涂抹压片法观察减数分裂和小孢子发育进程,用半定量RT-PCR技术分析不育与可育条件下不同发育时期的幼穗中腺嘌呤磷酸核糖基转移酶基因(APRT)的表达水平。结果表明,通过调整播种期改变雄性发育的温光条件,C412S表现出完全不育—高不育—半不育—正常可育的育性转换特性。C412S花粉败育的高峰在单核小孢子晚期,主要表现圆败型不育。C412S的育性敏感期是从花粉母细胞形成期到成熟花粉期,其中最敏感的时段是花粉母细胞形成期到减数分裂期。与对照相比,C412S的APRT1基因序列有个别碱基变异,但编码氨基酸序列没有变化。在花粉母细胞形成期至单核期,与晚播可育条件相比,早播不育的C412S幼穗中APRT基因转录水平下调,因此认为,其育性转换与幼穗中APRT基因转录水平有一定关系。