Haploid plant regeneration from unpollinated ovule cultures of niger (<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.)

Haploid plants were regenerated in vitro from unpollinated ovules of niger (Guizotia abyssinica (L. f.) (Cass.) on Murashige and Skoog nutrient medium (MS) supplemented with 10 μM naphthaleneacetic acid or 10 μM NAA + 1.5 μM kinetin and 30 g/l sucrose. Gamborg (B5) medium was the best for plant regeneration (in comparison with MS, Nitsch and Nitsch (NN), and Chu (N6) media) from cultured ovules, and 6.66 and 7.33 ovules of JNC-6 and Ootacamund cultivars were involved in direct plant regeneration on this medium. Matured ovules (ovules collected one day before anthesis or on the day of anthesis) only responded to cultural regimes and involved in direct plantlet development. Cytological preparation of root tips and chloroplast counts in the guard cells of leaf stomata of regenerated plants confirmed their haploid nature. This text was submitted by the authors in English.     

Aneuploidy and polyploidization in haploid tissue cultures of Larix decidua

Haploid embryogenic lines of Larix decidua which had been maintained on medium free of plant growth regulatiors were screened for chromosomal abnormalities. Chromosome squashes were prepared from fast-growing suspension culture. alternatively, a method using burst protoplasts was devised for improving chromosome spreads. One line showed polyploidization with chromosome mumbers ranging from haploid to tetraploid. This line is presently predominantly diploid. Aneuploidization also occurred. The most effective method for counting chromosomes was from burst protoplasts. It is recommended that long-term cultures of conifer tissues be checked for genetic stability.     

Functional Divergence of Multiple Duplicated Foxl2 Homeologs and Alleles in a Recurrent Polyploid Fish

Evolutionary fates of duplicated genes have been widely investigated in many polyploid plants and animals, but research is scarce in recurrent polyploids. In this study, we focused on foxl2, a central player in ovary, and elaborated the functional divergence in gibel carp (Carassius gibelio), a recurrent auto-allo-hexaploid fish. First, we identified three divergent foxl2 homeologs (Cgfoxl2a-B, Cgfoxl2b-A, and Cgfoxl2b-B), each of them possessing three highly conserved alleles and revealed their biased retention/loss. Then, their abundant sexual dimorphism and biased expression were uncovered in hypothalamic–pituitary–gonadal axis. Significantly, granulosa cells and three subpopulations of thecal cells were distinguished by cellular localization of CgFoxl2a and CgFoxl2b, and the functional roles and the involved process were traced in folliculogenesis. Finally, we successfully edited multiple foxl2 homeologs and/or alleles by using CRISPR/Cas9. Cgfoxl2a-B deficiency led to ovary development arrest or complete sex reversal, whereas complete disruption of Cgfoxl2b-A and Cgfoxl2b-B resulted in the depletion of germ cells. Taken together, the detailed cellular localization and functional differences indicate that Cgfoxl2a and Cgfoxl2b have subfunctionalized and cooperated to regulate folliculogenesis and gonad differentiation, and Cgfoxl2b has evolved a new function in oogenesis. Therefore, the current study provides a typical case of homeolog/allele diversification, retention/loss, biased expression, and sub-/neofunctionalization in the evolution of duplicated genes driven by polyploidy and subsequent diploidization from the recurrent polyploid fish.     

New microsatellite loci confirm hybrid origin,parthenogenetic inheritance,and mitotic gene conversion in the gynogenetic Amazon molly (Poecilia formosa)

We describe the first microsatellite loci for the gynogenetic Amazon molly, Poecilia formosa, an all‐female species arisen through hybridization of the bisexual species Poecilia mexicana and Poecilia latipinna. The loci showed one to six alleles and an expected heterozygosity between zero and 0.75. As expected with parthenogenetic inheritance, most loci were either constantly homozygous (five loci) or constantly heterozygous (eight loci). For six loci, both heterozygotes and homozygotes occurred. This and the fact that some loci only showed alleles of one of the ancestral species could indicate genome homogenization through mitotic gene conversion. Our new loci conformed to the hybrid origin of Amazon molly and are also applicable to both ancestral bisexual species.     

Masculinization of the gynogenetic juvenile ship sturgeon (Acipenser nudiventris Lovetsky, 1828) using 17α‐methyl testosterone

In sturgeons, the induction of gynogenesis and sex reversal could be important for potential production of neomale sturgeon and all‐female progeny for caviar production. The aim of this study was sex reversal of ship sturgeon (Acipenser nudiventris Lovetsky, 1828) gynogen into male sex. Five‐month‐old gynogens were sex reversed into male by including 17α‐methyl testosterone in their food for 7 months. Three treatments were considered as follows: (a) without treated (gynogen control), (b) 10 mg MT/kg diet, and (c) 50 mg MT/kg diet. All treatments (60 individuals) were sampled both the 30 and 36 months old and their sex was determined using classical histology method of gonad. The sex ratio of the progenies in the gynogen control were 73.3% female and 26.7% male. In treatment of 10 mg MT/kg feed, 18 specimens were studied that half of them (50%) showed pseudo‐testicular structure in the female gonad. That is 50% of the specimens were intersex, 27.7% were male and 22.3% were female. All of the fish fed by 50 mg MT/kg feed had been sex reversed to male. Sexual maturation of these fish had been recognized in stage III at 36 months old. In conclusion, 50 mg MT/kg feed found as effective dose for successful sex reversal in gynogenetic ship sturgeon.     

锦鲤4个人工雌核发育家系的微卫星标记研究

利用Crooijmans et al.(1997)分离的包含CA重复单元的普通鲤鱼（Cyprinus carpio L.)的8个微卫星DNA标记,对从锦鲤（Cyprinus carpio L.)的红白,大正和昭和3个不同品系中所获得的4个不同人工雌核发育家系的20尾个体进行PCR扩增。电泳结果表明,8对引物在20尾个体中均能重复稳定地扩增出相应的同源序列。随引物不同,各等位基因数为1－11个,大小在68－264bp。在MFW4,MFW7,MFW19,MFW20,MFW23和MFW24 6个微卫星的扩增结果中,20尾个体的扩增图谱呈现了高度的遗传多态性,不同雌核发育家系内个体的遗传异质性也较大。其中大正（TaS)和红白1（RW1）的个体不仅花色分化显著,而且个体间的平均遗传距离分别高达0．28。通过对微卫星等位基因和基因型分析发现,由于锦鲤品系中的每一个体是通过不断地杂交选育而获得,基因组来源复杂,基因高度杂合。因此,只进行1代的人工雌核发育,其家系内仅部分个体的部分座位出现纯合。所获得的人工雌核发育锦鲤为后续的色素遗传调控机制研究提供了必要的实验材料;同时,所鉴定的微卫星分子标记为进行锦鲤的分子标记育种的基因组作图提供了理想的工具。     

银鲫复合种外源遗传物质整入的RAPD分析

在优化ＲＡＤＰＤ检测条件的基础上,采用４０对随机引物,比较分析了银复合物,异育银鲫和兴国红鲤相互间扩增ＤＮＡ片段的异同性。总的来说,银鲫复合种和异育银鲫具有基本一致的扩增产物,而与兴国红鲤的扩增产物多数不同,相似率分析表明,银鲫复合种与兴国红鲤之间的相似率为３１．６％,异育银鲫与兴国红鲤之间的相似率为２８．６％。在分析中,除发现银鲫复合种,异育银鲫与兴国红鲤间共有扩增片段外,还发现了银鲫复合种与兴     

Flow cytometry for assessing the efficacy of interspecific gynogenesis induction in sturgeon

The efficacy of ploidy analysis for separating progeny of Siberian sturgeon Acipenser baerii after induced gynogenesis was demonstrated using sperm of a paternal species differing in ploidy level from the maternal species. Gynogenesis was induced in tetraploid A. baerii with UV‐C irradiated sperm from the diploid sterlet Acipenser ruthenus and vice‐versa. The success of sperm UV irradiation and diploidy restoration by heat‐shock was estimated based on the ploidy level of progeny, confirmed by microsatellite parentage assignment. Hatching rates of interspecific gynogenotes were comparable with rates reported for gynogenesis induction using sperm and eggs of the same species. Juvenile mortality was similar to that observed in the control hybrids. The efficiency and reliability of this method may foster its use for production of gynogenotes in aquaculture, potentially allowing interspecific gynogenesis to replace intraspecific.     

HYBRIDOGENESIS AND ANDROGENESIS IN THE STICK-INSECT BACILLUS ROSSIUS-GRANDII BENAZZII (INSECTA,PHASMATODEA)

In northwestern Sicily interspecific hybrid females between Bacillus rossius and B. grandii benazzii (Insecta, Phasmatodea) are sympatric with facultatively parthenogenetic demes of the former and bisexual populations of the latter. Preliminary observations suggested that hybrid females are maintained by hybridogenetic reproduction, not by current F₁ hybrid production nor through parthenogenesis. Being hybridogens, a complex of hemiclonal lineages, we informally refer to them as B. rossius-grandii benazzii, according to Schultz's proposal. In this study B. rossius-g. benazzii females were crossed with males of B. g. benazzii, B. g. grandii, B. g. maretimi, and B. rossius. Allozyme analysis of the progeny showed that the great majority of them were actually produced by hybridogenesis with a hemiclonal inheritance of the maternal B. rossius genotype (Br^m) and actual syngamy with a sperm from the fathering male, so that Br^m-gb^p, Br^m-gg^p, Br^m-gm^p, and Br^m-r^p offspring were obtained in the respective crosses. All-paternal progeny (androgenetics) were also produced (Bgb^pgb^p, Bgm^pgm^p, Br^pr^p) and two gynogenetic descendants were observed. Cytological investigations on virgin eggs that failed to hatch revealed in most of them a haploid-diploid blocked blastoderm; this rudimentary parthenogenesis appears to be an important prerequisite for further evolution of this hybridogen. Reproductive modes of descendants were also analyzed; although Br^m-g^p hybrids are still able to reproduce by hybridogenesis, a progressive disruption of the hybridogenetic-androgenetic system takes place in synthetic B. rossius (Br^m-r^p, Br^pr^p) and abundant thelytokous parthenogenetic offspring are obtained from females of androgenetic origin. The evolutionary role of these hybridogens appears to be linked to their shift towards parthenogenesis; this has apparently occurred in the southeastern Sicilian hybrid B. whitei (=B. rossius/g. grandii), which exhibits both hybridogenesis and parthenogenesis.     

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan. × Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blanco × Citrus sinensis L. Osb) is reported here for the first time.