Fertilization and Uniparental Chromosome Elimination during Crosses with Maize Haploid Inducers

Producing maternal haploids via a male inducer can greatly accelerate maize (Zea mays) breeding process. However, the mechanism underlying haploid formation remains unclear. In this study, we constructed two inducer lines containing cytogenetic marker B chromosome or alien centromeric histone H3 variant-yellow fluorescent protein vector to investigate the mechanism. The two inducer lines as the pollinators were crossed with a hybrid ZhengDan958. B chromosomes were detected in F1 haploids at a low frequency, which was direct evidence to support the occurrence of selective chromosome elimination during haploid formation. We found that most of the inducer chromosomes were eliminated in haploid embryonic cells during the first week after pollination. The gradual elimination of chromosomes was also detected in the endosperm of defective kernels, although it occurred only in some endosperm cells as late as 15 d after pollination. We also performed a genome-wide identification of single nucleotide polymorphism markers in the inducers, noninducer inbred lines, and 42 derived haploids using a 50K single nucleotide polymorphism array. We found that an approximately 44-Mb heterozygous fragment from the male parent was detected in a single haploid, which further supported the occurrence of paternal introgression. Our results suggest that selective elimination of uniparental chromosomes leads to the formation of haploid and possible defective kernels in maize as well, which is accompanied with unusual paternal introgression in haploid cells.Doubled haploid (DH) technology is widely used in maize (Zea mays) breeding. Although the life cycle of most sexually reproducing plant species alternates between a diploid sporophytic phase and a highly reduced gametophytic haploid phase, it is possible to obtain haploid plants containing the same number of chromosomes in their somatic cells as do the normal gametes of the species (Dunwell, 2010). Haploids can be obtained by in vitro or in vivo approaches. Anther and microspore culture are the most commonly used in vitro approaches; however, many species and genotypes are recalcitrant to these processes (Forster et al., 2007). Interspecies cross, which is an in vivo approach, induces haploids via chromosome elimination (Forster et al., 2007). For example, the cross between Hordeum vulgare and Hordeum bulbosum produces haploids in barley (Kasha and Kao, 1970; Sanei et al., 2011); maize and pearl millet (Pennisetum glaucum) are used as pollinators to produce haploids in wheat (Triticum aestivum; Laurie and Bennett, 1988; Gernand et al., 2005). There are two approaches for in vivo haploid induction in maize. One is using an ig mutant to generate both maternal and paternal haploids (Kermicle, 1969; Evans, 2007), and the other is using Stock6-derived inducers to produce maternal haploids only. The application of Stock6-derived inducers has become the foundation for modern DH technology (Prigge and Melchinger, 2012; Xu et al., 2013). The haploid induction rate (HIR) of inducer line Stock6 is 1% to 2% (Coe, 1959), which is 10 to 20 times higher than the spontaneous HIR in maize. The haploid-inducing capacity of inducers can be improved by selection (Sarkar et al., 1972). New inducers with improved HIRs have been developed, such as WS14 (2%–5% induction rate; Lashermes et al., 1988), RWS (Röber et al., 2005), and CAU5 (Xu et al., 2013).The first haploid inducer line Stock6 was discovered 50 years ago, and DH technology based on in vivo induction of maternal haploids has been widely used in maize breeding (Geiger, 2009). The mechanism underlying haploid formation remains unclear. Two hypotheses have been proposed for the mechanism. The first hypothesis is that one of the two sperm cells fails to fuse with an egg cell, but instead triggers haploid embryogenesis. In the second hypothesis, the two sperm cells fuse with an egg cell and a central cell, and the chromosomes from the inducer degenerate and are eliminated stepwise in the primordial cells during subsequent cell divisions.Evidence supporting the first hypothesis was reported by Bylich and Chalyk (1996). They found that 6.3% of the pollen grains of the ZMS haploid inducer line have two sperms showing different morphology. Thus, they proposed that the morphological defects of one of the sperms interfere in the sperm’s function, causing single fertilization. Another abnormality of inducer lines was described by Chalyk et al. (2003). They found that the frequency of aneuploid microsporocytes is much higher in haploid inducers than that in normal maize.Wedzony et al. (2002) studied the ovaries of inducer line RWS during the first 20 d after self-pollination. They found that about 10% of the embryos contain micronuclei with various sizes in the cytoplasm of every cell of shoot primordium. Their result supports the second hypothesis, which is selective chromosome elimination. Moreover, Fischer (2004) speculated that maternal haploids might possess small fractions of inducer genome. Consistently, previous studies (Zhang et al., 2008; Li et al., 2009) demonstrated morphological and molecular evidence for paternal DNA introgression in haploids, indicating the mechanism of chromosome elimination.Robust evidence to support either hypothesis is still missing due to lack of cytogenetic makers to trace chromosomes from inducers. In this study, we used B chromosomes and centromeric histone H3 variant (CENH3)-yellow fluorescent protein (YFP) as cytogenetic markers to discover direct evidence supporting selective chromosome elimination in both embryo and endosperm during haploid formation. In addition, we performed a genome-wide identification of single nucleotide polymorphism (SNP) markers in inducers, noninducer inbred lines, and 42 haploids using a 50K SNP array and detected unusual DNA introgression from inducer lines during haploid formation.     

Comprehensive Chromosome End Remodeling during Programmed DNA Elimination

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Genetic Control of Chromosome Pairing in Hexaploid Oats

THE common oat, Arena sativa, is a hexaploid (2n=6x=42) whose chromosomes form exclusively bivalents during meiosis. The bivalents are formed by the pairing of exact homologues resulting in disomic inheritance. Gene duplications and triplications were revealed by early inheritance studies¹ and homoeologous relationships of the three genomes were later supported by the good tolerance of deficiency for single and for pairs of chromosomes².     

Mitotic Chromosome Loss in a Disomic Haploid of SACCHAROMYCES CEREVISIAE

Experiments designed to characterize the incidence of mitotic chromosome loss in a yeast disomic haploid were performed. The selective methods employed utilize the non-mating property of strains disomic for linkage group III and heterozygous at the mating type locus. The principal findings are: (1) The frequency of spontaneous chromosome loss in the disome is of the order 10^-4 per cell; this value approximates the frequency in the same population of spontaneous mitotic exchange resulting in homozygosity at the mating type locus. (2) The recovered diploids are pure clones, and thus represent unique events in the disomic haploid. (3) Of the euploid chromosomes recovered after events leading to chromosome loss, approximately 90% retain the parental marker configuration expected from segregation alone; however, the remainder are recombinant for marker genes, and are the result of mitotic exchanges in the disome, especially in regions near the centromere. The recombinant proportion significantly exceeds that expected if chromosome loss and mitotic exchange in the disome were independent events. The data are consistent with a model proposing mitotic nondisjunction as the event responsible for chromosome loss in the disomic haploid.     

The Formation of Ribulose Diphosphate Carboxylase Protein during Chloroplast Development in Barley

Ribulose 1,5-diphosphate carboxylase is synthesized in barley leaves growing in the dark. Upon illumination there is a marked increase in the rate of synthesis of the enzyme. The specific activity of the enzyme expressed as cpm incorporated into phosphoglyceric acid per μg of fraction I protein, after isolation shows no change either during dark growth or greening. During early stages of illumination of 7 day dark grown leaves with 320 foot-candles the enzymic activity in the water soluble protein fraction of the leaf shows a short term decline after 15 min which lasts for 30 min. Leaves greening at 2 foot-candles show a similar decline which is shifted to a time between the fourth and eighth hr after the onset of illumination.     

Association between Rdna Alleles and Quantitative Traits in Doubled Haploid Populations of Barley

Doubled haploids (DH) were generated from reciprocal F1 hybrids which were heterozygous for alleles at the Nor-H3 locus on chromosome 5H of barley. The r-DNA alleles did not deviate significantly from the expected 1:1 ratio and the DH progenies were classified into two groups based on the allelic constitution of the Nor-H3 locus. The DHs were grown in a randomized, replicated field experiment and a range of agronomic and quality traits were recorded. The Nor-H3 locus was associated with a significant portion of the genetic variation for: yield, thousand corn weight, water sensitivity and milling energy requirement of the grain. However, the magnitude of the differences between groups was dependent on the direction of the cross. The milling energy requirement of the grain was consistently associated with alleles at the Nor-H3 locus. These results are presented in relation to the dynamics of rDNA evolution and variability. The potential of molecular markers in conjunction with doubled haploids to map quantitative traits in barley is also discussed.     

对用球茎大麦法诱导的大麦加倍单倍体及其后代表现的研究

用栽培大麦１０个品种间杂交组合的Ｆ１代与球茎大麦３个品系杂交,获得胚乳退化的杂效种子４８６７粒,解剖出幼胚２７６２个,再生植株４７６棵,成活植株３８２棵,加倍单倍体结实植株１２９棵,并对这１２９棵植株进行了后代观察。结果表明,杂交结实率可达９６。０％;再生植株加倍处理结实率在与球茎大麦ＰＢ１的组合中平均为６５。６％,与Ｃｂ２９２０／４的组合中平均为６１．３％,与２ｘ的组合中平均为２．９％。     

Genetic Control of Chromosome Synapsis in Mice Heterozygous for a Paracentric Inversion

Frequencies of formation of inversion loops and their relative sizes were studied in laboratory mice heterozygous for paracentric inversion In1(1)Rk in chromosome1, depending on the genetic background. Homozygotes In1/In1 were crossed with mice from five inbred strains (A/HeJ, BALB/cJ, C3H/HeJ, C57BL/6J, DBA2/J). The frequency of formation of inversion loops, their relative sizes, and the dependence of these parameters on the stage of pachytene were analyzed on electron-microscopic slides of spread spermatocytes in first-generation hybrids. It was shown that the genetic background and cross direction statistically significantly influenced the duration of individual pachytene stages and the frequency of inversion loops, but not relative loop size. Using a database on SNP distribution in the inbred strains examined, we carried out in silico mapping of genes affecting the genotype-dependent characters. We have found that the efficiency of synapsis in the inversion does not depend on interstrain differences in homology of the chromosome 1 region involved in the inversion. Genes controlling the inversion loop frequency in the inversion heterozygotes were mapped to chromosome 7, and genes controlling the duration of individual pachytene stages, to chromosomes 2 and 5.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 746–752.Original Russian Text Copyright © 2005 by Borodin, Ladygina, Rodionova, Zhelezova, Zykovich, Axenovich.     

Association of Chromosome Loss with Centromere-Adjacent Mitotic Recombination in a Yeast Disomic Haploid

Experiments designed to characterize the association between disomic chromosome loss and centromere-adjacent mitotic recombination were performed. Mitotic gene convertants were selected at two heteroallelic sites on the left arm of disomic chromosome III and tested for coincident chromosome loss. The principal results are: (1) Disomic chromosome loss is markedly enhanced (nearly 40-fold) over basal levels among mitotic gene convertants selected to arise close to the centromere; no such enhancement is observed among convertants selected to arise relatively far from the centromere. (2) Chromosome loss is primarily associated with proximal allele conversion at the centromere-adjacent site, and many of these convertants are reciprocally recombined in the adjacent proximal interval. (3) Partial aneuploid exceptions provisionally identified as carrying left arm telocentrics have been found. A testable model is proposed suggesting that centromere involvement in genetic recombination may precipitate segregational disfunction leading to mitotic chromosome loss.     

大麦胶含量的遗传分析

本文通过Hayman等人的双列分析系统对籽粒大麦胶含量进行了遗传研究。结果表明,控制大麦胶含量的基因系统符合加性-显性模型。大麦胶含量受微效多基因控制,加性效应起着主要的作用,也存在显性效应,而上位效应不明显。显性方向不十分一致,但总的趋势是低大麦胶含量为部分显性。环境对大麦胶含量有一定影响,但基因型—环境互作效应不显著。大麦胶含量与淀粉含量呈负相关(P<0.01);与株高呈弱正相关(P<0.05);而与千粒重、蛋白质含量无相关。     

应用基因组原位杂交及RFLP标记鉴定小麦中的大麦染色体

用生物素（Biotin-6-dUTP)标记的大麦Betzes基因组DNA作探针,以普通小麦中国春总DNA作封阻进行基因组原位杂交（Genomeinsituhybridization,简称GISH）,从13株小麦-大麦杂交后代中鉴定出2个含有3条大麦Betzes2H染色体的材料（2n＝43）;2个2H单体异代换系（2n＝42）;7个2H二体异代换系（2n＝42）。用已定位在小麦第2部分同源群短臂上的探针psr131进行RFLP分析,结果表明大麦Betzes、代换系A5有1条区别于小麦中国春的特异带,A     

The Genetic Control of Apomixis: Asexual Seed Formation

Apomixis (asexual seed formation) is the result of a plant gaining the ability to bypass the most fundamental aspects of sexual reproduction: meiosis and fertilization. Without the need for male fertilization, the resulting seed germinates a plant that develops as a maternal clone. This dramatic shift in reproductive process has been documented in many flowering plant species, although no major seed crops have been shown to be capable of apomixis. The ability to generate maternal clones and therefore rapidly fix desirable genotypes in crop species could accelerate agricultural breeding strategies. The potential of apomixis as a next-generation breeding technology has contributed to increasing interest in the mechanisms controlling apomixis. In this review, we discuss the progress made toward understanding the genetic and molecular control of apomixis. Research is currently focused on two fronts. One aims to identify and characterize genes causing apomixis in apomictic species that have been developed as model species. The other aims to engineer or switch the sexual seed formation pathway in non-apomictic species, to one that mimics apomixis. Here we describe the major apomictic mechanisms and update knowledge concerning the loci that control them, in addition to presenting candidate genes that may be used as tools for switching the sexual pathway to an apomictic mode of reproduction in crops.     

Genetic Control of the Formation and Reorganization of Chromocenter in Drosophila

The chromocenter integrates the entire Drosophilagenome into a unit. The formation and reorganization of chromocenter are genetically determined. Currently, several mutations affecting the structure of chromocenter have been described. In this work, I present evidence on the time of the formation and reorganization of chromocenter in mitotic and meiotic cells of females of the wild type and the ff16mutant line obtained by selection of mosaic clones produced from mitotic recombination of chromosomes in the dividing embryo cells. In females homozygous for this mutation, the second stage of the formation of chromocenter (joining two groups of nonhomologous chromosomes X-4 and 2-3 into a united ring structure =X=2=3=4=) is disturbed. The differences between the mitotic and meiotic reorganization of chromocenter and the role of chromocenter in the control of chromosome segregation are discussed.     

Genetic Analysis of Chemosensory Control of Dauer Formation in Caenorhabditis Elegans

Dauer larva formation in Caenorhabditis elegans is controlled by chemosensory cells that respond to environmental cues. Genetic interactions among mutations in 23 genes that affect dauer larva formation were investigated. Mutations in seven genes that cause constitutive dauer formation, and mutations in 16 genes that either block dauer formation or result in the formation of abnormal dauers, were analyzed. Double mutants between dauer-constitutive and dauer-defective mutations were constructed and characterized for their capacity to form dauer larvae. Many of the genes could be interpreted to lie in a simple linear epistasis pathway. Three genes, daf-16, daf-18 and daf-20, may affect downstream steps in a branched part of the pathway. Three other genes, daf-2, daf-3 and daf-5, displayed partial or complex epistasis interactions that were difficult to interpret as part of a simple linear pathway. Dauer-defective mutations in nine genes cause structurally defective chemosensory cilia, thereby blocking chemosensation. Mutations in all nine of these genes appear to fall at a single step in the epistasis pathway. Dauer-constitutive mutations in one gene, daf-11, were strongly suppressed for dauer formation by mutations in the nine cilium-structure genes. Mutations in the other six dauer-constitutive genes caused dauer formation despite the absence of functional chemosensory endings. These results suggest that daf-11 is directly involved in chemosensory transduction essential for dauer formation, while the other Daf-c genes play roles downstream of the chemosensory step.     

三个小黑麦花粉株系的染色体组成分析与抗白粉病鉴定

对来自小黑麦与小麦杂种的３个花粉株系,ＤＨ２２０－４,ＤＨ２２０－５和ＤＨ２２０－１４进行了形态性状观察,染色体组成分析和抗白粉病鉴定。经过染色体形态和数目观察、原位杂交、Ｃ－分带、同工酶等电聚焦和贮藏蛋白的ＳＤＳ－聚丙烯酰胺凝胶电泳分析,证明其中两个株系,ＤＨ２２０－４和ＤＨ２２０－５是６Ｒ／６Ｄ代换系,另一个株系ＤＨ２２０－１４是１Ｒ／１Ｄ代换系。经人工接种鉴定,两个６Ｒ／６Ｄ代换系高抗白粉病。从而进一步证明黑麦的６Ｒ染色体上存在抗白粉病的基因。同时还对小麦遗传背景下异源染色体的识别及６Ｒ染色体的利用价值等问题进行了讨论。