Mating patterns and pollen dispersal in a Japanese larch(Larix kaempferi) clonal seed orchard: a case study

Pollination dynamics highly determines the genetic quality of seed orchard crops. However, there is less research about the effect of mating patterns on seed productivity of orchard crops. So far, clonal seed orchards have been producing genetically improved seedlings used for most Japanese larch(Larix kaempferi(Lamb.) Carr.) plantations in China. In the present study, a total of 17 highly variable simple sequence repeat(SSR) markers were used for genotyping a progeny trial population consisting of 647 open-pollinated progenies germinated from seeds which were collected from 63 maternal clones with 140 potential paternal clones in a Japanese larch clonal seed orchard in China. Paternity analysis was used in the present case study in order to evaluate the level of paternal gametic contribution, estimate pollen contamination and selfing rates, and investigate pollination patterns,pollen dispersal patterns and the impact of mating patterns on seed productivity of orchard crops. We observed 93.7% of the success rate of the parental assignment, unequal paternal gametic contribution(0–12.4%) with 6.3% of the progenies derived from pollen contamination or unsampled pollen donors, and absence of evidence for selfing. We also found that pollination rate highly depended on the distance between pollen donors and maternal parents, the majority of the identified crossing(65.7%)occurred between clones within a 150-m radius, and large variations in growth performance existed among the paternal halfsiblings. Progeny growth performance(diameter at breast(DBH) and height(HGT)) was measured at Age-20 in order to investigate the impact of mating patterns on timber production of orchard crops. As either the paternal or maternal, two clones(i.e., clones Z38 and Z62) were identified to have produced progenies with higher average stem volume breeding values than that of all of the progenies. Specifically, the genetic gains for volume were 3.53% for the two clones as paternal parents, and 8.26% as the maternal parents at Age-20. Thus, both elite clones were ideal candidates for the construction of next-generation clonal seed orchards due to their synchronous reproductive phenology with greater crossing rate and higher genetic gain. These results improved the pedigree information to provide solid evidence of mating patterns for future design and effective management of seed orchards and for the development of viable long-term breeding strategies for other coniferous species.     

DNA指纹图带与鸡的蛋重性状的遗传相关分析

用蛋重大的和蛋重小的北京白鸡做亲本交配获得F1代个体,再用F1代个体交配得125只F2代个体。以人源小卫星DNA33.6做探针以上述125只鸡进行D NA指纹分析,发现其中有两条指纹图带与蛋重性状有关,经简单线型相关分析表明,这两条图带(D6.2和D6.3)的出现频率与蛋重的相关系数分别为-0.80和-0.81。 Abstract:Beijing White Chickens laying larger eggs and those laying smaller eggs were used as parental individuals for mating to produce the F1 progeny and then the F1 progeny individuals were mated to produce 125 individuals of the F2 progeny.Two bands associated with the egg weight trait were identified by DNA fingerprints of the 125 individuals generated with human minisatellite probe 33.6. The simple linear correlation analysis showed that the coefficients of correlation between frequencies of the two bands(D6.2 and D6.3)and egg weights are –0.8 and –0.81.     

Genetic Mapping of Laminaria japonica and L. Iongissima Using Amplified Fragment Length Polymorphism Markers in a ＂Two-Way Pseudo- Testcross＂ Strategy

With a ＂two-way pseudo-testcross＂ mapping strategy, we applied the amplified fragment length polymorphism （AFLP） markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F1 cross family （Laminaria iongissima Aresch. × L. Japonica Miyabe） with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1：1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3：1 Mendelian segregation ratio. Among the loci with 1：1 segregating ratios, 79 loci were ordered in 14 linkage groups （648.6 cM） of the paternal map, and 72 loci were ordered in 14 linkage groups （601.9 cM） of the maternal map. The average density of loci was approximately 1 per 8 cM. To Investigate the homologies between two parental maps, we used 58 loci segregated 3：1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.     

Establishment of a multi-color genomic in situ hybridization technique to simultaneously discriminate the three interspecific hybrid genomes in gossypium

To identify alien chromosomes in recipient progenies and to analyze genome components in polyploidy, a genomic in situ hybridization （GISH） technique that is suitable for cotton was developed using increased stringency conditions. The increased stringency conditions were a combination of the four factors in the following optimized state： 100：1 ratio of blocking DNA to probe, 60% formamide wash solution, 43 ℃ temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum （C1 C1 ） as a probe, strong hybridization signals were only observed on chromosomes from the C1 genome in somatic cells of the hybrid F1 （G. hirsutum x G. sturtianum） （AtDtC1）. Therefore, GISH was able to discriminate parental chromosomes in the hybrid. Further, we developed a multi-color GISH to simultaneously discriminate the three genomes of the above hybrid. The results repeatedly displayed the three genomes, At, Dt, and C1, and each set of chromosomes with a unique color, making them easy to identify. The power of the multi-color GISH was proven by analysis of the hexaploid hybrid F1 （G. hirsutum x G. australe） （AtAtDtDtG2G2）. We believe that the powerful multi-color GISH technique could be applied extensively to analyze the genome component in polyploidy and to identify alien chromosomes in the recipient progenies.     

Arabidopsis RAN 1 Mediates Seed Development through Its Parental .Ratio by Affecting the Onset of Endosperm Cellularization

Although previous studies have demonstrated that endosperm development is influenced by its parental genome constitution, the genetic basis and molecular mechanisms that control parent-of-origin effects require further elucidation. Here we show that the Ras-related nuclear protein 1 （RAN1） regulates endosperm development in Arabidopsis thaliana. Reciprocal crosses between wild-type （WT） and transgenic lines misexpressing RAN1 （msRAN1） gave rise to small F1 seeds when RAN1 down-regulated/up-regulated individuals were used as a male/female parent; in contrast, F1 seeds were aborted when RAN1 down-regulated/up-regulated plants were used as a female/male parent, suggesting that seed development is affected by the parental genome ratio of RAN1. Whereas RAN1 expression in wild-type plants is reduced before the onset of endosperm cellularization, F1 seeds from reciprocal crosses between WT and msRAN1 showed abnormal endosperm cellularization and ectopic expression of RAN1. The expression of MINISEED3 （MINI3）-a gene that also controls endosperm cellularization-was also affected in these reciprocal crosses, and the misregulation of MINI3 activity rescued F1 seeds when msRAN1 plants were used in reciprocal crosses. Taken together, our results suggest that the parental ratio of RAN1 regulates the onset of endosperm cellularization through its genetic interaction with MINI3.     

Isozyme Analysis of F 5 and BC 1F 4 from Cultivated Barley (Hordeum vulgare)× Roegneria ciliaris

栽培大麦,纤毛鹅观草,属间杂种,酯酶,过氧化物酶 ISOZYME ANALYSIS OF F5 AND BCiF4 FROM CULTIVATED BARLEY ( HORDEUM VULGARE ) ~ ROEGNERIA CILIARIS LI Wan-Ji LI Yi-Ping L1U Fang Abstract Esterase and peroxidase isozymes were analysed in the variants including 4 types, 16 lines of Fs, BC1F4 and the parents derived from cultivated barley ( Hordeum vulgare cv. Arupo) x Roegneria ciliaris (Trin.) Nevski in young roots, shoots, spikes and seeds. The zymogram patterns of esterase and peroxidase demonstrated that the 16 lines of F5 and BC1F4 had all or most bands of the cultivated barley parent cv. "Arupo", 1 to 3 bands from the male R. ciliaris, and new hybrid isozyme bands in various amount. Some bands of parent "Arupo" were lost. It suggested that the genetic substances come from R. ciliaris were stably inherited to the progenies of selfing and backcrossing, and there were some variations among the lines. There was certain relationship between isozyme variance and plant characters. Thus, in identifying the translocation lines by isozyme analysis, it would be preferable to study the various organ-specific isozymes or to trace one type of isozyme pattern in consequence.     

Offspring diet supersedes the transgenerational effects of parental diet in a specialist herbivore Neolema abbreviata under manipulated foliar nitrogen variability

Diet quality influences organismal fitness within and across generations.For herbivorous insects,the transgenerational effecets of diet remain relatively underexplored.Usinga3×3×2 factorial experiment,we evaluated how N enrichment in parental diets of Neolemd abbreviata(Larcordaire)(C oleoptera:Chrysomelidae),a biological control agent for Tradescantia fluminensis Vell.(Commelinaceae),may influence life history and performance of Fi and F2 offspring under reciprocal experiments.We found limited transgenerational effects of foliar nitrogen variability among life-history traits in both larvae and adults.Larval weight gain and mortality were responsive to parental diet contrary to feeding damage,pupal weight and duration taken to pupate.There were significant parental diet x test interactions in larval feeding damage,weight gain,pupal weight and time to pupation.Generally,offspring from parents under high N plants performed better even under low N test plants.Adult traits including oviposition selection,feeding weight and longevity did not respond to the efects of parental diet nor its interaction with test diet as was the case in the larval stage.However,the main efects of test diet were more important in determining adult performance in both generations suggesting limited sensitivity to parental diet in the adult stage.Our results show conflicting responses to parental diet between larvae and adults ofthe same generation among an insec species with both actively feeding larual and adult life stagee These tranegeneratinonal efferte,or lack thereof,may have implications on the field performance of N.abbrevita under heterogencous nutritional landscapes.     

Behavior of meiotic chromosomes in Pinus wallichiana, P. strobus and their hybrid and nrDNA localization in pollen mother cells of the hybrid by using FISH

The complete process of meiosis was investigated in Pinus wallichiana, P. strobus and their artificial hybrid （F1） using microsporocytes. It is revealed that there were slightly lower chiasma frequency, lower ring bivalent frequency, lower meiotic index and distinctly higher frequency of aberrance （chromosomal bridges, fragments or micronuclei） in pollen mother cells （PMCs） of the hybrid （F1） than those of the parental species, which showed a certain degree of differentiation between homologous chromosomes of the two parents. However, relatively higher frequency of ring bivalents and higher meiotic index in all the three entities indicate the great stability of genomes of parental species, and the differentiation of genomes between the two parents must have been slight. Total nineteen signal loci of 18S rDNA were observed in nine bivalents of the hybrid （F1）, among which one bivalent bears two loci, while the others have only one. It is suggested that distinct differentiation at genetic level existed in homologous chromosomes of the two parental species, whereas only slight differentiation at karyotypic and genomic levels take place between the parent species.     

Inheritance and Expression of Copies of Transgenes 1Dx5 and 1Ax1 in Elite Wheat （Triticum aestivum L.） Varieties Transferred from Transgenic Wheat through Conventional Crossing

To study the inheritance and expression of multiple copies of transgenes from transgenic wheat lines, three crosses between transgenic wheat lines B72-8-11b and B102-1-2 and Chinese elite wheat varieties Chuan89-107 and Email 8 were carried out. Chuan89-107×B72-8-11b, Chuan89-107×B102-1-2 and Email 8×B72-8-11b, and F_1 plants were selfed or backcrossed to obtain different generation populations. Protein analysis in grains of F_1 and F_2 and backcross progenies of BC_1F_1, BC_1F_2, BC_1F_3, BC_2F_1, BC_2F_2 and BC_2F_3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the transgenes lDx5 and lAx1 were expressed and segregated in the target wheat according to Mendelian laws. A range of lDx5 expression levels were observed in the progenies of Chuan89-107×B72-8-11b and Emai 18×B72-8-11b, but the expression levels of lAx1 in progenies of Chuan89-107×B102-1-2 rarely changed. It suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way and the foreign lDx5 gene of 5-10 copies had the more complicated expression mechanism than the lAx1 gene of 4-5 copies.     

Bt水稻杂交育种中转基因的遗传分析

利用PCR、GUS染色和Western印迹杂交技术检测了Bt水稻杂交后代群体,发现在394株GUS阳性株中,共有392株表达Bt蛋白,协同表达株率达99.49%。由此表明,在杂交后代中报告基因Gus和目的基因crylAb紧密连锁遗传与表达。本试验还发现,在BC1、BC1F2和粳粳交F2群体中转基因呈单基因显性遗传,而在籼粳交F2群体中偏离3:1分离。 Abstract:Improved histochemical staining for GUS activity,PCR and Western blotting were used to detect the population of Bt rice crossed to conventional rice varieties.A total of 392 plants expressing Bt toxin protein were found in 394 GUS positive plants.The result demonstrated that cry1Ab gene closely inherited and expressed with reporter gene gus.Therefore,it is possible to develop GUS-assisted-selection to preliminarily identify the Bt gene and study the inheritance of transgenes in (back)cross breeding.Mendelian segragation of reporter gene Gus was observed in F2,BC1 and BC1F2 progenies.Thus indicated that transgenes inherited as a single dominant gene in the progenies of Bt rice crossed to conventional rice varieties.     

ISSR标记在黑木耳单核体遗传分析中的应用

采用原生质体单核化和单孢分离法分别获得黑木耳Auricularia auricula栽培菌株新科5号两个亲本单核体(T1、T2)和33个F1代孢子单核体。从73条ISSR引物中筛选出13条可区分两个亲本单核体(T1、T2)的引物,对新科5号及F1代孢子单核体进行扩增,共扩增出70条带,其中63条在供试菌株间表现出多态性,多态位点百分率为90.0%。根据扩增结果采用软件NTsys2.10e计算菌株间的遗传相似系数并进行聚类分析,36个菌株间的遗传相似系数(GS值)的变化范围为0.2500～0.8382,其中T1和T2之间的GS值最小,遗传相似程度最低;F1代33个孢子单核体中24个与T1聚为一类,其余9个与T2聚为另一类,但并非严格按照交配型进行归类。试验表明黑木耳子实体上各个担子在减数分裂中染色体交换极具多样性,F1代孢子单核体表现出偏向其中一个亲本的现象,ISSR标记在黑木耳杂交育种中具有潜在的应用价值。     

Identification of mating type loci and development of SCAR marker genetically linked to the B3 locus in Pleurotus eryngii

In order to estimate how diverse the mating types in Pleurotus eryngii from different regions are, pairings between monokaryons derived from inter- and intragroups were done. Sixteen and 15 alleles were identified at loci A and B from the 12 strains. In the P. eryngii KNR2312, widely used for commercial production, four mating loci, A3, A4, B3, and B4, were determined. Those loci, except A3, were found in 4 strains out of 12 strains. To improve breeding efficiency, especially in mating type determination, RAPD and BSA were performed to screen for a mating type specific marker. The SCAR marker 13- 2(2100) was developed based on the RAPD-derived sequence typing B3 locus. The sequence analysis of 13-2(2100) revealed that it contained a conserved domain, the STE3 superfamily, and consensus sequences like the TATA box and GC box. It seems likely that the SCAR marker region is a part of the pheromone receptor gene.     

黑木耳交配型的研究*

从黑木耳Auricularia auricula沪3菌株的子实体收集、分离、鉴定得到157株单孢子萌发的单核菌丝体,以其中73株孢子单核体为材料进行交配实验,经过三轮交配反应,结果表明,木耳的交配型符合四极性交配系统的分布规律。根据四种交配反应情况把73株孢子单核体分为四组,并从四组中挑选出B17、B177、B101、B65作为四种交配型的标准菌株;与此同时,从沪3菌株的双核菌丝体制备的原生质体中获得53株原生质体单核体,对这些单核体进行交配,确定出两种亲本交配型,以Y150(A1B1)和Y121(A2B2)作为标准菌株,将两个亲本交配型标准菌株与四个孢子单核体标准菌株进行交配反应,初步确定出四种孢子单核体的交配型分别为: B17 (A2B2)、B177(A1B1)、B101(A2B1)和B65(A1B2)。     

Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae.

The STE4 gene of Saccharomyces cerevisiae encodes the beta subunit of the yeast pheromone receptor-coupled G protein. Overexpression of the STE4 protein led to cell cycle arrest of haploid cells. This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells. The arrest occurred in haploid cells of either mating type but not in MATa/MAT alpha diploids, and it was suppressed by defects in genes such as STE12 that are needed for pheromone response. Overexpression of the STE4 gene product also suppressed the sterility of cells defective in the mating pheromone receptors encoded by the STE2 and STE3 genes. Cell cycle arrest mediated by STE4 overexpression was prevented in cells that either were overexpressing the SCG1 gene product (the alpha subunit of the G protein) or lacked the STE18 gene product (the gamma subunit of the G protein). This finding suggests that in yeast cells, the beta subunit is the limiting component of the active beta gamma element and that a proper balance in the levels of the G-protein subunits is critical to a normal mating pheromone response.     

木耳交配型混合集群RAPD分析

用RAPD-混合集群分析来检测异宗结合担子菌不同交配型间的多态性。来源于同一木耳Anriculariaauricula（L．exHook．）Underw,子实体的10个单孢萌发而成的单核体进行RAPD分析证实,它们之间具有极高的同源性。将单核体按其所属的交配型分为两类,每类各5个,构成A1,A2两个基因池。用33种单引物,20种双引物对其进行扩增、分析,发现引物OPE19号能在这两个基因池间产生多态性,其中一条特异性谱带在A2基因池扩增产物中存在,在A1基因池中不存在。推测这一条谱带可能是与A2交配型基因连锁的分子标记。此研究结果不仅为由单因子控制的二极性担子菌的交配型测定提供科学依据,而且为RAPD技术在异宗结合担子菌极性研究中的应用展示了可喜的前景。     

The yeast alpha-factor receptor: structural properties deduced from the sequence of the STE2 gene.

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a component of the receptor for the oligopeptide pheromone alpha-factor. We have cloned and determined the nucleotide sequence of the STE2 gene. A sequence involved in the control of cell-type expression of the STE2 gene was found 5' of an open reading frame that could encode a protein of 431 amino acids. The predicted STE2 protein contains seven hydrophobic segments, suggesting that the alpha-factor receptor is an integral membrane protein. No extensive homology at the primary sequence level was detected between the predicted STE2 protein and other available protein sequences.     

香菇B交配型位点的分子遗传学结构研究II.一个香菇单核体的B位点结构的分子解析

在先前的工作中,曾经运用简并PCR和染色体步行的方法从香菇中获得了1个信息素受体编码基因和1个信息素前体编码基因。根据香菇135菌株的原生质体单核体的全基因组测序信息,设计了4对引物,用于扩增香菇苏香菌株的原生质体单核体SUP2中的信息素受体编码基因STE-3的同源物及其侧翼保守基因。实验结果共获得了33,655bp的DNA序列,运用BlastX搜索对所获得的序列进行同源性分析后,发现了7个推定基因,其中有3个为信息素受体编码基因。再根据信息素前体所具有的保守基序特征,在2个信息素受体编码基因附近发现了4个信息素前体编码基因。首次对香菇的B交配型位点的分子遗传学结构有了比较全面的了解。     

RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor

We have identified a gene (SUPH) of S. cerevisiae that is required for both RAS function and mating by cells of a mating type. supH is allelic to ste16, a gene required for the production of the mating pheromone a-factor. Both RAS and a-factor coding sequences terminate with the potential acyltransferase recognition sequence Cys-A-A-X, where A is an aliphatic amino acid. Mutations in SUPH-STE16 prevent the membrane localization and maturation of RAS protein, as well as the fatty acid acylation of it and other membrane proteins. We propose the designation RAM (RAS protein and a-factor maturation function) for SUPH and STE16. RAM may encode an enzyme responsible for the modification and membrane localization of proteins with this C-terminal sequence.