Interspecific Hybridization between Cucumis ssp. Plants and SRAP Analysis of Hybrids

To understand the inter-hybridization between Cucumis ssp.plants,we used 150 melon varieties as female parents to cross with Cucumis metuliferus and Cucumis anguria.Only melon accessions V2 and V129 set fruits,but seeds from fruits V2（V129）×C.metuliferus were abortive.A few of seeds from the bottom of fruit V2（V129）×C.anguria were fertile.Sequence-related amplified polymorphism（SRAP）molecular markers were used to analyze the progenies of inter-specific hybridization between C.anguria and melon V129.One pair primer（E14/M2）was found effective in amplification on male parent characteristic bands from the hybrids,suggesting that some DNA exchange had happened between C.anguria and melon V129.This study provided data for analyzing the mechanism of inter-hybridization between Cucumis plants.

Progress on Transferring Elite Genes from Non-AA Genome Wild Rice into Oryza sativa through Interspecific Hybridization

The progress of research on transferring elite genes from non-AA genome wild rice into Oryza sativa through interspecific hybridization are in three respects, that is, breeding monosomic alien addition lines （MAALs）, constructing introgression lines （ILs） and analyzing the heredity of the characters and mapping the related genes. There are serious reproductive barriers, mainly incrossability and hybrid sterility, in the interspecific hybridization of O. sativa with non-AA genome wild rice. These are the ＇bottleneck＇ for transferring elite genes from wild rice to O. sativa. Combining traditional crossing method with biotechnique is a reliable way to overcome the reproductive barriers and to improve the utilizing efficiency of non-AA genome wild rice.

Interspecific Hybridization Between Crassostrea angulata and C. ariakensis

lnterspecific hybridization can generate heterosis, which is proven to be a useful tool in selective breeding programs for oyster culture. Crassostrea angulata and C. ariakensis are two important economic shellfish species in China. We conducted 2 × 2 reciprocal crosses to determine whether these two species can cross-fertilize and their hybrids can hatch, survive and perform hetero- sis. Fertilization was found symmetrical without delay. The rate of fertilization success of C. angulata ♀× C. ariakensis ♂ was lower than that of C. ariakensis ♀ x C. angulata ♂, and the success rate of both hybridizations was lower than that of two intraspecific crosses each. During the planktonic period, survival rate of the progeny was lower in the hybrid crosses than in the intraspecific crosses. On day 360, mean shell height of the progeny of C. angulata ♀ x C. angulata ♂ was highest, which was followed by that of C. angulata ♀ × C. ariakensis ♂, C. ariakensis ♀x C. ariakensis ♂ and C. ariakensis ♀× C. angulata ♂ in a descending order. Mor- phology of adults produced by the hybrid crosses was similar to that of C. angulata. Both hybrids underwent normal gonad devel- opment and produced mature gametes in the mating season. This study provided new insights into the quantitative traits in interspeci- fic crosses of Crassostrea species, thus being of guidance value for selective breeding of oyster.

Comparative Embryological Studies on Infertility of Interspecific Hybridizations Between Oryza sativa with Different Ploidy Levels and O. officinalis

As maternal parents, diploid （L202-2x） and autotetraploid （L202-4x） of Oryza sativa cv. L2O2 were crossed with O. officinalis. Embryo development and fertilization in these two crosses were comparatively studied. There were no mature hybrid seeds obtained because all the hybridized spikelets died 30 days after pollination. The main reasons for no seed set were abnormal fertilization and development of the embryos and endosperms in the interspecific hybrids. There were doublefertilization, egg cell single-fertilization and non-fertilization in these crosses. Although 59.45% and 54.87% of hybrid embryos produced in the crosses of L202-2x/O. officinalis and L202-4x/O. officinalis, respectively, hybrid embryos ceased to develop or degenerated and plenty of free endosperm nuclei were in disaggregating state without developing cellular endosperms three days after pollination. Besides, some embryological differences in these two crosses were found, that is, the rate of double-fertilization and total rate of doubleand single-fertilization in L202-2x/O. officinalis were higher than those in L202-4x/O. officinalis. The embryo and endosperm of hybrids developed more slowly, and embryos and free endosperm nuclei were more severely degenerated in L202-4x/O. officinalis than in L202-2x/O. officinalis. Five days after pollination, a few of embryos in L202-2x/O. officinalis developed into pear-shaped ones, however, embryos in L202-4x/O. officinalis were all degenerated. Therefore, it is more difficult to obtain interspecific hybrids by wide crosses between autotetraploid of O. sativa and O. officinalis.

Cytogenetic Analysis of the Primary Amphidiploid Derived from Interspecific Hybridization in Cucumis and Its Selfed Progenies

Studies on the reproduction and cytogenetic characterization of a primary amphidiploid Cucumis species C. hytivus Chen and Kirkbride (2n = 4x = 38) indicated that a more comprehensive cytogenetic analysis of this species and its first selfed progeny would increase its potential utility in cucumber improvement. With tendrils used as source materials for mitotic analysis, chromosome numbers in all selfed progenies were 2n = 38, confirming chromosomal stability in this synthetic amphidiploid species. Detailed meiotic processes were described by comparing the primary and the selfed amphidiploids. Meiotic abnormalities, such as chromosome lagging, unequal separation, chromosome multi-polarization and polyads were observed frequently in all amphidiploid plants except for the selfed no.8, in which meiosis was arrested prior to metaphase Ⅰ. Generally, the frequency of multivalents was higher and the configurations were more complex in the selfed progenies, demonstrating a more extensive genetic exchange between cucumber and C. hystrix Chakr. Genome separation between cucumber and C. hystrix was observed through prophase Ⅰ to anaphase Ⅰ in both generations of the amphidiploids. Consequently, in addition to n = 19, a new gamete with n = 7 was produced, which was confirmed by the chromosome counts 2n = 14 in the backcrossing progenies from cucumber × amphidiploid mating. Fertility varied among the selfed amphidiploid plants. The selfed plant no.1 was found to have an improved fertility (e.g., pollen staining ability 40.8% and 25.6 seeds per fruit) and then was used as source germplasm in further introgression and gene exchange experiments.

INTERSPECIFIC SOMATIC HYBRIDS IN ACTINIDIA

Protoplasts isolated from cotyledon-derived calli of Actinidia chinensis var. chinensis (2n = 2x=58) were fused by the PEG method with cotyledon-callus protoplasts of A. deliciosa var. deliciosa (2n = 6x = 174) or with mesophyll protoplasts of A. kolomikta (2n = 2x = 58), respectively. Randomly amplified polymorphic DNA (RAPD) markers and flow cytometry was used to confirm the occurrence of somatic hybrids. RAPD results with some primers surveyed indicated that one clone (A. chinensis + A. deliciosa) and four clones (A. chinensis + A. kolomikta) had RAPD banding patterns which combined the parental banding profiles. Ploidy levels of the (A. chinensis + A. deliciosa) clone were deduced as octoploid (2n = 8x), and the (A. chinensis + A. kolomikta) clones were tetraploid (2n = 4x), triploid (2n = 3x) or pentaploid (2n = 5x). The clones were confirmed as interspecific somatic hybrids in Actinidia.

Contemporary Asymmetric Genetic Introgression Between Two Pelophylax Species in Shanghai

Human disturbances are considered to break reproduction barriers among species.Significant increases in hybridization events have been reported among a large number of taxonomic groups in anthropogenic environments,providing novel insights into species evolution mechanisms and conservation management in the Anthropocene.The Eastern Golden Frog(Pelophylax plancyi)and BlackSpotted Frog(P.nigromaculatus)are two sympatric anuran species with a long history of mitochondrial genome introgression in highly urbanized continental East Asia.However,there is only limited understanding of the pattern of their contemporary hybridization and factors influencing their interspecific relationship under anthropogenic disturbances.Here,interspecific hybridization between P.plancyi and P.nigromaculatus at the population level was investigated in Shanghai.All except two haplotypes obtained from both species in Shanghai were mixed together,and located in the introgression clade,implying multiple ancient mitochondrial introgression events occurred in the populations of our study area.Asymmetric genetic introgression was detected by microsatellite markers,with 0.7%of P.plancyi and 14.6%of P.nigromaculatus identified as contemporary admixed individuals.Consistent with the trend of population density,higher genetic diversity of neutral microsatellite loci was found in the more abundant P.plancyi;however,variation in mitochondrial(Cyt-b)and nuclear(POMC)genes was higher in relatively rare P.nigromaculatus.The population density of P.plancyi and number of water patches within local habitats were significantly positively correlated with both occurrences and proportions of admixed individuals in the populations of P.plancyi and P.nigromaculatus.Considering the prevalent transformation of habitats in urbanized areas,these results imply that a high population density in isolated artificially altered habitats is likely to increase interspecific hybridization.Thus,population monitoring and improvement of landscape connectivity between habitats would be needed to control the intensity of interspecific hybridization between P.plancyi and P.nigromaculatus in anthropogenic-disturbed environments.

Embryonic and Larval Development of Reciprocal Crosses between Clarias gariepinus (Burchell, 1822) and Clarias jaensis (Boulenger, 1909) in West Cameroon

Establishing intraspecific breeding and hybridization programs and determining genetic variability are two important issues for aquaculture. However, interspecific hybridization to improve growth and feeding efficiency is limited. For this purpose, the embryonic and larval development of reciprocal crosses of Clarias gariepinus (Burchell, 1822) and Clarias jaensis (Boulenger, 1909) were studied under laboratory conditions. The fertilization rate varied from 63.33% to 92%, while the hatching rate ranged from 55.68% to 76% with the highest value in hybrids ♀Cg × ♂Cj. Crosses between ♀Cj × ♂Cj, ♀Cg × ♂Cj and ♀Cj × ♂Cg had embryonic stages similar to those of the pure sib ♀Cg x ♂Cg. All crosses, however, had different timing for the various embryological stages. Hatching occurred at 32 h 15 min and 38 h for ♀Cj × ♂Cj and ♀Cj × ♂Cg, and 23 h and 23 h 30 min, respectively, for ♀Cg × ♂Cg and ♀Cg × ♂Cj. However, both crosses produced viable larvae until the first external feeding. The external morphological features of the larvae were completely formed by the 10th day after hatching. The body forms of the crosses at this time were indistinguishable from the pure sib. This study thus laid the groundwork for further comparative studies on hybrid performance and characterization.

Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis

Oryza officinalis is one of the important wild species in the tertiary gene pool of Oryza sativa. It has a number of elite genes for rice breeding in resistance or tolerance. However, breeding barriers are so serious that the gene transfer is much difficult by sexual cross method between O. sativa and O. officinalis. Characteristics of the breeding barriers were systemically studied in this paper. When both the diploid （AA, 2n=2x=24） and autotetraploid （AAAA, 2n=4x=48） cultivated rice were crossed as maternal parents with O. officinalis （CC, 2n=2x=24）, none F1 hybrid seeds were obtained. The young hybrid ovaries aborted at 13-16 d after pollinations （DAP）. By rescuing hybrid embryos, in vitro F1 plantlets were obtained in 2x×2x combinations with the crossabilities lower than 0.5%. Lower rates of double-fertilization and abnormal development of hybrid embryo and endosperm were mainly observed in both combinations of 2x×2x and 4x×2x. Free endosperm nuclei in hybrid degenerated early at 1 DAP in a large scale. Almost no normal endosperm cells formed at 3 DAP. Development of a lot of embryos ceased at globularor pear-shaped stage as well as some degenerated gradually. The hybrid plantlets were both male and female sterility. Due to the abnormal development, a diversity of abnormal embryo sacs formed in hybrids, and hybrid pollen grains were typically abortive. It showed that conflicts of genome A and C in hybrid induced abnormal meioses of meiocytes.

Identification and cell wall analysis of interspecific hybrids between Oryza sativa and Oryza ridleyi

Oryza ridleyi is an allotetraploid wild species with the HHJJ genome, and Oryza sativa is a diploid cultivated rice that has the AA genome. Although the wide hybrid between the two species is difficult to obtain, we overcome this difficulty by young embryo rescue. An obvious heterosis was primarily found for the plant height, tillering ability, vegetative vigor, etc. However, the hybrid panicle and culm traits were found to resemble that of the wild rice parent, O. ridleyi, for the long awns, exoteric purple stigma, grain shattering, dispersed panicles, and culm mechanical strength. Genomic in situ hybridization （GISH） analysis was subsequently performed on the mitotic metaphase chromosome of the root tips, and we determined that the hybrid is an allotriploid with 36 chromosomes and its genomic constitution is AHJ. Chemical analyses conducted on the culm of O. sativa, O. ridleyi, and their interspecific hybrids showed that major changes occurred in the xylose, glucose, and arabinose concentrations, which are correlated with the specific hemicellulose polymer and cellulose components that are important in the primary cell walls of green plants. Meanwhile, the culm anatomical analyses indicated that additional large vascular bundles and an extra sclerenchyma cell layer were found in O. ridleyi. Additionally, further thickening of the secondary cell walls of the cortical fiber sclerenchyma cells and the phloem companion cells was discovered in O. ridleyi and in the interspecific hybrids. These results imply that there may be a potential link between culm mechanical strength and culm anatomical structure.

Molecular Cytogenetic Analysis of Spontaneous Interspecific Hybrid Between Oryza sativa and Oryza minuta

Genomic in situ hybridization (GISH) is a powerful tool to characterize parental chromosomes in interspecific hybrids, including the behaviour of autosynapsis and chromosome pairing. It was used to distinguish the chromosomes of Oryza sativa from wild species in a spontaneous interspecific hybrid and to investigate the chromosome pairing at metaphase I in meiosis of the hybrid in this study. The hybrid was a triploid with 36 chromosomes according to the chromosome number investigated in mitosis of root tips. During metaphase I of meiosis in the hybrid, less chromosome pairing was observed and most of the chromosomes existed as univalent. Based on GISH and FISH (Fluorescent in situ hybridization) analyses, the chromosomes of the hybrid were composed of genomes A, B and C. Thus, it was believed that the hybrid was the result of natural hybridization between cultivated rice and wild species O. minuta which was planted in experimental fields.

A hybrid sterile locus leads to the linkage drag of interspecific hybrid progenies

Interspecific hybridization plays an important role in rice breeding by broadening access to desirable traits such as disease resistance and improving yields.However,interspecific hybridization is often hindered by hybrid sterility,linkage drag,and distorted segregation.To mine for favorable genes from Oryza glaberrima,we cultivated a series of BC4 introgression lines(ILs)of O.glaberrima in the japonica rice variety background(Dianjingyou 1)in which the IL-2769(BC4F10)showed longer sterile lemmas,wider grains and spreading panicles compared with its receptor parent,suggesting that linkage drag may have occurred.Based on the BC5F2 population,a hybrid sterility locus,S20,a long sterile lemma locus,G1-g,and a new grain width quantitative trait locus(QTL),qGW7,were mapped in the linkage region about 15 centimorgan(cM)from the end of the short arm of chromosome 7.The hybrid sterility locus S20 from O.glaberrima eliminated male gametes of Oryza sativa,and male gametes carrying the alleles of O.sativa in the heterozygotes were aborted completely.In addition,the homozygotes presented a genotype of O.glaberrima,and homozygous O.sativa were not produced.Surprisingly,the linked traits G1-g and qGW7 showed similar segregation distortion.These results indicate that S20 was responsible for the linkage drag.As a large number of detected hybrid sterility loci are widely distributed on rice chromosomes,we suggest that hybrid sterility loci are the critical factors for the linkage drag in interspecific and subspecific hybridization of rice.

Cytological Analyses on Development of Male and Female Gametophytes in an Interspecific Hybrid F_1 from Cucumis hystrix Chakr. × Cucumis sativus L.

An interspecific hybrid F1 of Cucumis hystrix Chakr. x Cucumis sativus L. （NC4406） was used to establish the developmental sequence and to characterize the male and female gametophytes at cytological level for further understanding of the phylogenic relationship and the mechanism of fertility or sterility in the interspecific hybrid F1 The development of male and female gametophytes was studied through meiotic analysis and paraffin section observation technique, respectively. Meanwhile, the fertility level was assessed through hybrid F1 backcrossing to cultivated cucumber 4406. Variable chromosome configurations were observed in the pollen mother cells （PMCs） of hybrid F1 at metaphase Ⅰ , e.g., univalents, bivalents, trivalents, quadravalents, etc. At anaphase Ⅰ and Ⅱ, chromosome lagging and bridges were frequently observed as well, which led to the formation of polyads and only a partial number of microspores could develop into fertile pollen grains （about 23.3%）. Observations of the paraffin sections showed numerous degenerated and abnormal embryo sacs during the development of female gametophytes, and only 40% of the female gametophytes could develop into normal eight-nuclear megaspore. On an average, 22.8 and 6.3 seeds per fruit could be obtained from the reciprocal backcross. The interspecific hybrid F1 of C. hystrix x NC4406 was partially fertile; however, the meiotic behaviors of hybrid F1 showed a high level of intergenomic recombination between C. hystrix and C. sativus chromosomes, which indicated that it plays an important role for introgression of useful traits from C. hystrix into C. sativus.

Genetic Variation and Heritability Estimates for Bunch Yield, Bunch Components and Vegetative Traits in Oil Palm Interspecific Hybrids

Progeny testing of oil palm interspecific hybrids was conducted in four trials in Kluang, Ulu Paka, Teluk Intan and Carey Island, Malaysia. The interspecific hybrids （O × P） were created according to the North Carolina Model I （NCM I） mating design, using Elaeis oleifera （0） and Elaeis guineensis var. pisifera （P） as the maternal and paternal parents, respectively. Differences among O, P and O-within-P were determined by the performance （bunch yield, components and vegetative traits） of the progenies. There were significant differences among P for fresh fruit bunch （FFB） yield, bunch number （BN） and average bunch weight （ABW） in Kluang, Ulu Paka and Teluk Intan, but not in Carey Island. FFB yield was generally higher on coastal soils （Teluk Intan and Carey Island） than inland soils （Kluang and Ulu Paka）. Heritability was calculated based on the intraclass correlation. Heritability estimates for these three yield components were variable, depending on the breeding material and environment in which the materials were tested. Fruit to bunch （F/B） and oil to bunch （O/B） of parthenocarpic fruits were important in determining the overall O/B of the interspecific hybrids. The O x P hybrids in Kluang showed the lowest height increment with only a mean of 14.0 cm/year, whereas in Ulu Paka and Teluk Intan, the values were higher at 24.0 cm/year and 25.0 cm/year, respectively. The study showed that the FFB yields ofoil palm interspecific hybrids performed better in coastal soils than inland soils.

Inheritance of the Anatomy--Morphological Structure of the Stalk by Interspecific Hybrids of the Glycine L.

The research focuses on the study of anatomical and morphological stalk structure ofsoya interspecific hybrids of the third generation （F3） between （Glycine max （L.） Merr.） and G. soja Sieb. et Zucc. in comparison with parent plant species. The parent plant species and interspecific hybrids were sowed and grew under similar conditions. The similarity of the anatomic structure of stalks of cultivated plants and Glycine soja （wild soya） proves the hypothesis the studied species have the same origin. However, the obtained results show the considerable degree of phylogenetic dissociation between the studied soya species. Interspecific hybrids inherit from G. soja the ability to high intensive growth. The G. soja use in practical selective breeding is of great interest.

Use of Interspecific Hybrids in the Texas A&M University Cotton Improvement Laboratory

Integrating alleles from Gossypium species into G.hirsutum is important for the enhancement of genetic variability and for creating polymorphism useful in molecular mapping studies.Through collaborative efforts,several species including G.barbadense,G.tomentosum,and G.mustelinum

Combination of Homoeologous Pairing Gene phKL and Ph2-deficiency in Common Wheat and Its Meiotic Behaviors in Hybrids with Alien Species

The combined lines having both phKL and Ph2-deficiency were obtained in the genetic background of common wheat (Triticum aestivum L.) landrace. These lines had normal fertility. In the wheat combined lines X Aegilops variabilis Eig. (or rye), a significant increase in the chiasmata of homoeologous pairing was shown by the phKL+Ph2(-) plants with respect to their phKL+Ph2 sibs, which indicates that Ph2-deficiency and phKL showed an additive effect on promoting pairing. The effects were shown in the increment of rod bivalents, ring bivalents and trivalents and reduction of univalents, of which, reduction of univalents was mainly due to the increment of rod bivalents. The combined lines are probably more desirable materials for alien gene transferring than phKL or Ph2(-) lines alone. In comparison with that of ph1b X Ae. variabilis (or rye), phKL+Ph2(-) X Ae. variabilis (or rye) show higher (or similar) numbers of rod bivalents, while the total chromosome pairing level significantly reduced that ascribed to the decrement in ring bivalents and multivalents. These results probably indicate the different genetic mechanisms for Ph1 and Ph2 or phKL on controlling homoeologous pairing.

Analysis of Genetic Differences between Luffa acutangula(L.) Roxb. and Luffa cylindrica(L.)Roem. by ISSR Markers

The genetic differences among Luffa acutangula （L.） Roxb., Luffa cylindnca （L.） Roem. and their F1 hybrid and F2 population were analyzed by ISSR technolo- gy. The results showed that the genomic DNA of test materials showed a high diversity; the genetic similarity coefficient between both parents was only 0.395; the bands of F1 hybrid mainly consisted of both parents＇; neither FI nor F2 was inherit- ed singly from male or female parent; there was greater genetic variation among F2 population.

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.

Induction of Tetraploid Male Sterile Tagetes erecta by Colchicine Treatment and Its Application for Interspecific Hybridization

Tagetes erecta is an annual multifunctional plant which can be cultivated under a broad range of climatic conditions. Polyploidization and interspecific hybridization are applied to facilitate breeding cultivars of T. erecta with improved ornamental qualities. Colchicine treatment to the germinating seeds was proved to be a useful tool for chromosome doubling of the male sterile two-type line ‘M525AB', with the resulting frequency of polyploid seedlings ranging from 88.89%(following 0.05% w/v colchicine applied for a 3–6 h exposure period) to a maximum of100.00%(following 0.1% for 3–6 h, or 0.2% for 3 h). Morphological observation, stomatal size and density analysis, flow cytometric analysis and chromosome counting were conducted to identify the tetraploid plants. Distinctive morphological changes were observed in a notable proportion of polyploid plants. The colchicine-treated polyploid T. erecta plants showed dwarfed and more robust growth, thicker, larger and greener leaves, larger inflorescences and florets. The mutant plants identified through morphological observation all aligned as polyploid plants, thus morphological observation could be an effective method for the detection of polyploidy. The polyploid plants had significant larger stomata size over the abaxial leaf surface, whereas the density of stomata distribution was remarkably reduced. The survival rate of tetraploid cuttings(i.e. 38%)was greatly reduced compared to that of diploid plants. The fertility of tetraploid plants was also decreased, as shown by cross-pollination yields.Interspecific hybridizations between colchicine-induced tetraploid plants of a male sterile T. erecta line and the naturally tetraploid fully fertile Tagetes patula species resulted in hybrid progeny. Most of these hybrids displayed the dwarfed growth stature and compact, larger-flower morphology which is the typical ideotype of herbaceous flowers. Thus, polyploidization may be employed effectively as a means to facilitate interspecific hybridization, thereby contributing significantly to the improvement of quantitative traits of Tagetes spp.