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.

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.

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.

Ways of Developing Plants in Interspecific Hybridization of Cotton

It is known,that there are various barriers to fertilization,development of embryos,and endosperm because of different number of chromosomes in parents in the interspecific hybridization of cotton.Thus the factors providing normal cell fission of a germ and endosperm are necessary.It is necessary to culture embryos in vitro on the artificial environments containing various phytohormones,or

Fiber Quality Improvement by Interspecific Hybridization in Upland Cotton

In order to transfer genes for extra fiberstrength to existing cotton cultivars,weintroduced 207 interspecific materials obtainedby outcrossing where G. hirsutum,G.barbadense,G.sturtianum,G.thurberi,G.harknessii and G.somalense etc.were involved asparents respectively.Under stringent

Phylogenetic Relationship of Dendranthema （DC.） Des Moul. Revealed by Fluorescent In Situ Hybridization

: Phylogenetic relationships of the different species in the genus Dendranthema (DC.) Des Moul. were estimated based on chromosome fluorescent in situ hybridization (FISH) with 18S-26S rDNA of Arabidopsis and genomic DNA of Dendranthema as probes. The results revealed that there was no positive correlation between the number of nuclear organization region (NOR) loci and the ploidy of Dendranthema. The exact cytogenetic information of NORs about 14 operational taxonomic units (OTUs) indicated that D. vestitum (Hemsl.) Ling et Shih was closer to the cultivars than other putative species, whereas D. zawadskii (Herb.) Tzvel. was the most distinct. The ambiguously distributed signals of genomic in situ hybridization (GISH) with genomic DNA of lower ploidy species as probes suggested that different genomes among Dendranthema were mixed. The result also indicated the limitation of GISH in studies on the phylogenetic relationships of the different species in this genus Dendranthema and on the origin of cultivated chrysanthemums. Based on these results and previous research, the origin of Chinese cultivated chrysanthemum is discussed.

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.

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.

Crossability Barriers in the Interspecific Hybridization between Oryza sativa and O.meyeriana

Oryza meyeriana Baill （GG genome） is a precious germplasm in the tertiary gene pool of cultivated rice （AA genome）, and possesses important traits such as resistance and tolerance to biotic and abiotic stress. However, interspeciflc crossability barrier, a critical bottleneck restricting genes transfer from O. meyeriana to cultivars has led to no hybrids through conventional reproduction. Therefore, the reasons underlying incrossability were investigated in the present report. The results showed that： （i） at 3-7 d after pollination （DAP）, many hybrid embryos degenerated at the earlier globular-shaped stage, and could not develop into the later pear-shaped stage. Meanwhile, free endosperm nuclei started to degenerate at 1 DAP, and cellular endosperm could not form at 3 DAP, leading to nutrition starvation for young embryo development; （ii） at 11-13 DAP, almost all hybrid ovaries aborted. Even though 72.22% of hybrid young embryos were produced in the interspecific hybridization between O. sativa and O. meyeriana, young embryos were not able to further develop into hybrid plantlets via culturing in vitro. The main reason for the incrossability was hybrid embryo inviability, presenting as embryo development stagnation and degeneration since 3 DAP. Some possible approaches to overcome the crossability barriers in the interspecific hybridization between O. sativa and O. meyeriana are discussed.

Interspecific hybridization between Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease, which has greatly damaged forests and ecosystems in countries of East Asia and Europe.Bursaphelenchus mucronatus is closely related to B. xylophilus in morphology and host plant specificity. A longrunning debate has existed regarding whether these two species can successfully produce hybrid offspring. In the present study, we performed in the laboratory, hybridization of two B. xylophilus nematode isolates from China and Japan and three B. mucronatus isolates from China, Japan and France. Nematode isolates of B. xylophilus were successfully crossed with B. mucronatus isolates, and the rate of hybridization was relatively high; however, some hybrid offspring died. Successful hybridization occurred between B. xylophilus and B. mucronatus isolates from China, and 22 generations of hybrids were produced. All F1 hybrids could be backcrossed with their parents and produce offspring. Variation in mucro length among the hybrid offspring and their parents was observed. The hybrid offspring and their parents were inoculated into 3-months-old black pine(Pinus tunbergii) seedlings. Weaker pathogenicity of hybrid offspring was observed compared with that of their parents, and significantly fewer offspring nematodes than parents were reisolated from pines. Therefore, the offspring of B. xylophilus and B. mucronatus may exist in the forest and could influence disease epidemics.

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.

Interpretation of the biological species concept from interspecific hybridization of two Helicoverpa species

The biological species concept defines species in terms of interbreeding. Interbreeding between spe-cies is prevented by reproductive isolation mechanisms. Based on our results of interspecific hybridi-zation between Helicoverpa armigera and Helicoverpa assulta, reproductive isolation mechanisms of the two species are analyzed. A combination of prezygotic factors (absent sex attraction and physical incompatibility of the genitalia) and postzygotic factors (female absence and partial sterility in F1 hy-brids) causes reproductive isolation of the two species. In addition, the role of interspecific hybridiza-tion in speciation is discussed.

Creation of the technique of interspecific hybridization for breeding in cotton

The new technique of interspecific hybridization was created in Gossypium, which could remarkably overcome abortion of interspecific hybridization and hybrid sterility of F1. A large number of germplasm resources were obtained from seventy cross combinations among the cultivated species and between the cultivated and the 14 wild species, respectively. 8 varieties have been developed, of which 4 were from the cross combination of G . hirsutum×G . arboretum and the other are the first breed from the hybrids between G . hirsutum and 4 wild species, respectively. Of them Shiyuan 321 (jimian 24) is a new variety which had the highest increase in the national Yellow River Valley Regional test, with planting area added up to 933333 ha in the recent three years. The breeding system of interspecific hybridization was established.

Interspecific Hybridization between <i>Arisaema sikokianum</i>and <i>A. serratum</i>(Araceae) Confirmed through Nuclear and Chloroplast DNA Comparisons

A morphologically intermediate plant between Arisaema sikokianum Franch. et Sav. and A. serratum (Thunb.) Schott has been newly found in Kochi Prefecture, Shikoku, Japan. The putative hybrid has the intermediate morphological characteristics of the parental species. Molecular analysis using PCR-RFLP of internal transcribed spacer (ITS) in nuclear DNA (nrDNA) indicates that the putative hybrid has a combined pattern of the two putative parent species. Moreover, the sequence result of chloroplast DNA (cpDNA) of the putative hybrid was identical to that of A. sikokianum. These results suggest that the putative hybrid is a hybrid between A. sikokianum and A. serratum and that it was formed by interactive gene exchanging via pollens from A. serratum to A. sikokianum. It is the first record of a hybrid between A. sikokianum and A. serratum.

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.

Development of a set of SSR markers for genetic polymorphism detection and interspecific hybrid jute breeding

Corchorus capsularis(white jute) and C. olitorius(dark jute) are the two principal cultivated spedes of jute that produce natural bast fiber of commercial importance. We have identified 4509 simple sequence repeat(SSR) loci from 34,163 unigene sequences of C. capsularis to develop a non-redundant set of 2079 flanking primer pairs. Among the SSRs, trinudeotide repeats were most frequent(60%) followed by dinucleotide repeats(37.6%). Annotation of the SSR-containing unigenes revealed their putative functions in various biological and molecular processes, including responses to biotic and abiotic signals. Eighteen expressed gene-derived SSR(eSSR) markers were successfully mapped to the existing single-nucleotide polymorphism(SNP) linkage map of jute,providing additional anchor points. Amplification of 72% of the 74 randomly selected primer pairs was successful in a panel of 24 jute accessions, comprising five and twelve accessions of C.capsularis and C. olitorius, respectively, and seven wild jute spedes. Forty-three primer pairs produced an average of 2.7 alleles and 58.1% polymorphism in a panel of 24 jute accessions. The mean PIC value was 0.34 but some markers showed PIC values higher than 0.5, suggesting that these markers can efficiently measure genetic diversity and serve for mapping of quantitative trait loci(QTLs) in jute. A primer polymorphism survey with parents of a wide-hybridized population between a cultivated jute and its wild relative revealed their efficacy for interspecific hybrid identificatioii. For ready accessibility of jute eSSR primers, we compiled all information in a user-friendly web database, JuteMarkerdb(http://jutemarkerdb.icar.gov.in/) for the first time in jute.This eSSR resource in jute is expected to be of use in characterization of germplasm, interspecific hybrid and variety identification, and marker-assisted breeding of superior-quality jute.

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.

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.