The use of cytogenetic tools for studies in the crop-to-wild gene transfer scenario

Interspecific hybridization in plants is an important evolutionary phenomenon involved in the dynamics of speciation that receives increasing interest in the context of possible gene escapes from transgenic crop varieties. Crops are able to cross-pollinate with a number of wild related species and exchange chromosome segments through homoeologous recombination. In this paper, we review a set of cytogenetic techniques that are appropriate to document the different steps required for the stable introgression of a chromosome segment from a donor species (i.e., the crop) into a recipient species (i.e., the wild). Several examples in hybrids and derivatives are given to illustrate how these approaches may be used to evaluate the potential for gene transfer between crops and wild relatives. Different techniques, from classical chromosome staining methods to recent developments in molecular cytogenetics, can be used to differentiate genomes and identify the chromosome regions eventually involved in genetic exchanges. Some clues are also given for the study of fertility restoration in the interspecific hybrid forms.     

Alien introgression in rice

Rice (Oryza sativa L.) productivity is affected by several biotic and abiotic stresses. The genetic variability for some of these stresses is limited in the cultivated rice germplasm. Moreover, changes in insect biotypes and disease races are a continuing threat to increased rice production. There is thus an urgent need to broaden the rice gene pool by introgressing genes for such traits from diverse sources. The wild species of Oryza representing AA, BB, CC, BBCC, CCDD, EE, FF, GG and HHJJ genomes are an important reservoir of useful genes. However, low crossability and limited recombination between chromosomes of cultivated and wild species limit the transfer of such genes. At IRRI, a series of hybrids and monosomic alien addition lines have been produced through embryo rescue following hybridization between rice and several distantly related species. Cytoplasmic male sterility and genes for resistance to grassy stunt virus and bacterial blight have been transferred from A genome wild species into rice. Similarly, genes for resistance to brown planthopper, bacterial blight and blast have also been introgressed across crossability barriers from distanly related species into rice. Some of the introgressed genes have been mapped via linkage to molecular markers. One of the genes Xa-21 introgressed from O. longistaminata has been cloned and physically mapped on chromosome 11 of rice using BAC library and flourescence in-situ hybridization. RFLP analysis revealed introgression from 11 of the 12 chromosomes of C genome species into rice. Introgression has also been obtained from other distant genomes (EE, FF, GG) into rice and in majority of the cases one or two RFLP markers were introgressed. Reciprocal replacement of RFLP alleles of wild species with the alleles of O. sativa indicates alien gene transfer through crossing over. The rapid recovery of recurrent phenotypes in BC₂ and BC₃ generations from wide crosses is an indication of limited recombination. Further cytogenetic and molecular investigations are required to determine precisely the mechanism of introgression of small chromosome segments from distant genomes in the face of limited homoeologous chromosome pairing. Future research should focus on enhancing recombination between homoeologous chromosomes. Introgression of QTL from wild species should be attempted to increase the yield potential of rice.     

Genome‐specific introgression between wheat and its wild relative Aegilops triuncialis

Introgression of sequences from crop species in wild relatives is of fundamental and practical concern. Here, we address gene flow between cultivated wheat and its widespread polyploid relative, Aegilops triuncialis, using 12 EST‐SSR markers mapped on wheat chromosomes. The presence of wheat diagnostic alleles in natural populations of the barbed goatgrass growing in proximity to cultivated fields highlights that substantial gene flow occurred when both species coexisted. Furthermore, loci from the A subgenome of wheat were significantly less introgressed than sequences from other subgenomes, indicating differential introgression into Ae. triuncialis. Gene flow between such species sharing nonhomeologous chromosomes addresses the evolutionary outcomes of hybridization and may be important for efficient gene containment.     

天然杂交与遗传渐渗对植物入侵性的影响

生物入侵给全球生态环境与社会经济都带来了严重危害, 对其入侵机制的研究非常重要。生物入侵是一个适应性进化的过程, 天然杂交与遗传渐渗可以改变外来物种对环境的适应性并提高其入侵能力, 使其进化成为入侵种。因此了解杂交-渐渗在促进生物入侵过程中的遗传作用, 将有助于我们采取有效措施来控制生物入侵及其危害。本文从杂交-渐渗对生物适应性进化和物种形成影响的角度, 阐明外来种如何通过杂交-渐渗在新的生境中改变其适应性、生存竞争能力和入侵能力。杂交-渐渗可以导致物种发生多倍体水平和同倍体水平的进化, 虽然二者的进化过程不尽相同, 但均能使杂种群体在遗传上产生较大变化, 进而影响杂种群体的适合度, 这一过程可能促使外来种在新的生境中的成功入侵进而转变为入侵种。随着转基因生物技术的迅速发展, 大量转基因作物进入环境释放和商品化种植, 具有特定功能的转基因可能通过杂交-渐渗进入野生近缘种群体, 也可能使之成为入侵性强的农田杂草, 带来难以预测的生态后果。总之, 生物入侵是一个复杂的进化和生态过程, 利用杂交-渐渗的理论来解释植物的入侵性, 仅从一个方面反映了入侵生物学的研究, 杂交-渐渗与其他理论的结合, 将从更深的层次来解释外来种的入侵机制。     

Targeted introgression of a wheat stem rust resistance gene by DNA marker-assisted chromosome engineering

Chromosome engineering is a useful strategy for transfer of alien genes from wild relatives into modern crops. However, this strategy has not been extensively used for alien gene introgression in most crops due to low efficiency of conventional cytogenetic techniques. Here, we report an improved scheme of chromosome engineering for efficient elimination of a large amount of goatgrass (Aegilops speltoides) chromatin surrounding Sr39, a gene that provides resistance to multiple stem rust races, including Ug99 (TTKSK) in wheat. The wheat ph1b mutation, which promotes meiotic pairing between homoeologous chromosomes, was employed to induce recombination between wheat chromosome 2B and goatgrass 2S chromatin using a backcross scheme favorable for inducing and detecting the homoeologous recombinants with small goatgrass chromosome segments. Forty recombinants with Sr39 with reduced surrounding goatgrass chromatin were quickly identified from 1048 backcross progenies through disease screening and molecular marker analysis. Four of the recombinants carrying Sr39 with a minimal amount of goatgrass chromatin (2.87-9.15% of the translocated chromosomes) were verified using genomic in situ hybridization. Approximately 97% of the goatgrass chromatin was eliminated in one of the recombinants, in which a tiny goatgrass chromosome segment containing Sr39 was retained in the wheat genome. Localization of the goatgrass chromatin in the recombinants led to rapid development of three molecular markers tightly linked to Sr39. The new wheat lines and markers provide useful resources for the ongoing global effort to combat Ug99. This study has demonstrated great potential of chromosome engineering in genome manipulation for plant improvement.     

Genome‐specific histories of divergence and introgression between an allopolyploid unisexual salamander lineage and two ancestral sexual species

Quantifying introgression between sexual species and polyploid lineages traditionally thought to be asexual is an important step in understanding what drives the longevity of putatively asexual groups. Here, we capitalize on three recent innovations—ultraconserved element (UCE) sequencing, bioinformatic techniques for identifying genome‐specific variation in polyploids, and model‐based methods for evaluating historical gene flow—to measure the extent and tempo of introgression over the evolutionary history of an allopolyploid lineage of all‐female salamanders and two ancestral sexual species. Our analyses support a scenario in which the genomes sampled in unisexual salamanders last shared a common ancestor with genomes in their parental species ～3.4 million years ago, followed by a period of divergence between homologous genomes. Recently, secondary introgression has occurred at different times with each sexual species during the last 500,000 years. Sustained introgression of sexual genomes into the unisexual lineage is the defining characteristic of their reproductive mode, but this study provides the first evidence that unisexual genomes have undergone long periods of divergence without introgression. Unlike other sperm‐dependent taxa in which introgression is rare, the alternating periods of divergence and introgression between unisexual salamanders and their sexual relatives could explain why these salamanders are among the oldest described unisexual animals.     

Nucleotide diversity in the two co-resident genomes of allopolyploid cotton

Genetic diversity within and among populations lies at the heart of evolution. Unraveling the extent to which each intrinsic or extrinsic factor determines levels of diversity among genes, populations, and species is challenging, given the difficulty of isolating any single potentially important variable from all others. Allopolyploid species provide an opportunity to disentangle external and intrinsic factors, as the two (or more) homoeologous genomes co-occur in the same nucleus, often exhibiting high collinearity along homoeologous chromosomes. Here we evaluate the pace of molecular evolution and intraspecific, intragenomic diversity in two species of allopolyploid Gossypium, G. hirsutum and G. barbadense, using several hundred genes sequenced from multiple accessions of each species. Genic diversity in both species is low, having been influenced both by the polyploid bottleneck and a domestication bottleneck (for cultivated accessions), but with a directional bias in homoeolog diversity favoring the same genome in both allopolyploids. Total diversity is remarkably similar for the two homoeologous genomes overall, but the two copies of many gene pairs have accumulated statistically different diversity levels, and in a biased fashion with respect to genome. Domesticated accessions show reduced diversity in both genomes, as expected, but with a much greater reduction in one of the two homoeologous genomes. Furthermore, this biased reduction affects opposite homoeologous genomes in the two species. Interspecific introgression has played a role in shaping diversity within each species. Introgression was only detected for certain accessions, and only from G. barbadense into G. hirsutum in one of the two co-resident genomes.     

小麦-鹅观草第一部分同源群染色体渗入系鉴定与基因组归属分析

鹅观草（Roegneria kamoji,2n=42,SSHHYY）是小麦异源六倍体野生近缘种,对小麦赤霉病具有良好抗性,是改良小麦赤霉病抗性的重要遗传资源。通过远缘杂交,将鹅观草第一部分同源群染色体上的抗赤霉病基因Fhb6导入普通小麦。由于第一部分同源群染色体包含1S、1H和1Y三条染色体,为研究这些同源染色体对小麦赤霉病抗性的影响,筛选出4个鹅观草第一部分同源群染色体特异分子标记,通过PCR扩增鹅观草属不同野生种的基因组DNA,明确了抗赤霉病Fhb6基因位于鹅观草1Y#1染色体。进一步利用分子细胞遗传学技术从中国春与鹅观草的后代中选育出5份涉及鹅观草1Y#2和1S#2染色体的渗入系材料。其中：21RK?1为二体异代换系DS1Y#2(1A),21RK?2为二体异代换系DS1S#2（1D）,21RK?3为二体异附加系DA1S#2,21RK?4为1S#2和TW·1S#2S的双单体附加系,21RK?5为纯合TW·1S#2S易位系。这些新种质为小麦抗赤霉病基因的发掘及遗传改良奠定了基础。     

Rapid Elimination of Low-Copy DNA Sequences in Polyploid Wheat: A Possible Mechanism for Differentiation of Homoeologous Chromosomes

To study genome evolution in allopolyploid plants, we analyzed polyploid wheats and their diploid progenitors for the occurrence of 16 low-copy chromosome- or genome-specific sequences isolated from hexaploid wheat. Based on their occurrence in the diploid species, we classified the sequences into two groups: group I, found in only one of the three diploid progenitors of hexaploid wheat, and group II, found in all three diploid progenitors. The absence of group II sequences from one genome of tetraploid wheat and from two genomes of hexaploid wheat indicates their specific elimination from these genomes at the polyploid level. Analysis of a newly synthesized amphiploid, having a genomic constitution analogous to that of hexaploid wheat, revealed a pattern of sequence elimination similar to the one found in hexaploid wheat. Apparently, speciation through allopolyploidy is accompanied by a rapid, nonrandom elimination of specific, low-copy, probably noncoding DNA sequences at the early stages of allopolyploidization, resulting in further divergence of homoeologous chromosomes (partially homologous chromosomes of different genomes carrying the same order of gene loci). We suggest that such genomic changes may provide the physical basis for the diploid-like meiotic behavior of polyploid wheat.     

Current knowledge of gene flow in plants: implications for transgene flow

Plant evolutionary biologists' view of gene flow and hybridization has undergone a revolution. Twenty-five years ago, both were considered rare and largely inconsequential. Now gene flow and hybridization are known to be idiosyncratic, varying with the specific populations involved. Gene flow typically occurs at evolutionarily significant rates and at significant distances. Spontaneous hybridization occasionally has important applied consequences, such as stimulating the evolution of more aggressive invasives and increasing the extinction risk for rare species. The same problems have occurred for spontaneous hybridization between crops and their wild relatives. These new data have implications for transgenic crops: (i) for most crops, gene flow can act to introduce engineered genes into wild populations; (ii) depending on the specific engineered gene(s) and populations involved, gene flow may have the same negative impacts as those observed for traditionally improved crops; (iii) gene flow's idiosyncratic nature may frustrate management and monitoring attempts; and (iv) intercrop transgene flow, although rarely discussed, is equally worthy of study.     

Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae

Key message

A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement.

Abstract

To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1U-6U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.     

Rapid identification of homozygosity and site of wild relative introgressions in wheat through chromosome‐specific KASP genotyping assays

For future food security, it is important that wheat, one of the most widely consumed crops in the world, can survive the threat of abiotic and biotic stresses. New genetic variation is currently being introduced into wheat through introgressions from its wild relatives. For trait discovery, it is necessary that each introgression is homozygous and hence stable. Breeding programmes rely on efficient genotyping platforms for marker‐assisted selection (MAS). Recently, single nucleotide polymorphism (SNP)‐based markers have been made available on high‐throughput Axiom^® SNP genotyping arrays. However, these arrays are inflexible in their design and sample numbers, making their use unsuitable for long‐term MAS. SNPs can potentially be converted into Kompetitive allele‐specific PCR (KASP?) assays that are comparatively cost‐effective and efficient for low‐density genotyping of introgression lines. However, due to the polyploid nature of wheat, KASP assays for homoeologous SNPs can have difficulty in distinguishing between heterozygous and homozygous hybrid lines in a backcross population. To identify co‐dominant SNPs, that can differentiate between heterozygotes and homozygotes, we PCR‐amplified and sequenced genomic DNA from potential single‐copy regions of the wheat genome and compared them to orthologous copies from different wild relatives. A panel of 620 chromosome‐specific KASP assays have been developed that allow rapid detection of wild relative segments and provide information on their homozygosity and site of introgression in the wheat genome. A set of 90 chromosome‐nonspecific assays was also produced that can be used for genotyping introgression lines. These multipurpose KASP assays represent a powerful tool for wheat breeders worldwide.     

Engineering of interstitial foreign chromosome segments containing the K+/Na+ selectivity gene Kna1 by sequential homoeologous recombination in durum wheat

Targeted homoeologous recombination mediated by the absence of the Ph1 locus is currently the most efficient technique by which foreign genes can be introgressed into polyploid wheat species. Because intra-arm homoeologous double cross-overs are rare, introgressed foreign genes are usually on terminal foreign chromosome segments. Since the minimum length of such a segment is determined by the position of a gene in the chromosome, large chromosome segments with undesirable genetic effects are often introgressed. Introgression of foreign genes on short interstitial segments based on two cycles of homoeologous recombination is described here. The utility of the technique is demonstrated by the introgression of the Kna1 locus, which controls K⁺/Na⁺ selectivity in T. aesivum L., on short interstitial segments of chromosome 4D into chromosome 4B of Triticum turgidum L. The level of recombination in a homoeologous segment is not significantly affected by a juxtaposed proximal homologous segment in the absence of the Ph1 locus.     

Genome differentiation in Aegilops. 1. Distribution of highly repetitive DNA sequences on chromosomes of diploid species.

Genome differentiation in 12 diploid Aegilops species was analyzed using in situ hybridization with the highly repetitive DNA sequences pSc119 and pAs1 and C-banding. Chromosomes of all these diploid Aegilops species hybridized with the pSc119 probe; however, the level of hybridization and labeling patterns differed among genomes. Only four species (Ae. squarrosa, Ae. comosa, Ae. heldreichii, and Ae. uniaristata) showed distinct hybridization with pAs1. The labeling patterns were species-specific and chromosome-specific. Differences in in situ hybridization (ISH) patterns, also observed by C-banding, exist between the karyotypes of Ae. comosa and Ae. heldreichii, suggesting that they are separate, although closely related, subspecies. The S genome of Ae. spelioides was most similar to the B and G genomes of polyploid wheats on the basis of both C-banding and ISH patterns, but was different from other species of section Sitopsis. These species had different C-banding patterns but they were similar to each other and to Ae. mutica in the distribution of pSc119 hybridization sites. Two types of labeling were detected in Ae. squarrosa with the pAs1 probe. The first resembled that of the D-genome chromosomes of bread wheat, Triticum aestivum L. em. Thell., while the second was similar to the D genome of some of the polyploid Aegilops species. Relationships among diploid Aegilops species and the possible mechanisms of genome differentiation are discussed. Key words : wheat, Triticum, Aegilops, in situ hybridization, C-banding, evolution.     

Chromosome isolation by flow sorting in Aegilops umbellulata and Ae. comosa and their allotetraploid hybrids Ae. biuncialis and Ae. geniculata

This study evaluates the potential of flow cytometry for chromosome sorting in two wild diploid wheats Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata. Flow karyotypes obtained after the analysis of DAPI-stained chromosomes were characterized and content of chromosome peaks was determined. Peaks of chromosome 1U could be discriminated in flow karyotypes of Ae. umbellulata and Ae. biuncialis and the chromosome could be sorted with purities exceeding 95%. The remaining chromosomes formed composite peaks and could be sorted in groups of two to four. Twenty four wheat SSR markers were tested for their position on chromosomes of Ae. umbellulata and Ae. comosa using PCR on DNA amplified from flow-sorted chromosomes and genomic DNA of wheat-Ae. geniculata addition lines, respectively. Six SSR markers were located on particular Aegilops chromosomes using sorted chromosomes, thus confirming the usefulness of this approach for physical mapping. The SSR markers are suitable for marker assisted selection of wheat-Aegilops introgression lines. The results obtained in this work provide new opportunities for dissecting genomes of wild relatives of wheat with the aim to assist in alien gene transfer and discovery of novel genes for wheat improvement.     

Phylogeny and molecular evolution of the DMC1 gene in the polyploid genus Leymus (Triticeae: Poaceae) and its diploid relatives

The level and pattern of nucleotide variation in duplicate genes provide important information on the evolutionary history of polyploids and divergent processes between homoeologous loci within lineages. Leymus, a group of allopolyploid species with the NsXm genomes, is a perennial genus with a diverse array of morphology, ecology, and distribution in Triticeae. To estimate the phylogeny and molecular evolution of a single-copy DMC1 gene in Leymus and its diploid relatives,DMC1 homoeologous sequences were isolated from the sampled Leymus species and were analyzed with those from 30 diploid taxa representing 18 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that: (i) different Leymus species might derive their Ns genome from different Psathyrostachys species; (ii) Pseudoroegneria has contributed to the nuclear genome of some Leymus species, which might result from recurrent hybridization or incomplete lineage sorting; (iii) the Xm genome origin of Leymus could differ among species; (iv) rapid radiation and multiple origin might account for the rich diversity, numbers of species, and wide ecological adaptation of Leymus species; and (v) the DMC1 sequence diversity of the Ns genome in Leymus species was lower than that in the Psathyrostachys diploids, while the level of DMC1 sequence diversity in Leymus was higher than that in diploid Pseudoroegneria. Our results provide new insight on the evolutionary dynamics of duplicate DMC1 genes, polyploid speciation, and the phylogeny of Leymus species.     

Genome editing of polyploid crops: prospects,achievements and bottlenecks

Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted.

     

Repetitive DNA variation and pivotal-differential evolution of wild wheats.

Several polyploid species in the genus Triticum contain a U genome derived from the diploid T. umbellulatum. In these species, the U genome is considered to be unmodified from the diploid based on chromosome pairing analysis, and it is referred to as pivotal. The additional genome(s) are considered to be modified, and they are thus referred to as differential genomes. The M genome derived from the diploid T. comosum is found in many U genome polyploids. In this study, we cloned three repetitive DNA sequences found primarily in the U genome and two repetitive DNA sequences found primarily in the M genome. We used these to monitor variation for these sequences in a large set of species containing U and M genomes. Investigation of sympatric and allopatric accessions of polyploid species did not show repetitive DNA similarities among sympatric species. This result does not support the idea that the polyploid species are continually exchanging genetic information through introgression. However, it is also possible that repetitive DNA is not a suitable means of addressing the question of introgression. The U genomes of both diploid and polyploid U genome species were similar regarding hybridization patterns observed with U genome probes. Much more variation was found both among diploid T. comosum accessions and polyploids containing M genomes. The observed variation supports the cytogenetic evidence that the M genome is more variable than the U genome. It also raises the possibility that the differential nature of the M genome may be due to variation within the diploid T. comosum, as well as among polyploid M genome species and accessions.