MOXEKymPHAa EHomrm, 2011, moM 45, № 2, c. 231-237
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IDENTIFICATION OF EXOTIC GENETIC COMPONENTS AND DNA METHYLATION PATTERN ANALYSIS OF THREE COTTON INTROGRESSION
LINES FROM Gossypium bickii © 2011 Shou-Pu He, Jun-Ling Sun, Chao Zhang, Xiong-Ming Du*
Key Laboratory of Cotton Genetic Improvement, Ministry of Agriculture; Cotton Research Institute, Chinese Academy of Agricultural Sciences, Anyang, Henan, 455004, China Received March 11, 2010 Accepted for publication May 11, 2010
The impact of alien DNA fragments on plant genome has been studied in many species. However, little is known about the introgression lines of Gossypium. To study the consequences of introgression in Gossypium, we investigated ~2000 genomic and ~800 epigenetic sites in three typical cotton introgression lines, as well as their cultivar (Gossypium hirsutum) and wild parents (Gossypium bickii), by amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP). The results demonstrate that an average of 0.5% of exotic DNA segments from wild cotton is transmitted into the genome of each introgression line, with the addition of other forms of genetic variation. In total, an average of 0.7% of genetic variation sites is identified in introgression lines. Simultaneously, the overall cytosine methylation level in each introgression line is very close to that of the upland cotton parent (an average of 22.6%). Further dividing patterns reveal that both hypomethylation and hypermethylation occurred in introgression lines in comparison with the upland cotton parent. Sequencing of nine methylation polymorphism fragments showed that most (7 of 9) of the methylation alternations occurred in the noncoding sequences. The molecular evidence of introgression from wild cotton into introgression lines in our study is identified by AFLP. Moreover, the causes of petal variation in introgression lines are discussed.
Keywords: Gossypium hirsutum, Gossypium bickii, introgression, AFLP, MSAP.
Interspecific hybridization occurs widely during the evolution history of a plant [1]. In breeding, interspecific hybridization can transfer adaptive traits into cultivar varieties and create novel genotypes/phenotypes [2]. Therefore, interspecific crossing and backcrossing have been widely used in the history ofplant breeding. Despite many difficulties existing in interspecific breeding (such as cross-incompatibility, F1 sterility, etc.), a large number of elite germplasm with exotic traits has been created and selected by this approach for decades [3].
Introgression was first mentioned and discussed by Anderson [4]. This is a phenomenon accompanied by interspecific hybridization occurring in most natural species [5]. In practical applications, introgression is used to describe the exchange of genes from donor species to target cultivars [2]. Therefore, cultivars selected from the progeny of interspecific hybridization are called "introgression lines." At present, the germplasm of cotton contains a large number of introgression lines created by several important cotton-improving programs in the past decades. For instance, Pee Dee lines are a series ofupland cotton with excellent fiber quality and high yield, which introgressed consanguinity of Gossypium ar-broeum, G. thurberi, and G. barbadense [6]. Moreover,
* E-mail: duxm@cricaas.com.cn
other cotton wild relatives are also used as donors for transferring useful traits in breeding [7]. In China, the study of interspecific hybridization of cotton has been conducted by several research institutes since the 1970s. In the following decades, a large quantity ofvarieties with excellent exotic properties was selected by breeders. Most of these varieties were later used as germplasm for further breeding programs.
DNA methylation is widely spread in all species, especially in high plants, and over 30% of cytosine residues are methylated [8, 9]. Most of the critical fundamental metabolic processes and phenotype variations are proven to be closely relevant with DNA methylation [10]. In high plants, gain and loss of DNA methylation at trans-poson sites is considered as a switch to control the activation process of transposons in plants [11]. In addition, DNA methylation shows the dynamic status in the different development stages of the plant. Ruiz-Garcia et al. [13] reported an increasing trend in DNA methylation from different stages of organs in Arabidopsis. This evidence implies that DNA methylation plays a critical role in the entire plant development.
Although the great improving effects of interspecific hybridization have been proven in many other species, leading to it being used as a common breeding method for many years [14—18], its mechanism is not well
K181
Gossypium bickii
Z2
Z8
Z9
Petal comparison of three ZYH introgression lines and their parents.
understood. In several recent studies, researchers attempted to assess the extensive genetic and epigenetic alterations in the initial stage of genome merging in plants by amplified fragment length polymorphism (AFLP) [19] and methylation-sensitive amplified polymorphism (MSAP) [20]. The results showed that almost all of the studied species are under genome-wide genetic and epigenetic regulation [21—25]. On the other hand, some studies also tried to explain introgression phenomena basing on molecular approaches. Exotic genome components were successfully detected in the introgression lines ofcoffee [26], rice [23], and cucumber [27] by AFLP technology. Consequently, all studies above demonstrated that AFLP and MSAP are feasible and efficient in analyzing genetic and epigenetic phenomena in plants.
In this paper, five samples, including a set of typical cotton introgression lines with Gossypium bickii consanguinity, and their parents were studied. The introgression lines possess the typical phenotype of G. bickii. There are two main purposes of this study: a) to determine whether exotic DNA components are integrated into the genome of these introgression lines and b) to estimate the genetic and epigenetic variations in cotton introgression lines and discuss their possible impacts.
EXPERIMENTAL
Plant material sampling and DNA extraction. Three cotton introgression lines with G. bickii consanguinity called " hirsutum-bickii red flower line" (HBRL), including Zhongyihong 2 (Z2), 8 (Z8), and 9 (Z9), which were bred and selected by Liang from 1989 to 1994 [28, 29], and their original parents, cultivar parent "Keyi181" (K181; G. hirsutum, also used as a backcross parent) and wild parent G. bickii, were used in this experiment. Distinguished from common upland cotton, the specific phenotype of these introgression lines was a pink petal with a purple spot inside, which looked more like the petal of their wild parent (Figure).
All five cotton germplasms were maintained by strictly selfing and then planted in the greenhouse of the Cotton Research Institute, Chinese Agricultural Academy of Sciences in Anyang China in 2008. The young seedlings
were sampled for DNA extraction by using the Cetyl tri-methylammonium bromid (CTAB) method [30].
AFLP analysis. As a highly sensitive technology, AFLP provided the richest polymorphism in fingerprinting analysis compared to other techniques [18, 31]. Standard AFLP analysis was performed to assess the genetic variations of introgression lines during introgression as compared to their parents by the protocol described by Vfos [19] with minor modifications for higher yield of PCR production. The purified DNA was digested in 37°C for 3 h (longer than the original procedure) with excessive amounts of restriction enzymes (since cotton has a more complex genome) for obtaining completely digested templates.
Ultimately, the denatured PCR products were separated on an 8% denaturing polyacrylamide gel for 1.5 h at 75 W The gels were stained by the silver stain method, and the band patterns were transformed to a "1, 0" matrix for statistics analysis ("1" indicates the presence of a band and "0" indicates the absence of a band).
MSAP analysis. We used the MSAP protocol described by Xu et al. [32]. The reaction components and procedures were exactly the same as for the AFLP analysis above. Technically, the only difference between AFLP and MSAP is the substitution of a pair of isoschizomers, Hpall and Mspl, for EcoRI.
MSAP fragment sequencing. Distinct and repeatable MSAP fragments were chosen for sequencing. Fragments were carefully cut out ofthe gel by a knife and then "smashed" into tiny splinters in a 1.5 ml centrifugal tube. 20 (l of double-distilled water were added to the tube and the mixture was heated in boiling water for 10 min. 5 (l of the solution in the tube were used as a template to ream-plify the target fragments using primer combinations. The fragments were separated in a 0.8% agarose gel, extracted, and ligated into the T-vector (Promega). The cloned DNA segments were sequenced with vector primers by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. Analysis of the similarity of se-quenced fragments was performed at the National Center for Biotechnology Information using the BLASTN program.
Table 1. Genetic variations in introgression lines based on AFLP
K181 Gossypium bickii Introgression lines Z2 Z8 Z9
+ + + 1134 1130 1128
+ - + 877 880 882
- + + 5 16 12
- - + 1 5 2
+ + - 0 2 1
Note: Here and in Table 2 "+"— indicates the presence of a band; "—" — indicates the absence of a band.
Table 2. Classification of the CCGG site status by different band patterns displayed on the gel
Classification Site status HpaII MspI E + H E + M
Class I CCGG Active Active + +
GGCC
Class II mCCGG Active Inactive + -
GGCC
Class III CmCGG Inactive Active - +
GGmCC
Not distinguishable mCCGGm CmCGG Inactive Inactive - -
GGCmC GGmCmC
Note: E + H — indicates that the DNA was digested by E
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