ГЕНЕТИКА, 2014, том 50, № 6, с. 692-699
ГЕНЕТИКА РАСТЕНИЙ
УДК 581.6
DEVELOPMENT OF 5Ns CHROMOSOME-SPECIFIC SCAR MARKERS FOR UTILIZATION IN FUTURE WHEAT BREEDING PROGRAMS
© 2014 J. Wang, L. M. Wang, W. L. Du, L. G. Chen, S. H. Liu, J. Wu, J. X. Zhao, Q. H. Yang, X. H. Chen
Shaanxi Key Laboratory of Genetic Engineering for Plant Breeding, College of Agronomy, Northwest A&F University, 712100 Yangling, Shaanxi, P.R. China e-mail: cxh2089@126.com Received November 14, 2013
In previous studies, we developed a wheat—Psathyrostachys huashanica Keng disomic addition line 3-8-10-2, which exhibited high stripe rust resistance and could be used as a donor source for introducing novel disease resistance gene(s) into wheat in future breeding programs. It was identified using cytology, genomic in situ hybridization (GISH), EST-SSR, EST-STS and morphological analyses. However, these techniques are not suitable for breeding programs that require the rapid screening of large numbers of genotypes because they are highly technical and time-consuming. In this study, three Ns genome-specific SCAR markers were developed via random amplified polymorphic DNA (RAPD) markers. These SCAR markers were further validated using a complete set of wheat—P. huashanica disomic addition lines, which segregated the 5Ns disomic addition line individuals. Our results indicated that the SCAR markers associated with the 5Ns chromosome of P. huashanica and they provide a low cost, high efficiency, alternative tool for screening 5Ns chromosomes in a wheat background. These newly developed SCAR markers that species-specificity of the markers was proved by analysis of a wide range of cereal species, and specific for 5Ns chromosome, which should be useful in marker-assisted selection for wheat breeders who want to screen genotypes that may contain 5Ns chromatin.
DOI: 10.7868/S0016675814060137
Molecular techniques, especially PCR-based markers, are useful for developing highly reliable, specific, sensitive, and rapid detection methods [1], while they also facilitate the mass screening of populations at a low cost [2]. Various molecular markers have been developed successfully for targeting specific chromosomes and genes. However, the use of AFLP markers is tedious and time-consuming because it involves many steps in a complex process [3], so it needs to be converted to produce more usable markers such as sequence-characterized amplified region (SCAR) markers. Random amplified polymorphic DNA (RAPD) markers are fairly simple, high speed, low cost, and they require no prior knowledge of the sequence so they can be produced using a small amount of template DNA [4, 5], but their main weakness is poor reproducibility [6]. To increase the reproducibility and reliability of PCR assays, SCAR markers have been developed from sequences of RAPD fragments. RAPD has the advantages of speed, low cost, and it supports high levels of polymorphism, so it has been used for the identification of crops [7].
Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) is a perennial cross-pollinating plant (subfamily Pooideae, tribe Triticeae, family Poaceae), which is distributed in the Huashan Mountains, Shaanxi Province, China [8—10]. It is an endemic species in China where it has been listed as an endangered and highly protected wild species [11]. It has many superior char-
acters, such as early maturity, resistance to biotic stresses (scab, stripe rust, take-all, and powdery mildew), and tolerance of abiotic stresses (cold, salinity, drought, and barren conditions)[10, 12—18].
Our research team successfully produced an F1 hybrid [12] and heptaploid hybrid [19] ofwheat—P. huashanica via embryo culture, two backcrossings, and selfing. Next, strict single-selfing was performed and, after testing based on cytology, genomic in situ hybridization (GISH), EST-SSR, EST-STS, and glutenin, gliadin and morphology analyses, a complete set of wheat—P. huashanica disomic addition lines (2n = 44 = = 22 II) were developed. One of these, the 5Ns disomic addition line, was found to resist a blend of different races of Puccinia striiformis f. sp. tritici (CYR31, CYR32, and SY11-14) as an adult plant, while it also had a better setting percentage than its female parent common wheat [17]. Therefore, we concluded that it had received novel gene(s) from P. huashanica. However, the process used to identify the 5Ns disomic addition line was complex, highly technical, expensive, and time-consuming. Thus, there is an urgent need to develop a rapid and simple method for detecting the 5Ns chromosome of P. huashanica.
The use of molecular markers can improve the efficiency of conventional plant breeding via the selection of a marker, or markers, associated with the trait of interest [20]. SCAR markers have obvious advantages
Table 1. Study species, ploidy level, genomic constitution, and origin
Species Ploidy Genome
7182 (Triticum aestivum L.) 6x AABBDD
Psathyrostachys huashanica Keng 2x NsNs
Triticum monococcum L. 2x AA
Triticum dicoccoides Korn. 4x AABB
Triticum araraticum Jakubz. 4x AAGG
Triticum zhukovskyi Men. et Er. 6x AAAAGG
Aegilops markgrafii (Greuter) Hammer 2x CC
Aegilops tauschii (Coss.) Schmal. 2x DD
Thinopyrum elongatum 2x EE
Hordeum violaceum 2x HH
Crithopsis delileana (Schult) Roshev 2x KK
Aegilops comosa Sm. in Sibth. & Sm. 2x MM
Agropyron cristatum Gaertn. 6x PPPPPP
Eremopyrum orientale 4x B'B'C'C'
Triticum timopheevii Zhuk. 4x AtAtGG
Secale cereale L. 2x RR
Aegilops speltoides Tausch 2 x SS
Roegneria ciliaris (Trin) Nevski 4x SSYY
Elymus rectisetus 6x SSYYWW
Pseudoroegneria strigosa A. Love 2x StSt
Roegneria grandiglumis Keng 6x StStPPYY
Origin
Common wheat cultivars
Rare species
Wild relative species
Our research group
The Chinese Academy of Agricultural Sciences
over RAPD markers, although they are not highly polymorphic [21, 22]. In this study, we analyzed RAPD patterns to develop three reliable SCAR markers to discriminate the 5Ns chromosome of P. huas-hanica in a wheat background. This is the first report of the development of specific sequences and molecular markers for the detection of the P. huashanica 5Ns chromosome. These SCAR markers were tightly linked to the 5Ns chromosome, so it solved the problem of its rapid detection in a large genetic pool. The identification of molecular markers is also necessary for marker-assisted selection (MAS). Furthermore, the development of SCAR markers for screening the 5Ns chromosome in wheat—P. huashanica populations can extend the industrial application of the molecular techniques used for current wheat germplasm research.
The main objective of this study was to develop and characterize three RAPD markers from the P. huashanica Ns genome and to convert them into SCAR markers to specifically detect the 5Ns chromosome in a complete set of wheat-P. huashanica disomic addition lines. These markers will be useful for wheat breeders who aim to improve wheat by utilizing genetic resources from the P. huashanica 5Ns chromosome.
MATERIALS AND METHODS
Plant materials. Nineteen distinct species and the two parental lines of common wheat cv. 7182 (AABBDD, 2n = 6x = 42) and Psathyrostachys huashanica Keng (accession number 0503383, NsNs, 2n = = 2x = 14) were analyzed in this study (Table 1), and also used as the controls in the RAPD analysis. These resources were provided partly by the Center for Crop Germplasm Resources Research (CGRR) at the Institute of Crop Science, CAAS, Beijing, China. A complete set of wheat—P. huashanica disomic addition lines (1Ns—7Ns, 2n = 44 = 22 II) were used for the chromosome validation of P. huashanica-specific SCAR markers, as shown in Table 2. These specimens were deposited at the Shaanxi Key Laboratory ofGenetic Engineering for Plant Breeding, College of Agronomy, Northwest A&F University, Shaanxi, China.
DNA extraction. Plant genomic DNA was extracted according to the standard cetyltrimethylammonium bromide (CTAB) methodology [23] and the samples were kept in sterilized double-distilled water. The quality and concentration of the DNA were checked using 1% agarose gels and an ultraviolet spectrophotometer (NanoDrop® ND-1000, USA), respectively.
RAPD analysis and marker selection. Two hundred decamer random primers (unpublished data) were used for RAPD screening to identify P. huashanica-
Table 2. The genetic constitution of the complete set of wheat—P. huashanica disomic addition lines (1Ns—7Ns, 2n = 44 = 22II) and its parents, common wheat cv. 7182 and P. huashanica
Plant code 2n Chromosome composition (P. huashanica homoelogous pair)
P. huashanica 14 14Ns
7182 42 42W
12-3 44 42W + 2Ns (1)
3-6-4-1 44 42W + 2Ns (2)
22-2 44 42W + 2Ns (3)
24-6-3 44 42W + 2Ns (4)
3-8-10-2 44 42W + 2Ns (5)
59-11 44 42W + 2Ns (6)
2-1-6-3 44 42W + 2Ns (7)
Note: Ns and W, P. huashanica and wheat chromosomes, respectively. The complete set of wheat—P. huashanica disomic addition lines were verified by morphology, cytology, genomic in situ hybridization (GISH), EST-SSR, EST-STS, glutenin and gliadin analyses.
specific markers based on the genomic DNA from 21 distinct species samples. The RAPD amplification reactions were performed in a 20 |L reaction mixture containing 2 |L 10x PCR buffer, 5 |L primer (2.5 |mol/mL), 5 |L DNA template (50-100 |g/|L), 1.6 |L dNTPs (2.5 |imol/mL), 1.6 |L MgCl2 (2.5 mmol/mL), 0.1 |L Taq polymerase (5 U/|L), and 4.7 | L ddH2O. The PCR program comprised 4 min at 94°C, 45 cycles at 94°C for 30 s, 45 s at 32°C, 1.5 min at 72°C, and a final 10 min extension at 72°C. The amplification products were separated by electro-phoresis using 1% agarose gels with 1x TAE buffer and the products were visualized using ethidium bromide.
Cloning and sequencing of P. huashanica-specific RAPD products, and sequence homology analyses. The P. huashanica-specific RAPD products amplified from 21 genome samples using 200 random primers were excised from the agarose gel and extracted
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.