научная статья по теме DEVELOPMENT OF BORON-EFFICIENT NEAR ISOGENIC LINES OF BRASSICA NAPUS AND THEIR RESPONSE TO LOW BORON STRESS AT SEEDLING STAGE Биология

Текст научной статьи на тему «DEVELOPMENT OF BORON-EFFICIENT NEAR ISOGENIC LINES OF BRASSICA NAPUS AND THEIR RESPONSE TO LOW BORON STRESS AT SEEDLING STAGE»

ГЕНЕТИКА, 2010, том 46, № 1, с. 66-72

ГЕНЕТИКА РАСТЕНИЙ

УДК 575.1:582.683.2

DEVELOPMENT OF BORON-EFFICIENT NEAR ISOGENIC LINES OF Brassica napus AND THEIR RESPONSE TO LOW BORON STRESS AT SEEDLING STAGE

© 2010 г. H. Zhao1,2, J. Liu1, L. Shi1,2, F. Xu1,2, Y. Wang2

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China;

e-mail: fangsenxu@mail.hzau.edu.cn 2Microelement Research Centre, Huazhong Agricultural University, Wuhan 430070, China

Received November 05, 2008

Rapeseed (Brassica napus) is sensitive to low boron (B) stress and plentiful variation exists in response to B deficiency. One major QTL, BE1, and three minor loci controlling B efficiency in Brassica napus were previously detected. To fine map and clone the B-efficient gene (s), the development of B-efficient NILs in Brassica napus was conducted, combining the identification of B efficiency at seedling stage with genetic background selection using random AFLP markers. The molecular marker assisted background selection proved its optimum and necessary in an early backcrossing generation to select the backcross individuals with high genetic background similarity to accelerate the construction of NILs. Based on B efficiency investigated at seedling stage under the low B conditions, the B-efficient backcross line can produce biomass twice about the B-inefficient parent's and show low B concentration and effective utilization of B under low B condition. Thus, the B efficiency of the B efficient NILs might be attributed to the higher B utilization efficiency or less demand for B.

Boron (B) has long been known to be an essential microelement for higher plant growth and development [1]. Boron deficiency significantly impairs cell elongation in growing tissues of plants [2]. Pollen germination and pollen tube growth are particularly sensitive to B deficiency [3]. A particular role of B in cell wall formation was found to be attributed to B cross-linking with rhamnogalacturonan II (RG-II) [4—6]. Recently, expression and function of NpGUTl (Nicoti-ana plumbaginifolia glucuronyltransferase 1) and its antisense gene demonstrated that the formation of borate cross-linked RG-II dimer (dRG-II-B) is required for the development of male and female tissues, which explains the molecular mechanism of B function in reproductive growth [7].

Boron deficiency is a worldwide nutritional problem in agricultural production and reduces crop yield in many areas of the world [8]. However, considerable genotypic variation in response to B deficiency exists among crop species or cultivars within a species [9]. Grain set of 21 advanced barley (Hordeum vulgare L.) lines ranged from 5% to 90% under sand culture conditions in the absence of B[10]. Response of wheat (Triticum aestivum L.) to low available B was expressed in large differences in grain set and pollen fertility, which was related to the ability for accumulation and distribution of B into the developing ear [11, 12]. Striking genetic differences of B efficiency in lentil (Lens culinaris medikus) was reported at low soil B, which has been proven to be associated with geographic origin [13]. Considerable genotypic variation in re-

sponse to B deficiency has been previously reported in rapeseed (Brassica napus L.) [14—16].

An efflux-type B transporter AtBORl is essential for protecting shoots from B deficiency with low external B supply [17, 18]. Recently, NLP5;1, a soybean NOD26-like major intrinsic protein (NIP), was proved to be crucial for the B uptake required for plant growth and development under B limitation [19]. Over-expression of AtBORl in Arabidopsis enhanced seed yield compared with the wild-type under B-defi-cient conditions as a result of increased production of an essential mineral nutrient transporter [20]. This suggested that breeding B-efficient genotypes has become a new practical approach by genetic improvement for maintaining crop yield potential in soils with low available B.

Rapeseed, one of the main oil crops in the world as well as in China, is sensitivity to B deficiency [21, 22]. However, considerable genotypic variation in response to B deficiency provides the possibility for genetic improvement. Eight B-efficient cultivars and two B-inef-ficient cultivars were identified from the 210 screening lines of Brassica napus through a two-step method [23, 24]. A major QTL, BE1, and three minor loci controlling the B-efficient trait in Brassica napus were detected by QTL mapping in a F2 population derived from a cross between a B-efficient cv. Qingyou 10 and a B-inefficient cv. Bakow [25]. The B efficiency in Brassica napus might be attributed to an enhanced ability for B uptake and reutilization when exposed to low available B soil [26, 27]. However, the comprehen-

sive physiological function for each QTL, especially the major QTL, and the relationship with B transporters in Arabidopsis are still unknown. Thus, cloning of B efficient QTLs and their function identification are necessary for unveiling the molecular mechanism of B efficiency in Brassica napus.

Near isogenic lines (NILs), a group of lines that are genetically identical except at one or a few QTL loci controlling target trait,were first put forward by Young [28]. According to theoretical value of the genetic background based on a formula for NILs construction, more than six successive backcrosses are required to acquire ideal NILs with about 95.0% of the expected genetic background of the recurrent parent. However, molecular marker assisted selection has been considered to be an efficient method to accelerate the NIL construction process [29, 30]. These near isogenic lines were precious genetic materials for fine mapping QTL and studying the effects of QTL and epista-sis [31, 32]. In the present paper, we reported the development of rapeseed B-efficient backcross lines by B efficiency phenotype identification at seedling stage as well as genetic background analysis assisted with random amplified fragment length polymorphism (AFLP) markers, and then analyzed B uptake and utilization, the segregation law of B efficiency trait at seedling stage of a B efficient backcross line responding to low-B stress. The developed B-efficient back-cross line and the recurrent parent are just a pair of B-efficient NILs, which would then be available for fine mapping and cloning of the B efficiency QTL.

MATERIALS AND METHODS

Plant materials. The BC populations were developed from the crosses between the B-inefficient cv. Bakow (recurrent parent) and B-efficient cv. Qingyou 10 (donor parent). The B efficiency of cv. Qingyou 10 and cv. Bakow has been previously identified [23, 24]. The B efficiency identification was conducted at seedling stage of each backcross generation using the method as described in the following part. Boron efficient individuals from each backcross generation populations were successively backcrossed with cv. Bakow to recovery the genetic background. The B-efficient backcross individuals with higher recovery of genetic background in BC2 and BC4 were selected after the assay genetic background using random AFLP markers.

The B-efficient individuals from BC4 with higher genetic background similarity (GBS) were self-pollinated to generate the BC4S1 population. The homozygous individuals from BC4S1 together with the recurrent parent are just a pair of B-efficient NILs.

Solution culture and B efficiency identification. To investigate the response of each backcross population to low B, hydroponic culture was employed. The nutrient solution components in hydroponic culture was as follows: KNO3 0.51 g L-1, KH2PO4 0.14 g L-1, MgSO4 • 7H2O 0.49 g L-1, Ca(NO3)2 • 4H2O 1.18 g L-1,

CuSO4 • 5H2O 0.08 mg L-1, ZnSO4 • 7H2O 0.22 mg L-1, MnCl2 • 4H2O 1.81 mg L-1,NaMoO4 • 2H2O 0.09 mg L-1, EDTA-Fe 0.025 g L-1. A low B level of 0.0025 mg L-1, confirmed through repeated experiments in our laboratory, was used for B efficiency identification at seedling stage using hydroponic culture. This is a B stress level at which only Bakow and B-inefficient individuals of backcross populations show typical signs of B deficiency.

Uniform seeds were selected and surface-sterilized using 0.5% NaClO (w/v) solution for 15 min and washed in deionized water, and then germinated on moistened gauze surface fixed on black plastic tray filled with deionized water and plants were grown at 22-24°C in an illumination room until cotyledons were fully developed after 5 days. The uniform seedlings were carefully transferred to a 10-L black plastic tray with one-fourth strength nutrient solution containing 0.0025 mg B/L using cv. Qingyou 10 and cv. Bakow as B-efficient and B-inefficient controls, respectively. The nutrient solution was replaced once a week with half strength first, and then followed by full strength nutrient solution. Each tray was randomly organized and positioned during replacement of nutrient solution to minimize any positional effects on plant growth. At the same time, cv. Qingyou 10 and cv. Bakow were grown at normal B level (0.5 mg B/L).

All plants were grown under a temperature of 24/22°C day/night, a relative humidity of 65%-80% and a 16 h day length of approximately 300-320 ^mol m-2 s-1. B-deficient symptoms were investigated after growing for ten days which could be used as an index to evaluate the backcross individuals response to low B stress compared with the growth of the parents grown at the low and normal levels. Backcross individuals without or with distinct B deficiency symptoms were defined as B-efficient or B-inefficient plants, respectively.

Sampling and B analysis. B efficient individuals of BC4S1 and BC5 population from two lines, along with cv. Qingyou 10 and cv. Bakow, were harvested after B efficiency investigation with cv. Qingyou 10 and cv. Bakow at the normal B levels (0.5mg B/L) as control. Samples were prepared from four plants in each category. All harvested samples were

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