Walnut (Juglans L.), a perennial woody plant, plays an important role in economic, ecological and social effects and other fields. Its distribution is wide. Xinjiang as the origin of one of the main walnut-growing regions has abundant resources of walnuts for studying and breeding, including early-bearing and late-bearing two kinds. Early-bearing walnuts produce seeds after planted 2~3 years, in normal situation, while the late-bearing walnuts need 8~10 years. Therefore, early-bearing walnuts were thought to be a rare resource in study woody plant’s flowering mechanism and would play a potential role in shortening juvenile phase of perennial woody plants, as well as in cultivars modification by genetic engineering (Gu et al., 1991; Yang et al., 1996; Zhang et al., 2007).

DNA molecular marker is an important way to study genetic background of walnut, which has been used in many researches, the origin, the interspecific relationship, construction of genetic maps and searching for genetic markers in target linkage of walnut. At present, some research on early-bearing walnut associated with molecular markers has been reported. Yang et al (2002) obtained the RAPD molecular marker OPB08-900 related to the early-bearing traits of walnut by BSA. Wang et al (2004) also used RAPD to analyze the related markers of early-bearing of walnut, and they thought the OPG15-710 may be the molecular marker related to early-bearing traits. We have obtained the RAPD molecular marker related to the early-bearing traits of walnut in Xinjiang and the molecular marker was transformed to SCAR successfully (Zhang et al., 2004). In this paper, we analyzed the heredity rule of SCAR molecular marker related to the early-bearing trait between maternal parents and F₁ generations of walnut in Xinjiang, which would give us some information to study the early-bearing traits of walnut in future.

1 Results and Analysis
1.1 Cloning and detecting the SCAR molecular marker of early-bearing
The fragment related with early-bearing was amplified based on the specific primers P1 and P2, and the size was about 750 bp in length (Figure 1). The PCR product was separated on 1.2% agarose gels for recovery, and then was cloned into PMD 19-T vector (TaKaRa Biotech, Dalian, China). The result of digestion and recycling showed that the size was same to the target fragment, which indicated that the fragments were transformed into the plasmids successfully (Figure 2). And the recombinant plasmid was sequenced by Sangon Biotech (Shanghai) Co., Ltd. The result showed that the length of the fragment was 762 bp, named as SCAR-762 (Figure 3).

1.2 The heredity rule of SCAR molecular marker related to early-bearing trait in maternal parents and F₁ generations
The DNA of four parents and a control were amplified based on primers P1 and P2 (Figure 4). The result showed that SCAR molecular marker could be obtained in early-bearing parents, but not in the control of late-bearing, which indicated that the SCAR molecular marker was related to early-bearing  trait in walnut. We also found that there was no difference among the early-bearing parents.

The F1 generations of the five parents were amplified based on the specific primers P1 and P2 (Figure 5; Figure 6). The results showed that 39 plants of 41 in Lun 1 had the SCAR molecular marker, 42 plants of 43 in Lun 2 had the SCAR molecular marker, 27 plants of 28 in Lun 3 had the SCAR molecular marker, 27 plants of 29 in Lun 4 had SCAR molecular marker, and 43 plants of 44 in Lun 5 had SCAR molecular marker, respectively. The percentage of this marker in the F₁ generations was 92.68%, 97.92% 96.43%, 93.10% and 97.73%, respectively (Table 1). The results indicated that early-bearing was an high heredity trait, which displayed the same result as Yang et al (2007).

2 Discussion
The stage of flowering was an important turning phase during the development process of fruit tree, the time of flowering would affect the fruit directly, To understand the molecular mechanisms of flowering, genetic and molecular biological approaches have been used to isolate the key genes that regulated flower development, such as, Carya cathayensis (Wang et al., 2006; 2008), Cictus (Liu et al., 2001), Prunus (An et al., 2008) and Malus domestica (Cao et al., 2003). Early-bearing walnut gene was a rare natural resources in studying flowering mechanism. So, isolation of gene and molecular markers related to early-bearing trait in walnut was important to shorten juvenile phase of woody plants (Yang et al., 2007). 

In this study, we cloned the SCAR molecular marker related to early-bearing traits based on the previous study of our laboratory. The length of the fragment was 762 bp, which reached 95.94% identity with the previous fragment (759 bp) isolated in our study by Zhang et al (2004). Based on this result, we attributed the differences to the sequencing probably. The results indicated that this molecular marker was stable, and it was related to the precocious trait in walnut. The SCAR molecular marker was used to analyze the maternal plants and F₁ generations. There were some differences between early-bearing and late-bearing parents, and all of the plants had a high heritability. The phenomenon indicated that the abundant resources of walnuts in Xinjiang provided rich materials for researching and breeding.

This research about the SCAR molecular marker would give us some information for studying the early-bearing trait, the juvenile mechanism and the flowering genes in woody plants in future. In the next step, we plan to clone the gene related to early-bearing of walnut, and analyze its function.

3 Materials and Methods
3.1 Plant materials
The materials were grown in the experiment-land of Shihezi University, a standard experiment-land, where there were many Xinjiang Luntai resources for studying. Five maternal plants and 150 F₁ generations were used in this study, maternal plants, planted and grafted by local farmers, were collected from Luntai National Research Station of Xinjiang Fruit Germplasm Resources named as Lun1, Lun2, Lun3, Lun4, and Lun5. The F₁ generations were planted with seeds which were collected from Luntai National Research Station of Xinjiang Fruit Germplasm Resources. The leaves of the maternal plants and F₁ generations were collected and stored at -80℃ for DNA isolation.

3.2 SCAR primers
According to the previous report of our laboratory, we designed a pair of primers for SCAR. They were synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The sequences of primers used in this experience were as follows: P1: 5'-ACTG GGACT CCA ATTGTATC-3'; P2: 5'-AC TGGGA CTC T CAAC TAT-3’.

3.3 DNA isolation
Total DNA of the maternal plants and F₁ generations were extracted by CTAB method descried by Zhang (2003). And it was detected on the 1% agarose gel electrophoresis and diluted to 20 ng/µL with TE, then compared with the Marker (GM335). And then stored at -20℃ for later use.

3.4 Cloning and sequencing
The volume of the reaction system for PCR was 20 µL as followins: 13.4 µL ddH₂O, 2 µL 10×buffer without Mg²⁺, 0.4 µL 10 µmol/L dNTPs, 1 µL primer, 2 µL DNA templates and 0.2 µL Taq polymerase. After mixed, the reaction solution was separated slightly in micro centrifuge, and then was placed in PCR thermal cycler. The PCR procedure was as follows: 94℃ for 3 min, and then 30 cycles at 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 80 s, with a final extension step at 72℃ for 7 min, and insulation work at 4℃. The PCR products were separated on 1.2% agarose gels electrophoresis for recovery. Then cloned into PMD 19-T vector, and finally transformed into XL1-Blue. After the production was induced by IPTG and X-gal, white colonies were identified by PCR and the positive colonies were sequenced.

3.5 Amplification of SCAR molecular marker in maternal plants and F₁ generations
The maternal plants and F₁ generations were amplified with P1 and P2, the amplification condition and system of PCR as 3.4.

Authors' contributions
CXY and JXN conceived the experimental design and objectives of all the experiments, conducted the data analyses, and wrote the manuscript. JQL, LW, RL and HPZ conducted a few data analyses and took an active part in experimental design method. All authors have read and approved the manuscript.

Acknowledgments
This research was supported by the National Natural Science Foundation of China (No. 30560090) and the important National Science and Technology Specific Projects of Xinjiang (No. 201130102-1-4).

Reference
An L.J., and Li T.H, 2008, Cloning, expression and production of polyclonal antibodies of peach PpLFY, Acta Horticulture Sinica, 35(11): 1573-1580

Cao Q.F., Masato W., Meng Y.P., Huang J., Sun Y., and Wang G.P., 2003, Cloning and expression analysis of LEAFY homologues genes from apple, Acta Horticulture Science, 30(3): 267-271

Gu R.S., and Xi R.T., 1991, The recent advance in early-bearing of walnut, Journal of Agricultural University of Hebei, 14(1): 100-103

Liu S.F., Cheng L.G., Cheng D.M., and Xu C.J., 2001, Cloning and analysis of LFY-like gene from Citrus, Journal of Zhejiang University (Agric. and Life Sci.), 27(3): 297-300

Wang G.A., Zhang H.P., Hu H.F., Niu J.X., and Ma B.G., 2004, Identification of a RAPD marker related to early-bearing characteristic of walnut seedlings, Journal of Fruit Science, 21(5): 485-487

Wang Z.J., 2006, Molecular marker analysis and flora gene CcLFY and its promoter cloning of Carya cathayensis, Disseration for Ph.D., Beijing Forestry University, Supervisor: Wang H.F., and Huang J.Q.

Wang Z.J., Huang Y.J., Xia G.H., Zheng B.S., Jin S.H., and Huang J.Q., 2008, Cloning and analysis of the APETALA1(AP1) homolog gene from Carye cathayensis, Journal of Zhejiang Forestry College, 25(4):427-430

Yang K.Q., Wang Y.J., Zhang Y.D., and Zhen X.Q., 2002, RAPD Analysis for the identification of the precocious trait in walnuts, Acta Horticulture Science, 29 (6) : 573-574

Yang K.Q., Ma M., Sun C.L., Sun M.G., and Guo Q.R., 2007, RAPD marker linked to precocious gene of walnut and its sequence analysis, Science Agriculture Sinica, 40(9): 2021-2027

Yang W.C., and Gao S.F., 1996, The early-bearing walnut of China, Journal of Hebei Forestry College, 10(11): 134-137

Zhang H.P., 2004, Studies on gene linked to character of early-bearing walnut with RAPD and SCAR markers, Thesis for M.S., Shihezi University, Supervisor: Niu J.X., pp.485-487

Zhang H.P., Niu J.X., Hu H.F., Ma B.G., and Wang G.A., 2007, Screening of a RAPD Marker related with precocious bearing trait in walnut and the SCAR marker conversion, Journal of Shihezi University (Nature Science), 25(4): 426-428
http://dx.doi.org/10.1007/s11741-007-0421-z