Design and Practice of a New Teaching Project of the Map-based Cloning Experiment in Genetics

doi:10.11983/CBB19107

Abstract

Abstract: Recombination, linkage and crossover of chromosomes are the key and difficult points in genetics teaching. For students’ comprehensively understanding these concepts in teaching, we designed a new genetic experiment named ‘Gene mapping using DNA molecular markers’ based on the principle of the map-based cloning method, using our previous studies on cloning of a rice lesion-mimic phenotype regulating gene SPOTTED LEAF 5 (SPL5) as an example. In this experiment, students are instructed to perform the linkage analysis, genetically map the spl5 mutation and align a genetic map by using an F₂ population generated by a cross between spl5 and wild-type plants and polymorphic molecu- lar markers. This teaching experiment can not only effectively promote students' understanding of the three Laws of genetics, but also be helpful to broaden their vision and improve their ability to solve problems and work in a team.

Key words: genetics, comprehensive designing experiment, map-based cloning, educational resources

Xifeng Chen,Yaping Liu,Bojun Ma. Design and Practice of a New Teaching Project of the Map-based Cloning Experiment in Genetics[J]. Chinese Bulletin of Botany, 2019, 54(6): 797-803.

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB19107

https://www.chinbullbotany.com/EN/Y2019/V54/I6/797

Figures/Tables 5

Figure 1 Recombination patterns between the target gene and the molecular markers during meiosis A and a represent a pair of dominant and recessive alleles, of which AA and Aa show dominant phenotypes and aa shows a recessive phenotype; M and m represent a given molecular marker at the same locus derived from the dominant parent (AA) and the recessive parent (aa), respectively.

Figure 2 The phenotype of the rice spl5 lesion mimic mutant and a schematic workflow of generation of the genetic mapping population (A) The plant phenotype of wild-type control Nipponbare, a Japonica rice cultivar, and the spl5 mutant; (B) The leaf phenotype of wild-type control and the spl5 mutant; (C) A workflow showing the generation of an F2 mapping population by crossing an Indica cultivar 9311 with the spl5 mutant.

Table 1 PCR primers of the SSR markers used in this study

SSR标记	正向引物 (5°-3°)	反向引物 (5°-3°)
RM21103	ACAAGTCCACAAGGACCACAACC	TGCTCCACCCAAAGATACAGAGC
RM21309	GAGTGGATTTACTGACAAGCTCTTCC	GATTCTCTTGGAGCCCATACTCC
RM21470	TCTTGCCATCACATAGCAACAGG	ACTCGGTGAGCATCCAATGTCC
RM22426	ATTTCGTCGTCGTGTCTGTCC	TTAAAGCAGCCTGATCGAAAGC
RM22648	AGCAGCTCCGATTGTGTTAGCC	AATGGCGTCTGTGGTACTTTGC
RM22925	CGATTACCAGGACAACAGATCACG	GTGGCTCTTGAGTGTACGTGTGC

Figure 3 Linkage analysis and genetic mapping of the SPL5 gene (A) A physical map of the SSR markers on chromosomes used in this study; (B) Analysis of the PCR products of the SSR markers by electrophoresis; (C) A genetic linkage map of the SPL5 gene.

Table 2 The Chi-square (χ2) test for the genotype separation ratio of the SSR markers in this study

分子标记	20个F₂突变型单株中各基因型的数量			χ²值	P值
分子标记	MM	Mm	mm	χ²值	P值
RM21103	0	0	20	60	<0.01
RM21309	0	3	17	38.7	<0.01
RM21470	1	7	12	13.9	<0.01
RM22426	4	11	5	0.3	>0.05
RM22648	5	9	6	0.3	>0.05
RM22925	7	10	3	1.6	>0.05

References 9

[1]	杜少陵, 徐思斌, 宫磊, 朱晓蕾, 汪萍, 林爱琴 ( 2010). 医学遗传学多元式教学的实践与探讨. 遗传 32, 1084-1088.
[2]	何俊平, 阮松林, 祝水金, 马华升 ( 2010). 图位克隆技术在农作物基因分离中的应用与评价. 遗传 32, 903-913.
[3]	姜树坤, 徐正进, 陈温福 ( 2008). 水稻QTL图位克隆的特征分析. 遗传 30, 1121-1126.
[4]	景润春, 黄青阳, 朱英国 ( 2000). 图位克隆技术在分离植物基因中的应用. 遗传 22, 180-185.
[5]	赵国珍, Jia YL, 严宗卜, Deren CW, Melissa HJ, 戴陆园 ( 2012). 一种高效便捷的水稻DNA提取法及其应用. 中国水稻科学 26, 495-499.
[6]	Chen XF, Hao L, Pan JW, Zheng XX, Jiang GH, Jin Y, Gu ZM, Qian Q, Zhai WX, Ma BJ ( 2012). SPL5, a cell death and defense-related gene, encodes a putative splicing factor 3b subunit 3 (SF3b3) in rice. Mol Breed 30, 939-949.
[7]	Chen XF, Pan JW, Cheng J, Jiang GH, Jin Y, Gu ZM, Qian Q, Zhai WX, Ma BJ ( 2009). Fine genetic mapping and physical delimitation of the lesion mimic gene spotted leaf 5 (spl5) in rice(Oryza sativa L.). Mol Breed 24, 387-395.
[8]	Coulson A, Sulston J, Brenner S, Karn J ( 1986). Toward a physical map of the genome of the nematode Caenorhabditis elegans. Proc Natl Acad Sci USA 83, 7821-7825.
[9]	International Rice Genome Sequencing Project ( 2005). The map-based sequence of the rice genome. Nature 436, 793-800.

[1]	YANG Ling, LIANG Si-Qi, PAN Jia-Ming, WEI Jin-Xin, DING Tao, JIANG Ri-Hong, SHAO Yi-Zhen, ZHANG Xian-Chun, LIU Yong-Bo, XIANG Qiao-Ping. Species delimitation and genetic conservation of the endangered firs Abies beshanzuensis and A. ziyuanensis [J]. Chin J Plant Ecol, 2023, 47(12): 1629-1645.
[2]	Xinyu Cai, Xiaowei Mao, Yiqiang Zhao. Methods and research progress on the origin of animal domestication [J]. Biodiv Sci, 2022, 30(4): 21457-.
[3]	Kairu Yang, Qiwei Jia, Jiayi Jin, Hanfei Ye, Sheng Wang, Qianyu Chen, Yian Guan, Chenyang Pan, Dedong Xin, Yuan Fang, Yuexing Wang, Yuchun Rao. Cloning and Functional Analysis of Rice Yellow Green Leaf Regulatory Gene YGL18 [J]. Chinese Bulletin of Botany, 2022, 57(3): 276-287.
[4]	Yanyan Liu, Chang Liu, Xiaoxin Wei. Current status of taxonomy, systematics and conservation of the white pines in China and adjacent regions [J]. Biodiv Sci, 2022, 30(2): 21344-.
[5]	Jiangyuan Shang, Yan Chun, Xueyong Li. Map-based Cloning and Natural Variation Analysis of the PAL3 Gene Controlling Panicle Length in Rice [J]. Chinese Bulletin of Botany, 2021, 56(5): 520-532.
[6]	Zhongdong Yu, Zhihe Yu, Shiyu Jin, Long Wang. Genetic diversity and toxin-producing characters of Aspergillus flavus from China [J]. Biodiv Sci, 2019, 27(8): 842-853.
[7]	Tu Weifeng,Zhang Yang,Tang Jie,Tu Yuqin,Xin Jiajia,Ji Hongli,Zhang Nanfeng,Zhang Tao. Comparison of taxonomic morphological characteristics between Rorippa indica and R. dubia [J]. Biodiv Sci, 2019, 27(2): 168-176.
[8]	Linfeng Li, Bao Liu. The roles of epigenetic variation in plant hybridization and polyploidization [J]. Biodiv Sci, 2017, 25(6): 600-607.
[9]	Jian-Feng Mao, Yongpeng Ma, Renchao Zhou. Approaches used to detect and test hybridization: combining phylogenetic and population genetic analyses [J]. Biodiv Sci, 2017, 25(6): 577-599.
[10]	Hang Sun, Tao Deng, Yongsheng Chen, Zhuo Zhou. Current research and development trends in floristic geography [J]. Biodiv Sci, 2017, 25(2): 111-122.
[11]	Wang Wenle, Feng Dan, Wu Jinxia, Zhang Zhiguo, Lu Tiegang. Gene Mapping of a Dwarf Gene WLD1 in Rice [J]. Chinese Bulletin of Botany, 2017, 52(1): 54-60.
[12]	Ruoyu Liu, Zhongmin Sun, Jianting Yao, Zimin Hu, Delin Duan. Genetic diversity of the habitat-forming red alga Gracilaria vermiculophylla along Chinese coasts [J]. Biodiv Sci, 2016, 24(7): 781-790.
[13]	Yongshu Liang, Junjie Zhou, Wenbin Nan, Dongdong Duan, Hanma Zhang. Progress in Rice Root System Research [J]. Chinese Bulletin of Botany, 2016, 51(1): 98-106.
[14]	Dexing Zhang. Unorthodox reflections on molecular ecology research in China [J]. Biodiv Sci, 2015, 23(5): 559-569.
[15]	Keping Ma. New opportunities for mainstreaming biodiversity conservation [J]. Biodiv Sci, 2015, 23(5): 557-558.