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Nanjing 南京市 | |
Sub-provincial city | |
Clockwise from top: 1. the city, Xuanwu Lake and Purple Mountain; 2. stone sculpture "bixie"; 3. Jiming Temple; 4. Yijiang Gate with the City Wall of Nanjing; 5. Qinhuai River and Fuzi Miao; 6. Nanjing Olympic Sports Centre; 7. the spirit way of Ming Xiaoling Mausoleum; 8. Sun Yat-sen Mausoleum | |
Location of Nanjing City jurisdiction in Jiangsu | |
Location in China | |
Coordinates: 32°03′N 118°46′E / 32.050°N 118.767°ECoordinates: 32°03′N 118°46′E / 32.050°N 118.767°E | |
Country | China |
Province | Jiangsu |
County-level | 11 |
Township-level | 129 |
Settled | 495 BC |
Government | |
• Type | Sub-provincial city |
• CPC Ctte Secretary | Yang Weize |
• Mayor | Miao Ruilin |
Area | |
• Sub-provincial city | 6,598 km2 (2,548 sq mi) |
Elevation | 20 m (50 ft) |
Population (2012) | |
• Sub-provincial city | 8,161,800 |
• Density | 1,237/km2 (3,183/sq mi) |
• Urban | 8,161,800 |
Demonym | Nanjinger |
Time zone | China Standard (UTC+8) |
Postal code | 210000–211300 |
Area code(s) | 25 |
GDP (Nominal) | 2013 |
- Total | US$131.4 billion |
- Per capita | US$ 16,210 |
- Growth | 11.25% |
- Total | US$224.1 billion |
- Per capita | US$ 27,646 |
Licence plate prefixes | 苏A |
Website | City of Nanjing |
Deodar Cedar (Cedrus deodara), Platanus × acerifolia[1] Méi (Prunus mume) |
This article needs additional citations for verification. (May 2014) |
Nanjing ( listen; Chinese: 南京; pinyin: Nánjīng; Wade–Giles: Nan-ching) is the capital of Jiangsu province in Eastern China.[2] It has a prominent place in Chinese history and culture, having been the capital of China for several periods.[3] Its present name means "Southern Capital" and was widely romanized as Nankin and Nanking until the pinyin language reform, after which Nanjing was gradually adopted as the standard spelling of the city's name in most languages that use the Roman alphabet.[4]
Located in the lower Yangtze River drainage basin and Yangtze River Delta economic zone, Nanjing has long been one of China's most important cities.[5][6][7] Having been the capital city of six different dynasties since 3 A.D., it is recognized as one of the Four Great Ancient Capitals of China.[8] It was the capital of Wu during the Three Kingdoms Period,[9] and the capital of the Republic of China prior to its flight to Taiwan during the Chinese Civil War.[10] Nanjing is also one of the fifteen sub-provincial cities in the People's Republic of China's administrative structure,[11] enjoying jurisdictional and economic autonomy only slightly less than that of a province.[12] Nanjing has long been a national centre of education, research, transport networks and tourism. It was the host city of the 2014 Summer Youth Olympics.[13]
With a total population of 8.16 million[14][15] and a urban population of 6.55 million,[16][17] Nanjing is the second-largest commercial centre in the East China region after Shanghai. It has been ranked seventh in the evaluation of "Cities with Strongest Comprehensive Strength" issued by the National Statistics Bureau, and second in the evaluation of cities with most sustainable development potential in the Yangtze River Delta. It has also been awarded the title of 2008 Habitat Scroll of Honour of China, Special UN Habitat Scroll of Honour Award and National Civilized City.[18]
Nanjing was one of the earliest established cities in what is now China. According to legend,[which?] Fuchai, King of the State of Wu, founded a fort named Yecheng (冶城) in today's Nanjing area in 495 BCE. Later in 473 BCE, the State of Yue conquered Wu and constructed the fort of Yuecheng (越城) on the outskirts of the present-day Zhonghua Gate. In 333 BCE, after eliminating the State of Yue, the State of Chu built Jinling Yi (金陵邑) in the western part of present-day Nanjing. Under the Qin and Han dynasties, it was called Moling (秣陵). Since then, the city had experienced destruction and renewal many times.[citation needed]
Nanjing first became a capital in 229 CE, where Sun Quan of the Wu Kingdom during the Three Kingdoms Period relocated its capital to Jianye (建業), a city he extended on the basis of Jinling Yi in 211 CE.[9] Although conquered by the Western Jin dynasty in 280, Nanjing and its neighbouring areas had been well cultivated and developed into one of the commercial, cultural and political centers of China during the rule of East Wu. This city would soon play a vital role in the following centuries.
Shortly after the unification of the region, the Western Jin dynasty collapsed. First the rebellions by eight Jin princes for the throne and later rebellions and invasion from Xiongnu and other nomadic peoples that destroyed the rule of Jin in the north. In 317, remnants of the Jin court, as well as nobles and wealthy families, fled from the north to the south and reestablished the Jin court in Nanjing, which was then called Jiankang (建康).
During the period of North–South division, Nanjing remained the capital of the Southern dynasties for more than two and a half centuries. During this time, Nanjing was the international hub of East Asia and one of the largest cities in the world.[20] Based on historical documents, the city had 280,000 registered households.[21] Assuming an average Nanjing household had about 5.1 people at that time, the city had more than 1.4 million residents.[22]
A number of sculptural ensembles of that era, erected at the tombs of royals and other dignitaries, have survived (in various degrees of preservation) in Nanjing's northeastern and eastern suburbs, primarily in Qixia and Jiangning District.[23] Possibly the best preserved of them is the ensemble of the Tomb of Xiao Xiu (475–518), a brother of Emperor Wu of Liang.[24][25] The period of division ended when the Sui Dynasty reunified China and almost destroyed the entire city, turning it into a small town.
The city of Nanjing was razed after the Sui took over it. It was reconstructed during the late Tang dynasty.[26] It was chosen as the capital and called Jinling (金陵) during the Southern Tang (937–976), a state that succeeded the Wu.[27] Jiankang's textile industry burgeoned and thrived during the Song dynasty despite the constant threat of foreign invasions from the north by the Jurchen Jin dynasty. Nanjing was the capital of Da Chu, a puppet state established by the Jurchens.[28] Da Chu came to an end scarcely a month after its creation.[29]
The Mongol Empire eventually occupied much of the territory that now constitutes China and further consolidated the city's status as a hub of the textile industry under what became known in China as the Yuan dynasty.[30]
The first emperor of the Ming dynasty Zhu Yuanzhang, the Hongwu Emperor, who overthrew the Yuan dynasty renamed the city Yingtian, rebuilt it, and made it the dynastic capital in 1368. He constructed a 48 km (30 mi) long city wall around Yingtian, as well as a new Ming Palace complex, and government halls.[31] It took 200,000 laborers 21 years to finish the project. The present-day City Wall of Nanjing was mainly built during that time and today it remains in good condition and has been well preserved.[32] It is among the longest surviving city walls in China.[33] the Jianwen Emperor ruled from 1398 to 1402.
It is believed that Nanjing was the largest city in the world from 1358 to 1425 with a population of 487,000 in 1400.[34] Nanjing remained the capital of the Ming Empire until 1421, when the third emperor of the Ming Dynasty, Zhu Di the Yongle Emperor, relocated the capital to Beijing.
Besides the city wall, other famous Ming-era structures in the city included the famous Ming Xiaoling Mausoleum and Porcelain Tower, although the latter was destroyed by the Taipings in the 19th century either in order to prevent a hostile faction from using it to observe and shell the city[35] or from superstitious fear of its geomantic properties.[36]
A monument to the huge human cost of some of the gigantic construction projects of the early Ming is the Yangshan Quarry (located some 15–20 km (9–12 mi) east of the walled city and Ming Xiaoling mausoleum), where a gigantic stele, cut on the orders of the Yongle Emperor, lies abandoned, just as it was left 600 years ago when it was understood it was impossible to move or complete it.[37]
As the center of the empire, early-Ming Nanjing had worldwide connections. It was home of the admiral Zheng He, who went to sail the Pacific and Indian Oceans, and it was visited by foreign dignitaries, such as a king from Borneo (Boni 渤泥), who died during his visit to China in 1408. The Tomb of the King of Boni, with a spirit way and a tortoise stele, was discovered in Yuhuatai District (south of the walled city) in 1958, and has been restored.[38]
Over two centuries after the removal of the capital to Beijing, Nanjing was destined to become the capital of a Ming emperor one more time. After the fall of Beijing to the Li Zicheng's rebels and then to Manchu Qing invaders, and the suicide of the last legitimate Ming emperor Zhu Youjian (the Chongzhen Emperor) in the spring 1644, the Ming Zhu Yousong, Prince of Fu was enthroned in Nanjing in June 1644 as the Hongguang Emperor.[39][40] His short reign was described by later historians as the first reign of the so-called Southern Ming dynasty.
Zhu Yousong, however, fared a lot worse than his ancestor Zhu Yuanzhang three centuries earlier. Beset by factional conflicts, his regime could not offer effective resistance to Manchu troops, when the Manchu army, led by Prince Dodo approached Jiangnan the next spring.[41] Days after Yangzhou fell to the Manchus in late May 1645, the Hongguang Emperor fled Nanjing, and the imperial Ming Palace was looted by local residents.[42] On June 6, Dodo's troops approached Nanjing, and the commander of the city's garrison, Zhao the Earl of Xincheng, promptly surrendered the city to them.[43][44] The Manchus soon ordered all male residents of the city to shave their heads in the Manchu queue way.[45] They requisitioned a large section of the city for the bannermen's cantonment, and destroyed the former imperial Ming Palace, but otherwise the city was spared the mass murders and destruction that befell Yangzhou.[46]
Under the Qing dynasty (1644–1911), the Nanjing area was known as Jiangning (江寧) and served as the seat of government for the Liangjiang Viceroy.[47] It had been visited by the Kangxi and Qianlong Emperors a number of times on their tours of the southern provinces.
Nanjing was invaded by British troops during the close of the First Opium War, which was ended by the Treaty of Nanking in 1842.
As the capital of the brief-lived Taiping Kingdom[48] in the mid-19th century, Nanjing was known as Tianjing (天京, "Heavenly Capital" or "Capital of Heaven") Both the Qing Viceroy and the Taiping king resided in buildings that would later be known as the Presidential Palace. When the Qing under Zeng Guofan retook the city in 1864, a massive slaughter occurred in the city with over 100,000 estimated to have committed suicide or fought to the death.[49] Since the rebellion began, Imperial forces allowed no rebels speaking its dialect to surrender.[50] This policy of mass murder of civilians occurred in Nanjing.[51]
The Xinhai Revolution led to the founding of the Republic of China in January 1912 with Dr. Sun Yat-sen as the first provisional president and Nanking was selected as its new capital. However, the Qing Empire controlled large regions to the North, so revolutionaries asked Yuan Shikai to replace Sun as president in exchange for the last-Qing Emperor's abdication. Yuan demanded the capital be Beijing (closer to his power base).
In 1927, the Kuomintang (KMT) under Generalissimo Chiang Kai-shek again established Nanking as the capital of the Republic of China, and this became internationally recognized once KMT forces took Beijing in 1928. The following decade is known as the Nanking decade.
In 1937, Japan started a full-scale invasion of China after invading Manchuria in 1931, beginning the Second Sino-Japanese War (often considered a theatre of World War II).[52] Their troops occupied Nanjing in December and carried out the systematic and brutal Nanking massacre (the "Rape of Nanking").[53] Even children, the elderly, and nuns are reported to have suffered at the hands of the Imperial Japanese army.[54] The total death toll, including estimates made by the International Military Tribunal for the Far East and the Nanjing War Crimes Tribunal, was between 300,000 and 350,000.[55] Nanjing city was also severely damaged during the massacre.[53] The Nanjing Massacre Memorial Hall was built in 1985 to commemorate this event.
A few days before the fall of the city, the National Government of China was relocated to the southwestern city Chungking (now Chongqing) and resumed Chinese resistance. In 1940, a Japanese-collaborationist government known as the "Nanjing Regime" or "Reorganized National Government of China" led by Wang Jingwei was established in Nanjing as a rival to Chiang Kai-Shek's government in Chongqing.[56] In 1946, after the Surrender of Japan, the KMT relocated its central government back to Nanjing.
On 21 April, Communist forces crossed the Yangtze River. On April 23, 1949, the People's Liberation Army conquered Nanjing.[57] The KMT government retreated to Canton (Guangzhou) until October 15, Chongqing until November 25, and then Chengdu before retreating to Taiwan on December 10. By late 1949, the People's Liberation Army was pursuing remnants of KMT forces southwards in southern China, and only Tibet was left. After the establishment of the People's Republic of China in October 1949, Nanjing was initially a province-level municipality, but it soon became the provincial capital of Jiangsu, and retains that status to this day.
Nanjing, with a total land area of 6,598 square kilometres (2,548 sq mi), is situated in one of the largest economic zones of China, the Yangtze River Delta, which is part of the downstream Yangtze River drainage basin. The Yangtze River flows past the west side of Nanjing City, while the Ningzheng Ridge surrounds the north, east and south side of the city. The city is 300 kilometres (190 mi) west-northwest of Shanghai, 1,200 kilometres (750 mi) south-southeast of Beijing, and 1,400 kilometres (870 mi) east-northeast of Chongqing.
Nanjing borders Yangzhou to the northeast, one town downstream when following the north bank of the Yangtze, Zhenjiang to the east, one town downstream when following the south bank of the Yangtze, and Changzhou to the southeast. On its western boundary is Anhui province, where Nanjing borders five prefecture-level cities.[58]
Nanjing is the intersection of Yangtze River—an east-west water transport artery and Nanjing–Beijing railway—a south-north land transport artery, hence the name "door of the east and west, throat of the south and north". Furthermore, the west part of the Ningzhen range is in Nanjing; the Loong-like Zhong Mountain is curling in the east of the city; the tiger-like Stone Mountain is crouching in the west of the city, hence the name "the Zhong Mountain, a dragon curling, and the Stone Mountain, a tiger crouching". Mr. Sun Yet-sen spoke highly of Nanjing in the "Constructive Scheme for Our Country", "The position of Nanjing is wonderful since mountains, lakes and plains all integrated in it. It is hardly [sic] to find another city like this."
Nanjing | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Climate chart (explanation) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Nanjing has a humid subtropical climate (Köppen Cfa) and is under the influence of the East Asian monsoon. The four seasons are distinct here, with damp conditions seen throughout the year, very hot and muggy summers, cold, damp winters, and in between, spring and autumn are of reasonable length. Along with Chongqing and Wuhan, Nanjing is traditionally referred to as one of the "Three Furnacelike Cities" along the Yangtze River (长江流域三大火炉) for the perennially high temperatures in the summertime.[60] However, the time from mid-June to the end of July is the plum blossom blooming season in which the meiyu (rainy season of East Asia; literally "plum rain") occurs, during which the city experiences a period of mild rain as well as dampness. Typhoons are uncommon but possible in the late stages of summer and early part of autumn. The annual mean temperature is around 15.46 °C (59.8 °F), with the monthly 24-hour average temperature ranging from 2.4 °C (36.3 °F) in January to 27.8 °C (82.0 °F) in July. The highest recorded temperature is 40.7 °C (105 °F), and the lowest −16.9 °C (2 °F).[61][62] On average precipitation falls 115 days out of the year, and the average annual rainfall is 1,062 millimetres (42 in). With monthly percent possible sunshine ranging from 37 percent in March to 52 percent in August, the city receives 1,983 hours of bright sunshine annually.
Nanjing is endowed with rich natural resources, which include more than 40 kinds of minerals. Among them, iron and sulfur reserves make up 40 percent of those of Jiangsu province. Its reserves of strontium rank first in East Asia and the South East Asia region. Nanjing also possesses abundant water resources, both from the Yangtze River and groundwater. In addition, it has several natural hot springs such as Tangshan Hot Spring in Jiangning and Tangquan Hot Spring in Pukou.
Surrounded by the Yangtze River and mountains, Nanjing also enjoys beautiful natural scenery. Natural lakes such as Xuanwu Lake and Mochou Lake are located in the centre of the city and are easily accessible to the public, while hills like Purple Mountain are covered with evergreens and oaks and host various historical and cultural sites. Sun Quan relocated his capital to Nanjing after Liu Bei's suggestion as Liu Bei was impressed by Nanjing's impeccable geographic position when negotiating an alliance with Sun Quan. Sun Quan then renamed the city from Moling (秣陵) to Jianye (建鄴) shortly thereafter.[63]
Climate data for Nanjing (1971–2000) | |||||||||||||
Month | Jan | Feb | Mar | Apr | May | Jun | Jul | Aug | Sep | Oct | Nov | Dec | Year |
Average high °C (°F) | 7.0 (44.6) | 8.8 (47.8) | 13.4 (56.1) | 20.3 (68.5) | 25.6 (78.1) | 28.8 (83.8) | 31.9 (89.4) | 31.7 (89.1) | 27.3 (81.1) | 22.2 (72) | 15.9 (60.6) | 10.0 (50) | 20.2 (68.4) |
Average low °C (°F) | −1.1 (30) | 0.6 (33.1) | 4.8 (40.6) | 10.6 (51.1) | 15.9 (60.6) | 20.7 (69.3) | 24.6 (76.3) | 24.2 (75.6) | 19.2 (66.6) | 12.9 (55.2) | 6.1 (43) | 0.4 (32.7) | 11.6 (52.8) |
Precipitation mm (inches) | 37.4 (1.472) | 47.1 (1.854) | 81.8 (3.22) | 73.4 (2.89) | 102.1 (4.02) | 193.4 (7.614) | 185.5 (7.303) | 129.2 (5.087) | 72.1 (2.839) | 65.1 (2.563) | 50.8 (2) | 24.4 (0.961) | 1,062.3 (41.823) |
Avg. precipitation days (≥ 0.1 mm) | 8.1 | 9.1 | 12.1 | 10.4 | 10.4 | 11.3 | 12.4 | 11.3 | 8.9 | 8.2 | 7.4 | 5.7 | 115.3 |
% humidity | 76 | 74 | 74 | 73 | 74 | 79 | 81 | 81 | 79 | 77 | 77 | 74 | 76.6 |
Mean monthly sunshine hours | 129.1 | 123.3 | 136.1 | 168.1 | 194.0 | 171.9 | 205.6 | 214.7 | 167.2 | 169.1 | 153.5 | 150.2 | 1,982.8 |
Percent possible sunshine | 41 | 40 | 37 | 44 | 46 | 41 | 47 | 52 | 45 | 48 | 49 | 48 | 44.8 |
Source: China Meteorological Administration[59] |
A dense wave of smog began in the Central and Eastern part of China on 2 December 2013 across a distance of around 1,200 kilometres (750 mi),[64] including Tianjin, Hebei, Shandong, Jiangsu, Anhui, Shanghai and Zhejiang. A lack of cold air flow, combined with slow-moving air masses carrying industrial emissions, collected airborne pollutants to form a thick layer of smog over the region.[65] The heavy smog heavily polluted central and southern Jiangsu Province, especially in and around Nanjing,[66] with its AQI pollution Index at "severely polluted" for five straight days and "heavily polluted" for nine.[67] On 3 December 2013, levels of PM2.5 particulate matter average over 943 micrograms per cubic metre,[68] falling to over 338 micrograms per cubic metre on 4 December 2013.[69] Between 3:00 pm, 3 December and 2:00pm, 4 December local time, several expressways from Nanjing to other Jiangsu cities were closed, stranding dozens of passenger buses in Zhongyangmen bus station.[66] From 5 to 6 December, Nanjing issued a red alert for air pollution and closed down all kindergarten through middle schools. Children's Hospital outpatient services increased by 33 percent; general incidence of bronchitis, pneumonia, upper respiratory tract infections significantly increased.[70] The smog dissipated 12 December.[71] Officials blamed the dense pollution on lack of wind, automobile exhaust emissions under low air pressure, and coal-powered district heating system in North China region.[72] Prevailing winds blew low-hanging air masses of factory emissions (mostly SO2) towards China's east coast.[73]
The full name of the government of Nanjing is "People's Government of Nanjing City". The city is under the one-party rule of the CPC, with the CPC Nanjing Committee Secretary as the de facto governor of the city and the mayor as the executive head of the government working under the secretary.
The sub-provincial city of Nanjing is divided into 11 districts.[74]
Map | Subdivision | Simplified Chinese | Population (2010) | Area (km2) | |
City Proper | |||||
1 | Xuanwu District | 玄武区 | 651,957 | 80.97 | |
2 | Qinhuai District | 秦淮区 | 1,007,922 | 50.36 | |
3 | Jianye District | 建邺区 | 426,999 | 82.00 | |
4 | Gulou District | 鼓楼区 | 1,271,191 | 57.62 | |
5 | Yuhuatai District | 雨花台区 | 391,285 | 131.90 | |
6 | Qixia District | 栖霞区 | 644,503 | 340.00 | |
Total | Nanjing | 南京 | 4,393,857 | 742.85 | |
Suburban | |||||
7 | Jiangning District | 江宁区 | 1,145,628 | 1,573.00 | |
8 | Pukou District | 浦口区 | 710,298 | 913.00 | |
9 | Luhe District | 六合区 | 915,845 | 1,485.50 | |
Rural | |||||
10 | Lishui District | 溧水区 | 421,323 | 983.00 | |
11 | Gaochun District | 高淳区 | 417,729 | 801.00 |
According to the Sixth China Census, the total population of the City of Nanjing reached 8.005 million in 2010. The statistics in 2011 estimated the total population to be 8.11 million. The birth rate was 8.86 percent and the death rate was 6.88 percent. The urban area had a population of 6.47 million people. The sex ratio of the city population was 107.31 males to 100 females.[76][77]
As in most of eastern China the ethnic makeup of Nanjing is predominantly Han nationality (98.56 percent), with 50 other minority nationalities. In 1999, 77,394 residents belonged to minority nationalities, among which the vast majority (64,832) were Hui nationalities, contributing 83.76 percent to the minority population. The second and third largest minority groups were Manchu (2,311) and Zhuang (533) nationalities. Most of the minority nationalities resided in Jianye District, comprising 9.13 percent of the district's population.[78]
Since the Three Kingdoms period, Nanjing has become an industrial centre for textile and mint owing to its strategic geographical location and convenient transportation. During the Ming dynasty, Nanjing's industry was further expanded, and the city became one of the most prosperous cities in China and even the world. It led in textile, mint, printing, shipbuilding and many other industries, and was the busiest business center in the Far East.
Into the first half of the twentieth century, Nanjing gradually shifted from a production hub into a heavy consumption city, mainly because of the rapid expansion of the wealthy population after Nanjing once again regained the political spotlight of China. A number of huge department stores such as Zhongyang Shangchang sprouted up, attracting merchants from all over China to sell their products in Nanjing. In 1933, the revenue generated by the food and entertainment industry in the city exceeded the sum of the output of the manufacturing and agriculture industry. One third of the city population worked in the service industry, while prostitution, drugs and gambling also thrived.
In the 1950s, the CPC invested heavily in Nanjing to build a series of state-owned heavy industries, as part of the national plan of rapid industrialization. Electrical, mechanical, chemical and steel factories were established successively, converting Nanjing into a heavy industry production centre of East China.[79] Overenthusiastic in building a "world-class" industrial city, leaders of Nanjing also made many disastrous mistakes during the development, such as spending hundreds of millions of yuan to mine for non-existent coal, resulting in the negative economic growth in the late 1960s.
The current industry of the city basically inherited the characteristics of the 1960s, with electronics, cars, petrochemical, iron and steel, and power as the "Five Pillar Industries". Some representative big state-owned firms are Panda Electronics, Jincheng Motors and Nanjing Steel. The tertiary industry also regained prominence, accounting for 44 percent of the GDP of the city. The city is also vying for foreign investment against neighboring cities in the Yangtze River Delta, and so far a number of famous multinational firms, such as Volkswagen Group, Iveco, A.O. Smith, and Sharp, have established their lines there. Since China's entry into the WTO, Nanjing has received increasing attention from foreign investors, and on average two new foreign firms establish offices in the city every day.
The city government is further improving the desirability of the city to investors by building large industrial parks, which now total four: Gaoxin, Xingang, Huagong and Jiangning. Despite the effort, Nanjing's Gross Domestic Product is still falling behind that of other neighbouring cities such as Suzhou, Wuxi and Hangzhou, which have an edge in attracting foreign investment and local innovation. In addition, the traditional state-owned enterprises find themselves incapable of competing with efficient multinational firms, and hence are either mired in heavy debt or forced into bankruptcy or privatization. This has resulted in large numbers of layoff workers who are technically not unemployed but effectively jobless.
In recent years, Nanjing has been developing its economy, commerce, industry, as well as city construction. In 2013 the city's GDP was RMB 801 billion (3rd in Jiangsu), and GDP per capita(current price) was RMB 98,174(US$16041), a 11 percent increase from 2012. The average urban resident's disposable income was RMB 36,200, while the average rural resident's net income was RMB 14,513. The registered urban unemployment rate was 3.02 percent, lower than the national average (4.3 percent). Nanjing's Gross Domestic Product ranked 12th in 2013 in China, and its overall competence ranked 6th in mainland and 8th including Taiwan and Hong Kong in 2009.[80]
Nanjing Baixia Hi-Tech Industrial Zone is a national hi-tech industrial zone with 16.5 square kilometres (6.4 sq mi) planned area. The zone is only 13.5 km (8.4 mi) away from Nanjing downtown and 50 km (31 mi) away from Nanjing Lukou Airport. Several expressways pass through here. It is well equipped with comprehensive facilities, and it provides a good investment environment for high-tech industries. Electronic industry, automobile, chemical, machinery, instruments and building materials are the encouraged industries in the zone.[81]
Established in 1992, Nanjing Economic and Technological Development Zone is a national level zone surrounded by convenient transportation network. It is only 20 km (12 mi) away from Nanjing Port and 40 km (25 mi) away from Nanjing Lukou Airport. It is well equipped with basic facilities like electricity, water, communication, gas, steam and so on. It has formed four specialized industries, which are electronic information, bio-pharmaceutical, machinery and new materials industry.[82]
On March 10, 2003 the State Council approved the establishment of this Export Processing Zone (EPZ) in Nanjing's Southern District. This EPZ is free from import/export duty area and provides 24-hour customs-bonded conditions. It has a planned area of 3 km2 (1.2 sq mi). The Central Government has given the special economic region preferential policies to attract more enterprises engaged in processing trade investment in the region. It is only 20 km (12 mi) from Nanjing Port and several expressways pass through here.[83]
Nanjing New and High-Tech Industry Development Zone was jointly founded by Jiangsu Provincial People's Government and Nanjing Municipal People's Government, and started to break ground of construction on September 1, 1988. It was established as a national new and high-tech industry development zone by the State Council on March 6, 1991. The zone is next to National Highway 104 and 312. Its pillar industries include electronic information, bio-engineering and pharmaceutical industry.[84]
Nanjing is the transportation hub in eastern China and the downstream Yangtze River area. Different means of transportation constitute a three-dimensional transport system that includes land, water and air. As in most other Chinese cities, public transportation is the dominant mode of travel of the majority of the citizens. The city now has four bridge or tunnel crossings spanning the Yangtze, which are tying districts north of the river with the city centre on the south bank.[85] See also Transport in Nanjing.
Nanjing is an important railway hub in eastern China.[86] It serves as rail junction for the Beijing-Shanghai (Jinghu) (which is itself composed of the old Jinpu and Huning Railways), Nanjing–Tongling (Ningtong), Nanjing–Qidong (Ningqi), and the Nanjing-Xian (Ningxi) which encompasses the Hefei–Nanjing Railway. Nanjing is connected to the national high-speed railway network by Beijing–Shanghai High-Speed Railway and Shanghai–Wuhan–Chengdu Passenger Dedicated Line, with several more high-speed rail lines under construction.
Among all 17 railway stations in Nanjing, passenger rail service is mainly provided by Nanjing Railway Station and Nanjing South Railway Station, while other stations like Nanjing West Railway Station, Zhonghuamen Railway Station and Xianlin Railway Station serve minor roles. Nanjing Railway Station was first built in 1968.[87] In 1999, On November 12, 1999, the station was burnt in a serious fire.[88] Reconstruction of the station was finished on September 1, 2005. Nanjing South Railway Station, which is one of the 5 hub stations on Beijing–Shanghai High-Speed Railway, has officially been claimed as the largest railway station in Asia and the second largest in the world in terms of GFA (Gross Floor Area).[89] Construction of Nanjing South Station began on 10 January 2008.[90] The station was opened for public service in 2011.[91]
As an important regional hub in the Yangtze River Delta, Nanjing is well-connected by over 60 state and provincial highways to all parts of China.
Express highways such as Hu–Ning, Ning–He, Ning–Hang enable commuters to travel to Shanghai, Hefei, Hangzhou, and other important cities quickly and conveniently. Inside the city of Nanjing, there are 230 km (140 mi) of highways, with a highway coverage density of 3.38 kilometres per hundred square kilometrs (5.44 mi/100 sq mi). The total road coverage density of the city is 112.56 kilometres per hundred square kilometres (181.15 mi/100 sq mi).[92] The two artery roads in Nanjing are Zhongshan Road and Hanzhong. The two roads cross in the city centre, Xinjiekou.
As of October 2014, Nanjing had five bridges and two tunnels over the Yangtze River.
National Highway (GXXX):
The city also boasts an efficient network of public transportation, which mainly consists of bus, taxi and metro systems. The bus network, which is currently run by 3 companies since 2011, provides more than 370 routes covering all parts of the city and suburban areas.[93] Nanjing Metro Line 1, started service on September 3, 2005, with 16 stations and a length of 21.72 km.[94] Line 2 and the 24.5 km-long south extension of Line 1 officially opened to passenger service on May 28, 2010.[95][96] The city is planning to complete a 17-line Metro and light-rail system by 2030.[97] The expansion of the Metro network will greatly facilitate the intracity transportation and reduce the currently heavy traffic congestion.
Nanjing's airport, Lukou International Airport, serves both national and international flights. In 2013, Nanjing airport handled 15,011,792 passengers and 255,788.6 tonnes of freight.[98] The airport currently has 85 routes to national and international destinations, which include Japan, Korea, Thailand, Malaysia, Singapore and Germany. The airport is connected by a 29-kilometre (18 mi) highway directly to the city center, and is also linked to various intercity highways, making it accessible to the passengers from the surrounding cities. A railway Ninggao Intercity Line is being built to link the airport with Nanjing South Railway Station.[99] Lukou Airport was opened on 28 June 1997, replacing Nanjing Dajiaochang Airport as the main airport serving Nanjing. Dajiaochang Airport is still used as a military air base.[100]
Port of Nanjing is the largest inland port in China, with annual cargo tonnage reached 191,970,000 t in 2012.[101] The port area is 98 kilometres (61 mi) in length and has 64 berths including 16 berths for ships with a tonnage of more than 10,000.[102] Nanjing is also the biggest container port along the Yangtze River; in March 2004, the one million container-capacity base, Longtan Containers Port Area opened, further consolidating Nanjing as the leading port in the region. In the 1960s the first Yangtze river bridge was completed, becoming almost the only solid connection between North and South in eastern China at that time. The bridge became a source of pride and an important symbol of modern China, having been built and designed by the Chinese themselves following failed surveys by other nations and the reliance on and then rejection of Soviet expertise. Begun in 1960 and opened to traffic in 1968, the bridge is a two-tiered road and rail design spanning 4,600 metres on the upper deck, with approximately 1,580 metres spanning the river itself. As of 2010, it operated six public ports and three industrial ports.[103]
Being one of the four ancient capitals of China, Nanjing has always been a cultural centre attracting intellectuals from all over the country. In the Tang and Song dynasties, Nanjing was a place where poets gathered and composed poems reminiscent of its luxurious past; during the Ming and Qing dynasties, the city was the official imperial examination centre (Jiangnan Examination Hall) for the Jiangnan region, again acting as a hub where different thoughts and opinions converged and thrived.
Today, with a long cultural tradition and strong support from local educational institutions, Nanjing is commonly viewed as a "city of culture" and one of the more pleasant cities to live in China.
Some of the leading art groups of China are based in Nanjing; they include the Qianxian Dance Company, Nanjing Dance Company, Jiangsu Peking Opera Institute and Nanjing Xiaohonghua Art Company among others.
Jiangsu Province Kun Opera is one of the best theatres for Kunqu, China's oldest stage art.[104] It is considered a conservative and traditional troupe. Nanjing also has professional opera troupes for the Yang, Yue (shaoxing), Xi and Jing (Chinese opera varieties) as well as Suzhou pingtan, spoken theatre and puppet theatre.
Jiangsu Art Gallery is the largest gallery in Jiangsu Province, presenting some of the best traditional and contemporary art pieces of China;[105] many other smaller-scale galleries, such as Red Chamber Art Garden and Jinling Stone Gallery, also have their own special exhibitions.
Many traditional festivals and customs were observed in the old times, which included climbing the City Wall on January 16, bathing in Qing Xi on March 3, hill hiking on September 9 and others (the dates are in Chinese lunar calendar). Almost none of them, however, are still celebrated by modern Nanjingese.
Instead, Nanjing, as a popular tourist destination, hosts a series of government-organised events throughout the year. The annual International Plum Blossom Festival held in Plum Blossom Hill, the largest plum collection in China, attracts thousands of tourists both domestically and internationally. Other events include Nanjing Baima Peach Blossom and Kite Festival, Jiangxin Zhou Fruit Festival and Linggu Temple Sweet Osmanthus Festival.
Nanjing Library, founded in 1907, houses more than 7 million volumes of printed materials and is the third largest library in China, after the National Library in Beijing and Shanghai Library. Other libraries, such as city-owned Jinling Library and various district libraries, also provide considerable amount of information to citizens. Nanjing University Library, owned by Nanjing University, with a collection of 4.2 million volumes, is the second largest university libraries in China after Peking University Library. More than 100 multimedia networked-computers are available to readers.
Nanjing has some of the oldest and finest museums in China. Nanjing Museum, formerly known as National Central Museum under KMT rule, is the first modern museum and remains as one of the leading museums in China having 400,000 items in its permanent collection,.[106] The museum is notable for enormous collections of Ming and Qing imperial porcelain, which is among the largest in the world.[107] Other museums include the China Modern History Museum in the Presidential Palace, the Nanjing Massacre Memorial Hall, the City Museum of Nanjing, the Taiping Kingdom History Museum, the Nanjing Customs Museum, the Nanjing City Wall Cultural Museum, and a small museum and tomb honoring the 15th century seafaring admiral Zheng He although his body was buried at sea off the Malabar Coast near Calicut in western India.[108]
Most of Nanjing's major theatres are multi-purpose, used as convention halls, cinemas, musical halls and theatres on different occasions. The major theatres include the People's Convention Hall and the Nanjing Arts and Culture Center.
Traditionally Nanjing's nightlife was mostly centered around Nanjing Fuzimiao (Confucius Temple) area along the Qinhuai River, where night markets, restaurants and pubs thrived.[109] Boating at night in the river was a main attraction of the city. Thus, one can see the statues of the famous teachers and educators of the past not too far from those of the courtesans who educated the young men in the other arts.
In the past 20 years, several commercial streets have been developed, hence the nightlife has become more diverse: there are shopping malls opening late in the Xinjiekou CBD and Hunan Road. The well-established "Nanjing 1912" district hosts a wide variety of pastime facilities ranging from traditional restaurants and western pubs to dance clubs. There are two major areas where bars are densely located; one is in 1912 block; the other is along Shanghai road and its neighbourhood. Both are popular with international residents of the city.
These days, the most comprehensive source of nightlife information (in English) can be found on HelloNanjing.net and NanjingExpat.com.
Local people still very much enjoy street food, such as Turkish Kebab. As elsewhere in Asia, Karaoke is popular with both young and old crowds.
Many of the city's local favorite dishes are based on ducks, including nanjing salted duck, duck blood and vermicelli soup, and duck oil pancake.[110]
The radish is also a typical food representing people of Nanjing, which has been spread through word of mouth as an interesting fact for many years in China. According to Nanjing.GOV.cn, "There is a long history of growing radish in Nanjing especially the southern suburb. In the spring, the radish tastes very juicy and sweet. It is well-known that people in Nanjing like eating radish. And the people are even addressed as 'Nanjing big radish', which means they are unsophisticated, passionate and conservative. From health perspective, eating radish can help to offset the stodgy food that people take during the Spring Festival".[111]
As a major Chinese city, Nanjing is home to many professional sports teams. Jiangsu Sainty, the football club currently staying in Chinese Super League, is a long-term tenant of Nanjing Olympic Sports Center. Jiangsu Nangang Basketball Club is a competitive team which has long been one of the major clubs fighting for the title in China top level league, CBA. Jiangsu Volleyball men and women teams are also traditionally considered as at top level in China volleyball league.
There are two major sports centers in Nanjing, Wutaishan Sports Center and Nanjing Olympic Sports Center. Both of these two are comprehensive sports centers, including stadium, gymnasium, natatorium, tennis court, etc. Wutaishan Sports Center was established in 1952 and it was one of the oldest and most advanced stadiums in early time of People's Republic of China. Nanjing hosted the 10th National Games of P.R.C. in 2005 and hosted the 2nd summer Youth Olympic Games in 2014.[112][113]
In 2005, in order to host The 10th National Game of People's Republic of China, there was a new stadium, Nanjing Olympic Sports Center, constructed in Nanjing. Compared to Wutaishan Sports Center, which the major stadium's capacity is 18,500,[114] Nanjing Olympic Sports Center has a more advanced stadium which is big enough to seat 60,000 spectators. Its gymnasium has capacity of 13,000, and natatorium of capacity 3,000.
On 10 February 2010, the 122nd IOC session at Vancouver announced Nanjing as the host city for the 2nd Summer Youth Olympic Games.The slogan of the Nanjing 2014 Youth Olympic Games is "Share the Games, Share our Dreams".The Nanjing 2014 Youth Olympic Games will encompass all 28 sports on the Olympic programme and will be held from 16 to 28 August. The Nanjing Youth Olympic Games Organising Committee (NYOGOC) will work together with the International Olympic Committee (IOC) to attract the best young athletes from around the world to compete at the highest level. Off the competition fields, an integrated culture and education programme will focus on discussions about education, Olympic values, social challenges, and cultural diversity. The YOG aims to spread the Olympic spirit and encourage sports participation.
Nanjing is one of the most beautiful cities of mainland China with lush green parks, natural scenic lakes, small mountains, historical buildings and monuments, relics and much more, which attracts thousands of tourists every year.
Because it was designated as the national capital, many structures were built around that time. Even today, some of them still remain which are open to tourists.
Nanjing has been the educational centre in southern China for more than 1700 years. Currently, it boasts of some of the most prominent educational institutions in the region, which are listed as follows:
Operated by Ministry of Education
Operated by Ministry of Industry and Information Technology
Operated by the joint Commission of the State Forest Administration and Public Order Ministry
Operated by the general sport Administration
民國十六年,國民政府宣言定為首都,今以臺北市為我國中央政府所在地。(In the 16th Year of the Republic of China [1927], the National Government established [Nanking] as the capital. At present, Taipei is the seat of the central government.)
(help)Wikimedia Commons has media related to Nanjing. |
Look up nanjing in Wiktionary, the free dictionary. |
Preceded by Beijing | Capital of China 1368–1420 | Succeeded by Beijing |
Preceded by Beijing | Capital of China 1928–1937 | Succeeded by Wuhan (wartime) |
Preceded by Chongqing | Capital of China 1945–1949 | Succeeded by Taipei for the Republic of China |
Succeeded by Beijing for the People's Republic of China |
Chuzhou (Anhui): Tianchang | ||||
Chuzhou: Urban area, Lai'an, Quanjiao, Hexian | Yangzhou: Yizheng Zhenjiang: Jurong Changzhou: Liyang | |||
| ||||
Ma'anshan (Anhui): Urban area, Dangtu | Xuancheng: Urban area | Xuancheng (Anhui): Langxi |
Largest cities or towns of China Sixth National Population Census of the People's Republic of China (2010) | |||||||||
Rank | Name | Province | Pop. | Rank | Name | Province | Pop. | ||
Shanghai Beijing | 1 | Shanghai | Shanghai | 22,315,426 | 11 | Chengdu | Sichuan | 7,123,697 | Chongqing Guangzhou |
2 | Beijing | Beijing | 18,827,000 | 12 | Hong Kong | Hong Kong | 7,055,071 | ||
3 | Chongqing | Chongqing | 15,294,255 | 13 | Harbin | Heilongjiang | 6,704,573 | ||
4 | Guangzhou | Guangdong | 12,700,800 | 14 | Xi'an | Shaanxi | 6,501,189 | ||
5 | Tianjin | Tianjin | 11,090,314 | 15 | Shenyang | Liaoning | 6,255,921 | ||
6 | Shenzhen | Guangdong | 10,357,938 | 16 | Hangzhou | Zhejiang | 6,241,971 | ||
7 | Wuhan | Hubei | 9,785,392 | 17 | Suzhou | Jiangsu | 5,349,093 | ||
8 | Dongguan | Guangdong | 8,220,937 | 18 | Shantou | Guangdong | 5,330,764 | ||
9 | Nanjing | Jiangsu | 8,004,680 | 19 | Changchun | Jilin | 4,647,644 | ||
10 | Foshan | Guangdong | 7,194,311 | 20 | Jinan | Shandong | 4,336,100 |


text:
Bulked segregant analysis (BSA) identifies molecular markers associated with a phenotype by screening two DNA pools of phenotypically distinct plants for markers with skewed allele frequencies. In contrast to gel-based markers, hybridization-based markers such as SFP, DArT or SNP generate quantitative allele-frequency estimates. Only DArT, however, combines this advantage with low development and assay costs and the ability to be deployed for any plant species irrespective of its ploidy level. Here we investigate the suitability of DArT for BSA applications using a barley array as an example.
In a first test experiment, we compared two bulks of 40 Steptoe/Morex DH plants with contrasting pubescent leaves (mPub) alleles on chromosome 3H. At optimized levels of experimental replication and marker-selection threshold, the BSA scan identified 433 polymorphic markers. The relative hybridization contrast between bulks accurately reflected the between-bulk difference in the frequency of the mPub allele (r = 0.96). The 'platform noise' of DArT assays, estimated by comparing two identical aliquots of a DNA mixture, was significantly lower than the 'pooling noise' reflecting the binomial sampling variance of the bulking process. The allele-frequency difference on chromosome 3H increased in the vicinity of mPub and peaked at the marker with the smallest distance from mPub (4.6 cM). In a validation experiment with only 20 plants per bulk we identified an aluminum (Al) tolerance locus in a Dayton/Zhepi2 DH population on chromosome 4H with < 0.8 cM precision, the same Al-tolerance locus that had been mapped before in other barley populations.
DArT-BSA identifies genetic loci that influence phenotypic characters in barley with at least 5 cM accuracy and should prove useful as a generic tool for high-throughput, quantitative BSA in plants irrespective of their ploidy level.
Bulked Segregation Analysis (BSA) is a widely used method for rapidly identifying molecular markers linked to a trait of interest. It involves genotyping two pools (bulks) of DNA samples from individual plants originating from an experimental cross. Plants are assigned to one of the two bulks based on the trait of interest. The bulks are screened with a large number of markers to identify those that distinguish the bulks and, by inference, must be genetically linked to the trait locus [1].
The success of the BSA approach relies on the dramatic reduction in the number of marker assays when compared to building a genetic map for the purpose of identifying markers associated with a phenotype. BSA 'collapses' the two-dimensional matrix of marker assays (DNA samples × markers) into a one-dimensional vector of genotypic differences between two DNA bulks. With gel-based marker technologies this vector is largely built up sequentially. Highly multiplexed, hybridization-based marker technologies such as SFP, DArT and SNP have the potential to further 'collapse' the vector of genotypic differences between bulks into a single (perhaps replicated) whole-genome assay [2-5].
Application of hybridization-based marker technologies to BSA not only reduces the genotyping effort, but has the additional benefit of producing quantitative raw data (hybridization intensities) which are only subsequently converted into discrete genotypes (allele calls) in the case of non-BSA applications. The raw hybridization data are a quantitative measure of allele frequencies [6-8] and thus should increase the accuracy of mapping a trait locus with BSA. Others have used this feature when performing BSA experiments using SFP typed on Affymetrix GeneChips [9,10].
While SFP are a powerful research tool for species with sufficient sequence information, their utility in the context of agricultural research and (pre)breeding appears to be fairly limited, not only because of technology-establishment and per-sample assay costs but also because polyploidy poses a serious barrier to any whole-genome hybridization approach. Polyploidy and the costs of technology establishment for new species are also limiting the widespread deployment of SNP among the approximately 150 crop species cultivated worldwide, many of which have large and/or polyploid genomes [11,12]. By contrast DArT arrays, already available for two dozens of plant and fungal species [4,13-18], can be rapidly developed for new species of any ploidy level for a small fraction of the investment required for SFP or SNP arrays [19].
In this paper we investigate the suitability of the DArT platform for BSA, using as an example a polymorphism-enriched barley array with 2,304 clones [20]. We first test 'DArT-BSA' using a DH population that segregates for a morphological marker with known map position (our model target trait). In this experiment we explore several technology features that are likely to be critical for routine deployment of DArT-BSA. In a subsequent validation experiment we deploy the optimized method to a different DH population segregating for Al tolerance to test the performance of DArT-BSA in a practical application.
Results and discussion
Test experiment
The Steptoe/Morex DH population segregates for 'pubescent leaf blades' (mPub), a morphological marker that has been incorporated into the genetic map of chromosome 3H in this population [21]. We used mPub to assemble contrasting bulks for a trait with known genetic location in order to evaluate the performance of DArT when applied to BSA. The bulks were compared by simultaneously assaying them on the same DArT array (replicated up to eight times) and measuring the contrast of hybridization intensity for markers that were identified as polymorphic in a comparison between the two parents on separate replicated arrays.
Selection of polymorphic markers
Markers differentiating between Steptoe and Morex were selected based on the contrast in hybridization intensity between the two cultivars (log2 [cy3/cy5]). Instead of applying an arbitrary threshold we measured the variability of hybridization-contrast estimates by comparing two identical aliquots of a 1:1 mixture of the two parents ('self comparison'). Polymorphic markers were then selected by applying to the hybridization contrast between the parents a normal distribution-based probability threshold derived from the self comparison.
As the stringency of this marker-selection threshold was increased from 10-2 to 10-5, the minimum parental hybridization contrast increased from 0.69 to 1.18 log units (Table 1). The latter caused a concomitant decrease in the number of selected polymorphic markers from 631 to 384. At the same time the proportion of markers previously mapped in the Steptoe/Morex population with an array containing a subset of markers [4] increased from 45 to 55% (Table 1). These numbers indicate a gradual enrichment of high-quality, 'mappable' markers at more stringent thresholds at the expense of excluding other possibly informative markers. A test of several threshold levels for their effectiveness in excluding outlier markers in a plot of relative hybridization contrast vs. map position (see section entitled Genome-wide linkage scan for an example) suggested that p < 0.0001 was an acceptable compromise between these two tendencies (data not presented). This threshold was used for the rest of this study.
Table 1. Effect of experimental settings on polymorphic-marker selection and linkage-detection thresholds in the mPub BSA scan
Experimental replicates
We next tested the effect of varying the number of replicate arrays between two and eight. Increasing the degree of replication resulted in the inclusion of markers that were previously not mapped, in part because the hybridization contrast between the parental alleles was small (Table 1) [20]. Only a small minority of the additional markers, however, were outliers with respect to a Loess curve in a plot of the relative hybridization contrast vs. map position (see section entitled Genome-wide linkage scan below for an example). This result suggests that most of these markers were still genetically informative. A high degree of experimental replication seems to improve the precision of quantifying markers with a smaller hybridization contrast between the parental alleles, thus resulting in the inclusion of more markers in a BSA scan. We considered four experimental replicates adequate for the purpose of this study.
Quantification of the allele-frequency difference between bulks
The contrast in hybridization intensity between the two allelic states varies from marker to marker and has to be taken into account when quantifying allele abundances in DNA pools. The log2 [cy3/cy5] values measured for polymorphic markers in the bulk comparison were therefore scaled by division by the log2 [cy3/cy5] values measured in the comparison between the parents.
To link the scaled or relative hybridization contrast of a marker to the allelic composition of the bulks, we computed from the segregation data of a Steptoe/Morex DArT map the frequency of the Steptoe allele in each bulk (see section entitled 'Allele-frequency determination and simulation' in Materials and Methods). The difference between the Steptoe allele frequencies in the two bulks was closely correlated to the relative hybridization contrast (r = 0.96; Figure 1). The degree of correlation was identical to the correlation obtained in other studies using the Affymetrix SNP genotyping platform [7,8]. This result confirms that hybridization intensities are proportional to the abundance of DArT alleles in DNA pools. The relative hybridization contrast, therefore, can be used as a measure of the between-bulk difference in the frequency of the allele from the trait-donor parent.
Figure 1. Quantitative precision of DArT-BSA. For each marker, there is a 1:1 relationship between the relative hybridization contrast (= log2 [cy3/cy5] between bulks as a percentage of log2 [cy3/cy5] between parents) and the allele-frequency difference between the bulks. This relationship makes interpretation of experimental results straightforward. The plot is based on a comparison of mPub bulks (size = 40), prepared from genomic representations of individual Steptoe/Morex DH plants (see corresponding genome scan in Figure 3). It includes all markers present in a previously published Steptoe/Morex DArT map [20] and reports the difference in the frequency of Steptoe alleles between bulks.
It follows from this data that previously identified limitations of dominant markers for BSA with certain population types [22] do not apply to DArT-BSA, although in a non-BSA context DArT markers are typically scored in a dominant manner [13]. Any type of population that segregates for a trait of interest should be amenable to DArT-BSA; be it DH, RIL, BC, F2 or more complex populations.
Linkage-detection threshold derived from 'platform noise'
Before scanning the genome for markers linked to mPub we quantified the basal 'platform noise' to obtain a significance threshold for detecting linkage. The dispersion of the apparent allele-frequency difference in a comparison between two identical aliquots of a 1:1 mixture of the parents was used to derive a genome-wide significance threshold (Table 1). The size of this threshold was a function of the extent to which markers with limited hybridization contrast between alternative alleles were incorporated in the analysis (Table 1), because the dispersion of allele-frequency estimates was larger for less well-separated markers (Additional File 1). More experimental replicates kept the threshold low even if poorly separated markers were included (Table 1).
Additional File 1. Influence of the hybridization contrast between parental alleles on the precision of estimating allele-frequency equality. PDF file with a chart displaying the relationship between the apparent allele-frequency differences, measured by comparing two identical aliquots of a 1:1 mixture of Steptoe and Morex, and the hybridization contrasts between alternative alleles. All markers present in a Steptoe/Morex DArT map [20] were included in this figure. The SD of groups of markers in allelic-contrast bins of 0.5 units on the log2 [cy3/cy5] scale are included.
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Linkage-detection threshold derived from 'pooling noise'
Besides quantifying the effect of platform-related noise, it is important to consider the expected variability caused by binomial sampling in the bulking process. As a result of the random assortment of chromosomes, the between-bulk difference in allele frequency may deviate from zero for markers that are not linked to the target locus, thus generating spurious linkage signals.
We established a genome-wide significance threshold by simulating the comparison of random bulks (see Materials and Methods for details). With increasing bulk sizes, the threshold asymptotically drops toward 0% allele-frequency difference (Figure 2). At 40 plants per bulk, for example, there is a 5% probability of detecting, by chance only, at least one genomic region with an allele-frequency difference greater than 37.5% (Table 1). The thresholds reported in Figure 2 are specific to the number of chromosomes and the type of population used in this study. For species with more chromosomes, for example, larger bulk sizes are required to achieve comparable thresholds (data not presented).
Figure 2. 'Pooling noise': the effect of bulk sizes on the amplitude of spurious linkage signals. Genome-wide significance thresholds for detecting spurious linkage between a marker and a target locus were derived from 10,000 simulated comparisons between bulks of F1-derived DH barley plants (see section entitled 'Allele-frequency determination and simulation' in Materials and Methods for details). Trait-linked markers should only be considered as statistically significant if the allele-frequency difference between bulks is superior to the value derived for the relevant combination of bulk size and desired probability level.
Genome-wide linkage scan
A plot of the allele-frequency difference between mPub bulks (40 plants each) vs. the chromosomal positions of the markers on a DArT consensus map [20] immediately confirmed that mPub was located on the long arm of chromosome 3H (Figure 3, top panel) [21]. The marker with the maximum allele-frequency difference (93.5%) was bPb-8978. A Steptoe/Morex map for chromosome 3H, built from markers overlapping between this and a previous study [20], confirmed that bPb-8978 was the closest marker to mPub at 4.6 cM distance. The Loess curve also peaked at this marker. These results underscore the mapping accuracy of DArT-BSA.
Figure 3. DArT-BSA genome scan for the 'pubescent leaves' (mPub) locus in the Steptoe/Morex DH population. The difference in the frequency of Steptoe alleles between different pairs of DNA pools is shown as a function of the cM positions of markers previously incorporated into a DArT consensus map for barley (375 of 418 polymorphic markers) [20]. The 'Bulks (mPub)' panel displays data from the comparison between bulks contrasting for the mPub locus on chromosome 3H (40 plants each). The black arrow indicates the position of the mPub locus on the DArT consensus map [20] according to which the DH plants had been distributed into bulks. The 'Random bulks' panel displays data from a comparison of two randomly assorted bulks of 40 plants each. The 'Self comparison' panel shows the result of a comparison of two identical aliquots of a 1:1 mixture of Steptoe and Morex. Vertical lines within each of the panels denote borders between individual chromosomes. Horizontal lines indicate two types of significance thresholds. The 'pooling noise' significance threshold was based on a simulation of the bulking process (see Figure 2). The 'platform noise' significance threshold was derived from an analysis of the distribution of values in the self comparison (bottom panel) and Bonferroni-adjusted for multiple comparisons (see section entitled 'Polymorphic marker identification' in Materials and Methods).
The mPub linkage signal was highly significant, peaking well beyond both the 'pooling noise' and the 'platform noise' threshold (Figure 3, top panel). A comparison of two randomly assembled bulks of 40 plants (Figure 3, middle panel) showed that bulks were not large enough to decrease the random fluctuations in allele frequencies to a level comparable to the apparent fluctuations caused by the baseline noise of the DArT platform (Figure 3 bottom panel). The SD of the allele-frequency difference was 12.0% in the random-bulk comparison, 12.5% in the mPub-bulk comparison (all chromosomes except 3H), but only 5.9% in the self comparison.
We conclude from this data that in the case of barley, it would be beneficial to bulk up to 150 DH plants for DArT-BSA. At this bulk size the extent of random fluctuations in allele frequencies introduced in the pooling process (Figure 2) become comparable to the approximate 'platform noise' of DArT-BSA (24% at p < 0.05 in the test experiment, but only 17% in the subsequent validation experiment).
Linkage decay in the vicinity of the target locus
The 'linkage signal' decayed as the distance between markers and the target locus increased (Figure 4). A linear-regression analysis using markers within ± 30 cM of mPub indicated a 2.3% decrease per cM distance from the target locus (see inset in Figure 4). It is therefore possible to estimate the approximate cM distance of DArT markers from the target locus based on the following formula: cM distance ≈ 0.43 × % allele-frequency difference – 43. This relationship could be useful for analyzing populations derived from genetically close parents. In such situations, lower marker densities are expected and the approximate position of the target locus could be estimated from moderately linked markers based on this relationship.
Figure 4. Decay of linkage as a function of the genetic distance between markers and a trait locus. The measured allele-frequency difference between two bulks (40 plants each) was plotted against the cM distance between chromosome-3H markers and the mPub locus. The inset displays a linear regression of the allele-frequency difference for markers within ± 30 cM from mPub on the cM distance from mPub. Black data points are significantly linked to mPub at the p < 0.05 level for 'pooling noise' (see Figure 2).
Gel-based markers (RAPD, AFLP and SSR) compromise the efficiency and precision of BSA by generating discrete allelic data in a somewhat arbitrary and hence error-prone process (i.e., alleles are called 'present' or 'absent'). The latter is problematic in situations where the bulks contain different proportion of both parental alleles, for example in case of less than perfectly linked makers or for QTL with moderate effects. The ability of DArT-BSA to quantify the degree of linkage between markers and a target locus is a clear advantage in such cases.
Marker density
The mPub genome scan comprised 433 polymorphic markers (Table 1), that is, approximately one marker every 2.7 cM. This resolution is lower than the resolution afforded by the Arabidopsis SFP array [9,23]. However, SFP hybridization data tend to be quite variable, which makes a high marker density an important prerequisite for calculating robust allele-frequency difference estimates for genomic regions. More importantly, the precision with which a target locus can be mapped by BSA not only depends on the marker density but also on the number of crossover in the vicinity of the target locus. In experimental populations that are the result of a limited number of meioses the 'linkage peaks' will simply be too broad to make effective use of more than approximately 500 to 1,000 markers.
Validation experiment
Having established that DArT-BSA accurately identifies the known genomic location of a model trait, we continued to validate the method by attempting to map an Al-tolerance locus in a Dayton/Zhepi2 DH population (Wang et al., submitted). For this purpose we 'relaxed' the conditions of analysis in two ways to accommodate more typical experimental designs. First, we pooled genomic-DNA samples (for a comparison against pooled genomic representations). Second, we only pooled 20 plants per bulk, not an uncommon bulk size in this sort of experiments.
Genomic-DNA samples can be bulked directly
Because DArT-BSA compares the abundance of alternative alleles between bulks in a quantitative manner (Figure 1), it is important to make sure that individual plants contribute equally to bulks, particularly when working with small bulk sizes. Presumably, the most robust way to achieve this is to bulk representations derived from individual samples of genomic DNA. Direct bulking of genomic-DNA samples, however, produces virtually identical estimates of allele-frequency differences (r = 0.91; Additional File 2). It may well be tolerable to bulk equal amounts of plant material before DNA extraction, although we did not test this method experimentally.
Additional File 2. Comparison between two bulking strategies. PDF file with a chart displaying, for each of 490 markers, the difference of the Dayton allele frequency between Al-tolerant and Al-sensitive bulks of 20 Dayton/Zhepi2 DH plants (Δ %), measured using two alternative methods: (1) by pooling representations prepared from individual DNA samples (horizontal axis), and (2) by preparing two representations from genomic-DNA pools. The dotted line denotes equality between the two alternative methods.
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DArT-BSA identifies an Al-tolerance locus on chromosome 4H
The DArT-BSA scan for Al tolerance in the Dayton/Zhepi2 population revealed a highly significant peak on chromosome 4H, despite the elevated 'pooling-noise' threshold (50%; p < 0.05) due to the small bulk sizes used (Figure 5). The position of the peak is consistent with the location of an Al-tolerance locus (Alt) on 4HL, which appears to mediate Al-activated citrate secretion from roots, and has already been identified in several barley populations [24-30].
The Alt locus was previously mapped between SSR markers Bmag490 on the proximal side and HVM68 on the distal side [29,30]. These two SSR markers have also been incorporated into a DArT linkage map of the Dayton/Zhepi2 population where they span a small 0.8-cM region (see Additional File 2 in [20]). The marker that showed the greatest allele-frequency difference in the DArT-BSA scan (101.7%; bPb-6872) co-segregated with Bmag490, which implies that it must have been closer than 0.8 cM from the Alt locus. The Loess curve peaked another 4.2 cM proximal of the Bmag490/bPb-6872 locus (Figure 5).
Figure 5. DArT-BSA genome scan for Al tolerance in the Dayton/Zhepi2 DH population. The difference in the frequency of the Dayton allele between two bulks with contrasting Al tolerance (20 plants each) is shown as a function of the cM positions of markers previously incorporated into a DArT consensus map for barley (446 of 490 polymorphic markers) [20]. The vertical lines within the panel denote borders between individual chromosomes. Horizontal lines indicate the 'pooling noise' and 'platform noise' significance thresholds as defined in Figure 3, using Dayton and Zhepi2 instead of Steptoe and Morex. The black arrow indicates the approximate position of the Alt locus which was identified in other populations as the principal locus conferring Al resistance in barley [24–30].
These results are consistent with the performance of DArT-BSA in the Steptoe/Morex model experiment conducted under technically more stringent experimental conditions (Figure 3). In both experiments the position of the target locus was mapped with at least 5 cM precision. A third experiment not reported here identified markers linked to a disease-resistance locus that was subsequently confirmed by conventional linkage analysis.
Given that DArT arrays are already available for two dozens of plant and fungal species [4,13-18] and can be rapidly developed for new species of any ploidy level with limited resources [19], we extrapolate from the results of this study that DArT should prove useful as a generic platform for quantitative BSA in plants. DArT markers in established arrays for a number of important crops are being sequenced, thus providing instant access to sequence anchor(s) for any DArT-BSA-tagged character when sufficient genomic resources become available in the future.
Populations and targeted traits
Two DH populations derived from the F1 of bi-parental crosses between barley cultivars were used for this study. The Steptoe/Morex population segregated for the morphological marker 'pubescent leaf blades' (mPub) [21]. The Dayton/Zhepi2 population segregated for Al tolerance (Wang et al., submitted). DNA was prepared from individual plants using a CTAB method [31,32].
Individual DH plants derived from the Steptoe/Morex cross were classified as 'pubescent' and 'hairless' according to their mPub allele [33] and assigned to two pairs of contrasting bulks (20 or 40 plants each). The DH progeny from the Dayton/Zhepi2 cross was assessed for Al tolerance using a nutrient solution-culture method [26]. The roots were exposed to 15 μM of Al for 3 days and stained with 0.1% (w/v) eriochrome cyanine R. The seedlings were then visually scored as 'sensitive' or 'tolerant' as described previously [29] and assigned to one of two contrasting bulks of 20 plants each.
'Representation bulks' were prepared by mixing the genomic representations prepared from individual genomic DNA samples (see section entitled DArT assays below). 'Genomic bulks' were prepared by mixing the genomic DNA samples (~1 μg each) themselves (Dayton/Zhepi2 population only).
Experimental design
A BSA experiment typically comprised four comparisons between individual DNA samples or DNA pools. First, phenotypically contrasting bulks (either of the 'genomic' or the 'representation' type) were compared by simultaneously assaying them on the same DArT array. Second, representations prepared from the two parents were compared on a separate array. Third, a pair of 'representation bulks' derived from two randomly assorted DNA pools ('random bulks') were assayed on another array to evaluate the impact of stochastic effects associated with bulking. Fourth, two identical aliquots of a 1:1 mixture of the two parents ('parent mixture') were compared against each other to quantify the platform (technical) noise of the DArT assays. Each pair of samples ('genomic bulks', 'representation bulks', 'parents', 'random bulks', 'parent mixture') was independently analyzed up to eight times on separate arrays. Half of these replicate assays were performed by swapping the cy3 and cy5 fluorescent dyes used to label the representation pairs compared against each other (see next section).
DArT assays
PstI/BstNI representations of genomic DNA samples (from individual plants or 'genomic bulks') were prepared as described previously [4]. The representations produced from individual DNA samples were either bulked according to mPub or Al tolerance ('representation bulks') or pooled randomly ('random bulks'). All representations and 'representation bulks' were purified, labeled with cy3 or cy5 and hybridized to DArT arrays together with the FAM-label polylinker of the vector that had been used to clone the fragments printed on the arrays [3,14]. The DArT arrays contained 48 sub-arrays with 2,304 polymorphism-enriched clones printed in duplicate and 384 control clones, each printed six times (6,912 array features in total) [20]. The arrays had been printed with a MicroGrid II arrayer (Biorobotics, Cambridge, UK) on SuperChip poly-L-lysine slides (Erie Microarray, Portsmouth NH, USA) using DArT-spotter, a thoroughly optimized buffer for heavy-duty microarray printing (Wenzl et al. in preparation). After an overnight hybridization at 62°C, the arrays were washed and scanned with 10-μm resolution at 543 nm (cy3), 633 nm (cy5) and 488 nM (FAM) on a LS300 confocal laser scanner (Tecan, Grödig, Austria) [3,14].
Array-data analysis
Array images were analyzed with DArTsoft 7.4 (Diversity Arrays Technology P/L, Canberra, Australia). The program automatically recognized array features using a seeded-region-growth algorithm and reported, for each fluorescent channel, the average and SD of pixel intensities within and around each array feature, the fraction of saturated pixels within each feature and the number of pixels of each feature, amongst other parameters (Cayla et al. in preparation). The logarithm of the ratio between the two background-subtracted averages of feature pixels in the cy3 and the cy5 channel (log2 [cy3/cy5]) was used as a measure of the difference in abundance of the corresponding DNA fragment in the two representations hybridized to an array. The log2 [cy3/FAM] and log2 [cy5/FAM] values, approximate measures of the amount of hybridization signal per amount of DNA spotted on the array, were used for quality-control purposes (see next section).
Whole array quality-filtering
Arrays were rejected if the average correlation of either the log2 [cy3/FAM] or log2 [cy5/FAM] values of non-polymorphic clones (as identified below) with the corresponding values from all other arrays in an experiment was smaller than 0.9. One out of 68 arrays (1.5%) was removed this way.
Array feature quality-filtering
Some array features hybridized weakly in both fluorescent channels, either because of an insufficient amount of DNA printed on the array or because the corresponding DNA fragments were not captured in the genomic representation of either parent. Therefore, features with signal-to-noise ratios (the background-subtracted average of feature pixels divided by the SD of local-background pixels) below 5 in both fluorescent channels were removed from further analysis. The remaining features were accepted if the coefficient of variation of their pixel intensities was smaller than 70% in at least one of the two channels, if less then 20% of feature pixels were saturated in both channels, if the SD of background pixels was smaller than 5 times the array median in both channels, and if their size (pixel number) was at least 30% of the array median. Overall, this quality-filtering procedure removed 12.3 ± 0.7% of all array features (mean ± SD across all experiments).
Hybridization-intensity normalization and averaging
The SD of the central 90% of all log2 [cy3/cy5] feature values on an array was scaled to the average SD of the group of arrays hybridized to the same type of DNA samples or pools. The average of the central 90% of all features on each array was adjusted to zero. Subsequently, the normalized/scaled log2 [cy3/cy5] values of replicate features (both within and across arrays hybridized with identical sample pairs) were averaged to obtain clone values (values derived from dye-swap arrays were multiplied by -1). For clones with at least 6 remaining replicate features, only the central 66% of values were averaged. Clones with less than 25% of replicate features left and the 3% of clones with the highest across-replicate-feature SD of log2 [cy3/cy5] were removed from further analysis. In this manner the best 92.9 ± 0.1% of all clones on the array were selected as potential markers for BSA analysis (mean ± SD across all experiments).
Polymorphic marker identification
Polymorphic clones (markers) were selected from the set of quality-filtered clones, using a two-tiered approach. In step one, a normal distribution-based significance threshold for log2 [cy3/cy5] clone values was established (typically at p < 0.0001) to detect outliers in the comparison between two aliquots of the parent mixture. Polymorphic markers were identified in the comparison between the parents based on this log2 [cy3/cy5] threshold, after it was adjusted by the ratio of the mean between-replicate-feature SD in the two types of comparisons. Potentially unstable polymorphic clones were identified by searching for polymorphic clones that were present in the tails of the log2 [cy3/cy5] distribution in the comparison between two aliquots of the parent mixture (p < 0.05). They were excluded from further analysis.
For step two, the log2 [cy3/cy5] values obtained in the 'representation/genomic bulks', 'random bulks' and 'parent mixture' comparisons were referenced against (divided by) the corresponding values measured in the comparison between parents. Ratios between two groups of log2 [cy3/cy5] values (derived from replicated array features) were computed using weighted jackknifing [34]. The resulting values for relative hybridization contrast (i.e., the hybridization contrast as a percentage of the contrast between the parents) were accepted if their standard error was smaller than 10%. The values obtained in the comparison of two aliquots of the parents mixture were screened for outliers using a normal distribution-based significance threshold of p = 0.05. Outlier markers were excluded.
Allele-frequency determination and simulation
A subset of the markers that were identified as polymorphic in the Steptoe vs. Morex comparisons had previously been incorporated into a linkage map for this population [20]. For each of these markers, the difference in the frequency of the Steptoe allele between the contrasting mPub bulks of 40 plants was calculated from the segregation data after inferring missing data and removing likely genotyping errors (see section entitled Distance of DArT markers from mPub below).
The probability of detecting spurious linkages when analyzing bulks of limited size was estimated by evaluating the distribution of the maximum difference in allele frequency in 10,000 comparisons between random bulks consisting of varying numbers of simulated barley DH genotypes (10, 20, 40 and 80). The latter were generated by randomly seeding seven chromosome telomeres with one of the two parental alleles, followed by propagating the seeded genotypes along 59 additional chromosomal loci based on a Markov chain with a constant 3% transition probability (i.e., assuming 60 equidistant loci on 7 chromosomes of approximately 180 cM length each).
Marker positions on linkage maps
Distance of DArT markers from mPub
Segregation data of DArT markers previously mapped to chromosome 3H in the Steptoe/Morex population were combined with the segregation pattern of mPub [20,33]. The 3H map was then re-optimized using the RECORD algorithm [35] and missing data were inferred from neighboring markers. Potential genotyping errors were identified as described previously [36] (LODerror > 4) and replaced with missing data (< 0.2% of allele calls). Map distances between mPub and all other markers on chromosome 3H were then computed by adding Kosambi cM distances between adjacent markers.
Genome-scan display
The relative contrast in hybridization intensity of a subset of markers that had previously been incorporated into a DArT consensus map for barley [20] were plotted against the markers' positions in the barley genome. A Loess curve was fitted to each of the chromosomes to visualize changes in allele frequency across chromosomes.
List of abbreviations
AFLP, amplified fragment length polymorphism; Al, aluminum; Alt, aluminum-tolerance locus; BC, backcross; BSA, bulked segregant analysis; CTAB, cetyl trimetyl ammonium bromide; DArT, diversity arrays technology; DH, doubled haploid; FAM, 5,6-carboxy-fluorescein; LODerror, logarithm of odds value for genotyping error; Loess, locally-weighted scatterplot smoothing; mPub, pubescent leaf blades locus; QTL, quantitative trait locus/loci; RAPD, random amplified polymorphic DNA; RIL, recombinant inbred line(s); SD, standard deviation; SFP, single feature polymorphism; SNP, single nucleotide polymorphism; SSR, simple sequence repeat
Authors' contributions
PW designed and performed the DArT-BSA assays, analyzed the data and drafted the manuscript. HR performed some Al-tolerance bioassays and supervised molecular and physiological work on the Dayton/Zhepi2 population. JW isolated DNA of the Dayton/Zhepi2 population, performed the Al-tolerance bioassays and assembled the Al-tolerant and sensitive bulks. MZ provided the Dayton/Zhepi2 DH population. EH provided ongoing intellectual input to the design and development of the barley DArT array and edited the manuscript. AK provided overall guidance during the development of DArT for barley, including its deployment in BSA and co-designed and edited the manuscript. All authors read and approved the final manuscript.
We thank the Australian Grains Research and Development Corporation (GRDC) for ongoing financial support and the Value Added Wheat Cooperative Research Centre (VAWCRC) for their commitment to our Triticarte P/L partnership.
Michelmore RW, Paran I, Kesseli RV: Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations.
Proc Natl Acad Sci USA 1991, 88:9828-9832. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
West MA, van Leeuwen H, Kozik A, Kliebenstein DJ, Doerge RW, St Clair DA, Michelmore RW: High-density haplotyping with microarray-based expression and single feature polymorphism markers in Arabidopsis.
Genome Res 2006, 16:787-895. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Jaccoud D, Peng K, Feinstein D, Kilian A: Diversity Arrays: a solid state technology for sequence information independent genotyping.
Nucl Acids Res 2001, 29:e25. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wenzl P, Carling J, Kudrna D, Jaccoud D, Huttner E, Kleinhofs A, Kilian A: Diversity arrays technology (DArT) for whole-genome profiling of barley.
Proc Natl Acad Sci USA 2004, 101:9915-9920. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Fan JB, Chee MS, Gunderson KL: Highly parallel genomic assays.
Nat Rev Genet 2006, 7:632-644. PubMed Abstract | Publisher Full Text
Meaburn E, Butcher LM, Schalkwyk LC, Plomin R: Genotyping pooled DNA using 100 K SNP microarrays: a step towards genomewide association scans.
Nucleic Acids Res 2006, 34:e28. Publisher Full Text
Kirov G, Nikolov I, Georgieva L, Moskvina V, Owen MJ, O'Donovan MC: Pooled DNA genotyping an Affymetrix SNP genotyping arrays.
BMC Genomics 2006, 7:27. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Steer S, Abkevich V, Gutin A, Cordell HJ, Gendall KL, Merriman ME, Rodger RA, Rowley KA, Chapman P, Gow P, Harrison AA, Highton J, Jones PBB, O'Donnell J, Stamp L, Fitzgerald L, Iliev D, Kouzmine A, Tran T, Skolnick MH, Timms KM, Lanchbury JS, Merrima TR: Genomic DNA pooling for whole-genome association scans in complex disease: empirical demonstration of efficacy in rheumatoid arthritis.
Genes Immun 2007, 8:57-68. PubMed Abstract | Publisher Full Text
Wolyn D, Borevitz J, Loudet O, Schwartz C, Maloof J, Ecker J, Berry C, Chory J: Light Response QTL Identified with Composite Interval and eXtreme Array Mapping in Arabidopsis thaliana.
Genetics 2004, 167:907-917. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Brauer MJ, Christianson CM, Pai DA, Dunham MJ: Mapping novel traits by array-assisted bulk segregant analysis in Saccharomyces cerevisiae.
Genetics 2006, 173:1813-1816. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
The Global Crop Diversity Trust [http://www.croptrust.org] webcite
Kilian A, Huttner E, Wenzl P, Jaccoud D, Carling J, Caig V, Evers M, Heller-Uszynska , Cayla C, Patarapuwadol S, Xia L, Yang S, Thomson B: The fast and the cheap: SNP and DArT-based whole genome profiling for crop improvement. In In the Wake of the Double Helix: From the Green Revolution to the Gene Revolution. Edited by Tuberosa R, Phillips RL, Gale M. Bologna, Italy: Avenue media; 2005:443-461.
Akbari M, Wenzl P, Vanessa C, Carling J, Xia L, Yang S, Uszynski G, Mohler V, Lehmensiek A, Kuchel H, Hayden MJ, Howes N, Sharp P, Rathmell B, Vaughan P, Huttner E, Kilian A: Diversity Arrays Technology (DArT) for high-throughput profiling of the hexaploid wheat genome.
Theor Appl Genet 2006, 113:1409-1420. PubMed Abstract | Publisher Full Text
Lezar S, Myburg AA, Berger DK, Wingfield MJ, Wingfield BD: Development and assessment of microarray-based DNA fingerprinting in Eucalyptus grandis.
Theor Appl Genet 2004, 109:1329-1336. PubMed Abstract | Publisher Full Text
Xia L, Peng K, Yang S, Wenzl P, de Vicente C, Fregene M, Kilian A: DArT for high-throughput genotyping of cassava (Manihot esculenta) and its wild relatives.
Theor Appl Genet 2005, 110:1092-1098. PubMed Abstract | Publisher Full Text
Wittenberg AHJ, van der Lee T, Cayla C, Kilian A, Visser RGF, Schouten HJ: Validation of the high-throughput marker technology DArT using the model plant Arabidopsis thaliana.
Mol Genet Genomics 2005, 274:30-39. PubMed Abstract | Publisher Full Text
Yang S, Pang W, Ash G, Harper J, Carling J, Wenzl P, Huttner E, Kilian A: Low level of genetic diversity in cultivated pigeonpea compared to its wild relatives is revealed by Diversity Arrays Technology (DArT).
Theor Appl Genet 2006, 113:585-595. PubMed Abstract | Publisher Full Text
Official website of Diversity Arrays Technology P/L [http://www.DiversityArrays.com] webcite
Wenzl P, Li H, Carling J, Zhou M, Raman H, Paul E, Hearnden P, Maier C, Xia L, Caig V, Ovesná J, Cakir M, Poulsen D, Wang J, Raman R, Smith KP, Muehlbauer GJ, Chalmers KJ, Kleinhofs A, Huttner E, Kilian A: A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits.
BMC Genomics 2006, 7:206. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Kleinhofs A, Kilian A, Saghai Maroof M, Byashev RM, Hayes PM, Chen F, Lapitan N, Fenwick A, Balkes TK, Kanazin V, Ananiev E, Dahleen L, Kudrna D, Bollinger J, Knapp SJ, Liu B, Sorrels M, Heun M, Franckowiak JD, Hoffman D, Skadsen R, Steffenson BJ: A molecular isozyme and morphological map of barley (Hordeum vulgare) genome.
Theor Appl Genet 1993, 86:705-71. Publisher Full Text
Mackay IJ, Caligari PDS: Efficiencies of F2 and backcross generations for bulked segregant analysis using dominant markers.
Hazen SP, Borevitz JO, Harmon FG, Pruneda-Paz JL, Schultz TF, Yanovsky MJ, Liljegren SJ, Ecker JR, Kay SA: Rapid array mapping of circadian clock and developmental mutations in Arabidopsis.
Plant Physiol 2005, 138:990-997. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Minella E, Sorrells ME: Inheritance and chromosome location of Alp, a gene controlling aluminium tolerance in 'Dayton' barley.
Plant Breeding 1997, 116:465-469. Publisher Full Text
Tang Y, Sorrells ME, Kochian LV, Garvin DF: Identification of RFLP markers linked to the barley aluminium tolerance gene Alp.
Raman H, Moroni JS, Sato K, Read BJ, Scott BJ: Identification of AFLP and microsatellite markers linked with an aluminium tolerance gene in barley (Hordeum vulgare L).
Theor Appl Genet 2002, 105:458-464. PubMed Abstract | Publisher Full Text
Raman H, Karakousis A, Moroni JS, Raman R, Read B, Garvin DF, Kochian LV, Sorrells ME: Development of allele diversity of microsatellite markers linked to the aluminium tolerance gene Alp in barley.
Aust J Agric Res 2003, 54:1315-1321. Publisher Full Text
Ma JF, Nagao S, Sato K, Ito H, Furukawa J, Takeda K: Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley.
J Exp Bot 2004, 55:1335-1341. PubMed Abstract | Publisher Full Text
Wang J, Raman H, Read B, Zhou M, Mendham N, Venkatanagappa S: Validation of an Alt locus for aluminium tolerance scored with eriochrome cyanine R staining method in barley cultivar Honen (Hordeum vulgare L).
Aust J Agric Res 2006, 57:113-118. Publisher Full Text
Wang JP, Harsh R, Zhang GP, Mendham N, Zhou MX: Aluminium tolerance in barley (Hordeum vulgare L): physiological mechanisms, genetics and screening methods.
J Zhejiang Univ Sci B 2006, 7:769-787. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Doyle JJ, Doyle JL: A rapid DNA isolation procedure for small quantities of fresh leaf tissue.
Plant DNA extraction protocol for DArT [http://www.DiversityArrays.com/pub/DArT_DNA_isolation.pdf] webcite
GrainGenes 2.0 [http://wheat.pw.usda.gov/GG2/index.shtml] webcite
Buonaccorsi JP, Liebhold AM: Statistical methods for estimating ratios and products in ecological studies.
Van Os H, Stam P, Visser RGF, van Eck HJ: RECORD: a novel method for ordering loci on a genetic linkage map.
Theor Appl Genet 2005, 112:30-40. PubMed Abstract | Publisher Full Text
Lincoln SE, Lander ES: Systematic detection of errors in genetic linkage data.
Genomics 1992, 14:604-610. PubMed Abstract | Publisher Full Text

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It doesn't take a dozen exceptional guitarists to extol the virtues of Jimi Hendrix. But having that many certainly makes for a great show, as was the case Sunday at the Orpheum with the "Experience Hendrix" concert.
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Curtis Wright and the PATHETIC ABLE PACSubmitted by Peter C. (not verified) on Thu, 03/31/2011 - 8:10am.
> Looking at the 30 minute video of Brian's interview yesterday -- it is clear he has an IQ that is pushing 80 at best -- that's why he needs ABLE to tell him what to do. He rambles and sounds confused the whole time. Thank Curtis for plugging this guy so much on the Big Talker -- NOT. Shame on Ben McCoy, Curtis Wright, Laura Triece, Carolyn Bordeaux, Tom Heitman and the rest of that sickening ABLE bunch ragtags. For those who don't know, ABLE is a tea party PAC that tries (and fails) to take over the New Hanover County GOP every year. Most of them are un-employed and they claim the other county commissioners are "crooks." Hell, I'd vote for a commie before I ever cast a ballet for you guys. »
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A wool coating fabric in midnight black, and with a twill weave - get a head start on sewing a new winter coat now - perhaps an elegant wrapcoat, or a dashing wool pea coat. 100% wool. 58" wide.$39.99 $31.99 -
NY Designer Pine Green & Black Plaid Twill Wool Blend
A NY designer midweight reversible plaid wool twill in deep pine green and black plaid on one side, and black on the reverse. This sturdy fabric would make a great tailored jacket or toasty flat front slacks. 2 3/4" repeat. 53% cotton and 47% wool. 60" wide.$49.99 $39.99 -
Luxurious Striped Peach & Black Fur Mohair Coating LIMITED STOCK
A luxurious whisper soft mohair coating fabric with 1" long pile. Mohair is known for it's warmth and luster; this piece is eminently pet-able and lovely! We love the crosswise stripes in smoke, light persimmon and black - perfect for a spiffy winter jacket, or even as an accent collar and cuffs. 100% mohair. 64" wide.$125.99 $100.79 -
GUEST BLOGGER PICK! Etro Olive & Pumpkin Plaid Wool (Made in Italy)
A designer mid to heavyweight wool plaid from Etro in shades of olive and leaf green, with pumpkin, coffee, and teal blue accents - perfect for a toasty fall swing coat, or even a vintage look alpine vest....anyone for cocoa and fondue while overlooking the alps? Made in Italy by Etro. 100% wool. This fabric is 60" wide.
Check out our Guest Blogger tutorial on fringing this lovely wool!
$89.99 $71.99