CN105075850A - New camellia oleifera high-yield cultivation molecular design method - Google Patents
New camellia oleifera high-yield cultivation molecular design method Download PDFInfo
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Abstract
The invention relates to a new camellia oleifera high-yield cultivation molecular design method which mainly comprises the following three main steps: (1) determining a Neigenetic distance range of optimal oiltea camellia pollination combination by virtue of an SSR molecular recognition method; (2) detecting a natural cultivation population and wild population mating system; and (3) determining a high-outcross pollination allocation pattern. The Neigenetic distance range of optimal oiltea camellia pollination combination, which is obtained by virtue of the SSR molecular recognition method provided by the invention, is 0.431-0.480, and the fruit-setting rate and the yield rate are respectively not less than 80% and 59%; and by virtue of combination with the high-outcross pollination allocation pattern, the fruit-setting rate and yield rate of oiltea camellia are greatly improved, and the problem of low oiltea camellia yield is solved.
Description
Technical field
The present invention relates to the high-yield culturing Planting pattern by molecular recognition design oil tea, belong to biological technical field.
Background technology
Oil tea and olive, oil palm, coconut be called the large traditional oil tree in the world four, have the cultivation history of more than 2300 year in China.The tea oil that tea seed squeezes is the green health edible oil of high-quality, is of high nutritive value, and FAO (Food and Agriculture Organization of the United Nation) has been classified as the health type table oil that emphasis is promoted.Oil tea economic worth is high, ecological functions are strong, is one of all good Good Indigenous Tree Species of the distinctive economic benefit of China and ecological benefits, occupies very consequence in southern economy Foresty industry.To 2014 the end of the year China oil tea area reach 5,095 ten thousand mu, and existing working ability is 4 times of raw material supply amount, for ensureing national edible oil safety, " the national camellia oleiferaindustry development plan (2009-2020) " of the State Administration of Forestry, reaches 7018.1 ten thousand mu to the year two thousand twenty camellia oleifera lam base.But China's existing large area camellia oleifera lam per unit area yield is low, per mu yield tea oil 5.79 kilograms in 2008, lacks science reliable fast-growing andhigh-yeilding trees Planting Techniques system simultaneously, is difficult to reach planning requirement.
Chinese scholar to be beared fruit and the internal cause of output and external cause have carried out a series of research on affecting oil tea, as flowering habit (Zeng Yanru etc., Zhejiang Forestry College, 2009, 26 (6): 802-809), pollination feature of blooming (Wang Xiangnan etc., Sino-South African Forestry University of Science and Technology's journal, 2013, 33 (12): 1-5), florescence weather is on impact (Zeng Yanru and Li Zhangju that bear fruit after spending, Zhejiang Forestry College, 2010, 27 (3): 323-328), pollination Process of Insemination (Liao Ting etc., forest-science, 2014, 50 (2): 51-55), utilize growth regulator flower and fruit protecting, plot is selected, breeding strong sprout, rationally plant, tending management, prune and fertilising (Chen Yanjing, agricultural and technology, 2014, (1): 81-83), the extermination of disease and insect pest and stand structure optimization (Li Yanming etc., Agriculture of Anhui science, 2013, 41 (33): 12898-12899) and (Deng Yinwei etc. such as grafting and cutting strunk renewal, China's Forest by-product and speciality, 2013, (6): 19-22).Result shows, from flower with homophyletic cross pollination, the age of tree are too small or excessively old, the quality of kind, damage by disease and insect and mechanical damage etc. are all the main causes causing oil tea fruit drop.
In addition, oil tea self-pollination setting ratio lower than geitonogamy setting ratio, geitonogamy setting ratio again lower than xenogamy setting ratio.Oil tea hat is large, Hua Duo, pollinator be everlasting homophyletic do not suit between access, easily cause homophyletic cross pollination and self-pollination, therefore normal self-incompatible phenomenon occur, cause shedding.But up to the present, do not form the technical standard of building oil tea outcrossing woods, thus cannot fundamentally improve tea-oil tree yield, realize the high-yield and high-efficiency of oil tea.
Prior art mainly adopts bud stock grafting seedling to build new camellia oleifera lam, if the adjacent plant of new camellia oleifera lam of according to said method building is the same gene type from affine (SI), selfing abortion more easily occurs, thus forms low production forest.Also fundamentally do not solve the method for the problem how avoiding camellia oleifera lam selfing abortion at present.Therefore, the affiliation between distinct oil tea kind matter and joining property, set up outcrossing fast-growing andhigh-yeilding trees construction pattern significant to raising tea-oil tree yield.
Summary of the invention
For the problems referred to above, the present invention with choiceness oil tea for material, different pollination combinations is designed according to the NeiShi genetic distance between matter not of the same race, and optimize the combination of the suitableeest kind of matter according to the Fruit-setting rate of difference pollination combination and setting ratio, again with high outcrossing pollination Planting pattern design High Yield Cultivation Pattern, realize tea-oil tree yield and improve.Technical scheme of the present invention is as follows: a kind of molecular design method of new camellia oleifera lam high-yield culturing, is made up of the step of following order:
(1) select different oil tea kind matter, extract its DNA respectively;
(2) screening is applicable to the SSR label primer of oil tea;
(3) utilize the SSR label primer filtered out, calculate the NeiShi genetic distance between different oil tea kind matter;
(4) NeiShi genetic distance is divided into 11 ranks, in each rank, designs different pollination combinations, carry out artificial pollination respectively;
(5) within the 30th day after artificial pollination, add up and calculate Fruit-setting rate, the 180th day statistics also calculating achievement rate; Obtain the oil tea NeiShi genetic distance scope of coordinate force the best;
(6) oil tea kind matter within the scope of NeiShi genetic distance is obtained for seedling with step (5), design camellia oleifera lam High Yield Cultivation Pattern.
Further, described step (1) extracts the method for DNA is modified CTAB method.
Further, the method for described step (2) screening SSR label primer comprises the steps:
1) the SSR marker sequence of RNA-Seq technological development oil tea is utilized;
2) PrimerPremier5.0 Software for Design special primer is adopted: parameter is: primer length (20 ~ 24nt), 3 ' end stability (-6.0 ~-9.0kal/mol), primer Tm (55 ~ 60 DEG C), GC content (45 ~ 55%), primer rating value > 90.With the DNA of different oil tea kind matter for template, carry out pcr amplification, according to polyacrylamide vertical gel electrophoresis result, filter out SSR label primer.
Further, the described camellia oleifera lam High Yield Cultivation Pattern of step (6), the seeding row spacing of oil tea is 2m × 3m, and the NeiShi genetic distance of planting between matter combination is 0.431-0.480.
Further, the described camellia oleifera lam High Yield Cultivation Pattern of step (6), the seeding row spacing of oil tea is 3m × 2m, and the NeiShi genetic distance of planting between matter combination is 0.431-0.480.
The present invention matches out the suitableeest pollination combination according to the NeiShi genetic distance between the different oil tea kind matter utilizing SSR marker to detect: NeiShi genetic distance is that the pollination of 0.431-0.480 is combined as the best, Fruit-setting rate and the setting ratio of outcrossing pollination reach more than 80% and 59% respectively, with the molecule Silvicultural pattern of the new camellia oleifera lam high-yield culturing of height different pollination Planting pattern successful design, for China's large area builds fresh oil tea economic woods, development camellia oleiferaindustry provides the technical foundation of high-yield culturing.
Beneficial effect of the present invention is as follows:
(1) Molecular identification methods of the present invention is simple to operate, easily promotes to expand to use;
(2) Molecular identification methods provided by the invention is used, the pollination filtering out NeiShi genetic distance scope 0.431-0.480 is combined as the best, Fruit-setting rate and the setting ratio of outcrossing pollination reach more than 80% and 59% respectively, greatly improve Fruit-setting rate and the setting ratio of oil tea;
(3) the invention provides the molecule Silvicultural pattern of new camellia oleifera lam high-yield culturing, for China's large area builds fresh oil tea economic woods, development camellia oleiferaindustry provides the technical foundation of high-yield culturing.
Embodiment
Below in conjunction with embodiment, the present invention is described further, but this does not limit the scope of the invention, embodiment with 49 oil tea improved seeds (clone) in jade screen oil tea kind matter base for material, be respectively: 3-18, 5-35, 5-35-1, 5-35-2, 5-36-1, 5-36-2, 5-36-2, 5-37, 5-37-2, 5-37-3, 5-38, 5-39, 5-39-2, 5-40, 5-41, 5-42, 5-42-2, 5-42-3, 5-44, 5-44-2-1, 5-44-3, 5-45-1, 5-45-2, 5-47, 6-48, 6-48-2, 6-49, 6-53, 6-53-2, 6-53-3, 6-54, 6-54-1, 6-55, 6-57, 6-58, 6-62, 6-62-2, 6-62-3, 6-63, Y-41, Y-44, ZB1, soft No. 11 of Cen, No. 1, only seed, Hunan 210, Hunan woods No. 5, Hunan woods 5, jade screen 1, long woods 27, the concentration of NaAc solution is 3mol/L, PH is 5.2.
Embodiment 1
One, the NeiShi genetic distance scope of best pollination of oil tea tree combination is selected
(1) with 49 Camellia Oleifera Clones kinds for material, utilize improvement CTAB method to extract its DNA respectively, step is as follows:
1) 10 grams, oil tea blade is put into mortar, after pouring appropriate liquid nitrogen grinding into, move into and be added with in advance in 700 μ L2 × CTAB centrifuge tubes, be placed in 65 DEG C of water bath processing 45 ~ 60min, centrifugal (4 DEG C, 1000rpm, 10min) obtain supernatant I;
2) get step 1) in centrifuge tube supernatant I600 μ L in new centrifuge tube, add phenol, chloroform and isoamyl alcohol mixed liquor (volume ratio is 25:24:1) that cumulative volume is 600 μ L, shake up rear centrifugal (4 DEG C, 1000rpm, 10min) and obtain supernatant II;
3) supernatant II 550 μ L is got in centrifuge tube, add 500 μ L10 × CTAB, shake up to be placed in 65 DEG C of water-baths and dissolve 2 ~ 3min, add the phenol that cumulative volume is 50 μ L again: chloroform: isoamyl alcohol mixed liquor (volume ratio is 25:24:1), shake up latter centrifugal (4 DEG C, 1000rpm, 10min), obtain supernatant III;
4) get the absolute ethyl alcohol that supernatant III adds its volume 2 times, then add the NaAc solution of its volume 1/10, leave standstill more than 2 hours, obtain precipitation;
5) by step 4) gained washing of precipitate post-drying, bake out temperature is 37 DEG C, and the time is 8 ~ 10min;
6) by step 5) dry after precipitation dissolve after normal temperature leave standstill 2 hours, obtain oil tea DNA sample;
(2) with oil tea blade for material, extract its RNA with any one method known, and adopt RNA-Seq technology to carry out RNA order-checking, then according to sequence search simple repeated sequence, 8564 SSR marker sequences detected altogether;
(3) PrimerPremier5.0 Software for Design special primer is adopted: parameter is: primer length (20 ~ 24nt), 3 ' end stability (-6.0 ~-9.0kal/mol), primer Tm (55 ~ 60 DEG C), GC content (45 ~ 55%), primer rating value > 90.With the DNA of 2 different oil tea kind matter for template, carry out pcr amplification, according to polyacrylamide vertical gel electrophoresis result, choose and can amplify band, locus band is clear, and have the primer pair of polymorphism, filter out 38 pairs of SSR label primer altogether, in table 1;
(4) the 38 pairs of SSR label primer utilizing step (3) to filter out are extracted to step (1) the oil tea DNA obtained respectively and are carried out pcr amplification, and amplified production utilizes 8% polyacrylamide gel vertical electrophoresis to detect;
(5) counting with the presence or absence of electrophoresis detection result band, be designated as " 1 ", without being designated as " 0 ", utilizing NTYsys2.0 software to calculate NeiShi genetic distance;
(6) NeiShi genetic distance is divided into 11 level ranges, is respectively 0.080-0.130,0.131-0.181,0.182-0.230,0.231-0.280,0.281-0.330,0.331-0.380,0.381-0.430,0.431-0.480,0.481-0.530,0.531-0.580,0.581-0.630; In each NeiShi genetic distance rank, design 8 pollination combinations, as following table 2; Each pollination combination adopts artificial outcrossing pollination process, each combined treatment 300 flowers;
(7) (number × 100% is spent in Fruit-setting rate=solid number/pollination process to the 30th day statistics Fruit-setting rate after artificial pollination process; 180th day statistics setting ratio (setting ratio=achievement number/solid number × 100%), the results are shown in Table 2;
38 pairs of SSR label primer that table 1 oil tea Nei genetic distance detects
The Fruit-setting rate of table 2 difference pollination combination and setting ratio
Known by table 2, between oil tea kind matter, NeiShi genetic distance is that the pollination of 0.431-0.480 is combined as the best, and Fruit-setting rate and the setting ratio of outcrossing pollination reach more than 80% and 59% respectively, greatly improve Fruit-setting rate and the setting ratio of oil tea.
Two, the mating system of oil tea nature cultivated population and wild population is detected
Naturally cultivate in jade screen oil tea and in wild population, respectively choose 5 sample prescriptions, quadrat size is all more than 2 mu, and the fructescence, random selecting 40 individual plants in each sample prescription, each individual plant distance, at more than 10m, collects individual plant fruit.Each sample prescription Stochastic choice 30 individual plants, mix its seed, and Stochastic choice 25 seeds are wherein used for DNA and extract.
Utilize the shown 38 pairs of SSR label primer of table 1, pcr amplification is carried out to 250 DNA sample in 10 sample prescriptions; Amplified production utilizes 8% Polyscrylamide gel vertical electrophoresis to detect; With band with or without counting, be designated as " 1 ", without being designated as " 0 ".
Oil tea is cultivated and the unit point outcrossing rate (t of wild population naturally to utilize MLTR3.2 software to estimate
s), various some outcrossing rate (t
m) and parents' inbreeding coefficient (t
m-t
s), parental inbreeding coefficient (F) and the multidigit point degree of correlation (r
pm), in table 3.
Table 3 oil tea is cultivated and wild population mating system naturally
| Population | t m | t s | t m-t s | r pm | F |
| Natural Populations 1 | 0.923 | 0.917 | 0.006 | 0.009 | 0.009 |
| Natural Populations 2 | 0.935 | 0.926 | 0.009 | 0.124 | 0.012 |
| Natural Populations 3 | 0.947 | 0.938 | 0.009 | 0.131 | 0.008 |
| Natural Populations 4 | 0.919 | 0.906 | 0.013 | 0.098 | 0.017 |
| Natural Populations 5 | 0.927 | 0.923 | 0.004 | 0.095 | 0.016 |
| Wild population 1 | 0.939 | 0.927 | 0.012 | 0.123 | 0.011 |
| Wild population 2 | 0.946 | 0.938 | 0.008 | 0.101 | 0.016 |
| Wild population 3 | 0.927 | 0.921 | 0.006 | 0.092 | 0.014 |
| Wild population 4 | 0.952 | 0.947 | 0.005 | 0.117 | 0.013 |
| Wild population 5 | 0.930 | 0.923 | 0.007 | 0.095 | 0.018 |
Known by table 3, oil tea naturally cultivate and wild population outcrossing rate all higher, respectively between 0.919 to 0.947 and 0.927 to 0.952, show that oil tea belongs to obligate outcrossing species.The multidigit point outcrossing rate of 10 oil tea population is all higher than unit point outcrossing rate, and site parent's degree of correlation is smaller, illustrates that in population, inbreeding is less.Meanwhile, each colony unit point degree of correlation and multidigit point degree of correlation difference less, show to there is not substructure in colony.
Three, high outcrossing pollination Planting pattern is determined
In October, 2014, in Dong Autonomous County of Yu ping oil tea base, at oil tea full-bloom stage, chooses the oil tea sample prescription of different plant and row spacing, observes the foraging insects of oil tea, pollinator's behavior and mechanics thereof.Observe bloom, the visiting frequencies of loose powder and pollinator time observation wind-force, record weather condition.The results are shown in Table 4.
Known by table 4: the seeding row spacing of high outcrossing pollination incidence is 2m × 3m or 3m × 2m, seeding row spacing is that between the different individual plant of same pollinator of 2m × 3m is spent, visiting frequencies is 0.028 time/min, and the same pollinator access frequency of the different perianth of homophyletic is 0.013 time/min; Seeding row spacing is that between the different individual plant of same pollinator of 3m × 2m is spent, visiting frequencies is 0.027 time/min, and the same pollinator access frequency of the different perianth of homophyletic is 0.008 time/min.
Table 4 different plant and row spacing oil tea pollinator mechanics
The seeding row spacing of incidence of pollinating according to pollination combination and high outcrossing, the high-yield tea-oil cultivation Silvicultural pattern of design " choiceness+high-combining ability+high outcrossing pollination Planting pattern ".In this mode, select NeiShi genetic distance to be the oil tea of 0.431-0.480, Fruit-setting rate and the setting ratio of outcrossing pollination reach more than 80% and 59% respectively, and design seeding row spacing is 2m × 3m or 3m × 2m, effectively can increase outcrossing pollination, realize tea-oil tree yield and improve.
Claims (4)
1. a molecular design method for new camellia oleifera lam high-yield culturing, is characterized in that, is made up of the step of following order:
(1) select different oil tea kind matter, extract its DNA respectively;
(2) screening is applicable to the SSR label primer of oil tea;
(3) utilize the SSR label primer filtered out, calculate the NeiShi genetic distance between different oil tea kind matter;
(4) NeiShi genetic distance is divided into 11 ranks, in each rank, designs different pollination combinations, carry out artificial pollination respectively;
(5) within the 30th day after artificial pollination, add up and calculate Fruit-setting rate, the 180th day statistics also calculating achievement rate; Obtain the oil tea NeiShi genetic distance scope of coordinate force the best;
(6) oil tea kind matter within the scope of NeiShi genetic distance is obtained for seedling with step (5), design camellia oleifera lam High Yield Cultivation Pattern.
2. the molecular design method of a kind of new camellia oleifera lam high-yield culturing as claimed in claim 1, is characterized in that, the method that described step (1) extracts DNA is modified CTAB method.
3. the molecular design method of a kind of new camellia oleifera lam high-yield culturing as claimed in claim 1, it is characterized in that, the described camellia oleifera lam High Yield Cultivation Pattern of step (6), the seeding row spacing of oil tea is 2m × 3m, and the NeiShi genetic distance scope of planting between matter combination is 0.431-0.480.
4. the molecular design method of a kind of new camellia oleifera lam high-yield culturing as claimed in claim 1, it is characterized in that, the described camellia oleifera lam High Yield Cultivation Pattern of step (6), the seeding row spacing of oil tea is 3m × 2m, and the NeiShi genetic distance scope of planting between matter combination is 0.431-0.480.
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