CN110896928B

CN110896928B - Chinese bee queen bee breeding method

Info

Publication number: CN110896928B
Application number: CN201911248607.0A
Authority: CN
Inventors: 杨明显; 吴贵辉; 李为常; 何伟东; 潘柳初; 黄伦; 王遂林
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2019-12-09
Filing date: 2019-12-09
Publication date: 2022-02-18
Anticipated expiration: 2039-12-09
Also published as: CN110896928A

Abstract

The invention discloses a method for breeding Chinese bee queens, which comprises the following steps: adopting a apis mellifera larva swarm as a feeding swarm to feed the apis cerana queen royal jelly, wherein the apis cerana queen is a natural mating apis cerana queen; the weight and the daily egg laying amount of the Chinese bee queens are remarkably improved, the egg weight, the egg length and the birth weight of the offspring worker bees are increased in different degrees, the morphology of the offspring worker bees is changed in different degrees, and the reproductive performance of the Chinese bee queens is improved.

Description

Chinese bee queen bee breeding method

Technical Field

The invention relates to the technical field of breeding, in particular to a breeding method of a Chinese bee queen bee.

Background

Chinese bee (Chinese bee for short) has the characteristics of cold resistance, heat resistance, low feed consumption, utilization of sporadic honey powder source and the like, and is particularly suitable for feeding in vast mountainous areas and hilly areas in China. The artificial breeding of the queen bee is one of key technologies for breeding Chinese bees and developing fine breed breeding of the Chinese bees. At present, the method for artificially culturing the queen bee of the Chinese bee by the bee-keeping worker is to feed the larva of the Chinese bee in the artificial queen bee platform by the worker bee of the Chinese bee to obtain the queen of the Chinese bee. However, the artificial queen bee breeding method has the defects that the breeding speed of the Chinese bee colony is slow, the colony vigor of the Chinese bee colony is weak, and the yield of the Chinese bee colony cannot meet the requirements of Chinese bee production and Chinese bee breeding due to the fact that the Chinese bees have strong wildness, the working bee plasma secretion capacity of the Chinese bee is poor, and the egg laying amount of the Chinese bee queen is low.

Disclosure of Invention

In view of the above, the present application provides a method for breeding a queen bee of a Chinese bee, wherein the queen bee weight and the daily egg laying amount of the Chinese bee bred by a feeding group consisting of apis mellifera broods are significantly increased, the egg weight, the egg length and the birth weight of the offspring worker bees are increased to different extents, and the morphology of the offspring worker bees is also changed to different extents. The reproductive performance of the Chinese bee queen is improved.

In order to solve the technical problem, the technical scheme provided by the application is a method for breeding a Chinese bee queen, which comprises the following steps: the apis mellifera larva bee colony is used as a feeding colony for feeding apis cerana queen royal jelly, and the apis cerana queen is a natural bee queen.

Preferably, the cultivation method further comprises: the preparation process of the Chinese bee queen comprises the following steps:

culturing a queen cell: selecting Chinese bee colonies as parent colonies and queen bee colonies, and culturing mature queen bee stands;

mating: and putting the mature queen bee platforms into a Chinese bee mating group, finishing mating until the Chinese bee queen bees emerge from the hive after the feathering, and laying eggs to obtain the standby Chinese bee queen bees.

Preferably, the amount of the cross-bred swarm is 1 splenic bee.

Preferably, the time of the ending process is as follows: 8-12 d.

Preferably, the cultivation method further comprises: a preparation process of the nursing group, the preparation process of the nursing group comprising: selecting Italian bee colony, rewarding for feeding, limiting oviposition space, after emerging from the room, putting into an artificial climate box for centralized emergence, and forming feeding colony by the Italian bee brood emerging from the room.

Preferably, the amount of nursing swarms is not less than 3 splenic bees.

Preferably, the oviposition limiting space is specifically to enable the apis mellifera colony to lay eggs on 1-2 empty spleens.

Preferably, the method specifically comprises: the novel beehive is internally provided with a apis cerana empty honeycomb, an apis cerana honeycomb with honey and pollen storage and a feeding group consisting of apis cerana larva bees, wherein the feeding group is shaken into the novel beehive, the apis cerana queen is transferred into the novel beehive, the apis cerana larva bee group is used as the feeding group to feed the apis cerana queen royal jelly, and a novel apis cerana empty honeycomb is used for replacing the apis cerana queen bee after the apis cerana queen bee on the apis cerana empty honeycomb is full of eggs.

Preferably, the feeding group is supplemented with 1 splenic apis mellifera baby bee every 7 days.

Preferably, the spleen relationship of the nursing swarm is to keep bees more than spleens.

Compared with the prior art, the detailed description of the application is as follows:

in beekeeping production, the colony vigor of the bee colony must be increased in order to increase the yield of honey. At present, the main methods for increasing the egg laying amount of queen bees in production comprise the following points: (1) when the queen bees are replaced, the new queen bees generally have more abundant egg laying amount and lay more eggs;

rewarding feeding, namely feeding bee colonies more actively by rewarding and feeding pollen and sugar water to the bee colonies, and also promoting the queen bee to lay eggs to a certain extent; (3) and (3) nutrient hybridization, wherein in the process of culturing the queen bees, larvae for culturing the apis cerana queen are fed in the apis cerana colony for 2-3 days, and then are put back into the apis cerana colony for continuous culture to obtain the apis cerana queen, and the apis cerana queen which grows after eating the apis cerana royal jelly in the larva stage is large in size and strong in oviposition. The current method for increasing the egg laying amount of queen bees in production mainly has the following defects: (1) for the replacement of the queen bees, because the queen bee cultivation needs a certain period and needs to be carried out in a specific season, and the risk that the queen bee is not successful in mating or mistakenly enters the queen bee colony to be killed exists, common beekeepers do not use the method except for some beekeepers who have rich experience and higher breeding level and technology; (2) for reward feeding, the reward feeding can improve the feeding enthusiasm of bee colonies and the egg laying amount of queen bees to a certain extent, but because the queen bees do not directly eat artificially fed food, worker bees are required to convert, the effect is limited;

according to the invention, the preparation process of selecting the Chinese bee queen is to place the mature queen platform into the Chinese bee mating group, the quantity of the mating group is 1 splenic bee, when the Chinese bee queen feather emerges from the hive, mating is completed until spawning is finished, and the standby Chinese bee queen is obtained. The reproductive structure of the newly emerged virgin queen bee is not developed completely, the sexual maturity can be reached only after the development, and the virgin queen bee begins to emerge from a nest for marriage and flight copulation. After natural mating, the ovary of the queen bee can rapidly develop, the volume and the weight can be greatly increased, and then the queen bee has the capability of spawning. The invention adopts the Chinese bee queen which is naturally mated to induce the feeding group consisting of the Italian bee brood, and can improve the egg-laying capacity.

The invention adopts the Chinese bee queen which is naturally mated to induce the feeding group consisting of the apis mellifera larvae, and realizes the normal production of the Chinese bee queen. The queen weight and the daily egg laying amount of the Chinese bees fed by the feeding group consisting of the apis mellifera larvae are obviously improved, the egg weight, the egg length and the birth weight of the offspring worker bees are increased in different degrees, and the morphology of the offspring worker bees is changed in different degrees.

The RNA-seq technology is utilized to carry out transcriptome sequencing on the ovary tissue of the queen bee to obtain transcriptome data of the queen bee ovary, and through analyzing the sequencing result, the reproductive performance of a feeding group formed by inducing the queen bee of the Chinese bee into apis mellifera brood by means of cross stimulation can be obviously improved.

Drawings

FIG. 1 is a diagram of worker bee morphological dissection specimen;

FIG. 2 is a graph comparing the body types of queen bees fed by Italian bees and Chinese bees;

FIG. 3 is a line graph of weight change and daily egg production for EF1, WF1, EF2, WF2, EF3, WF3 queen bees;

FIG. 4 is a line graph of weight change and daily egg laying amount of EF3, WF3, EF4 and WF4 queen bees, and a line graph of average weight change and average daily egg laying amount of queen bees in the test group and the control group;

fig. 5 is a comparison of egg shapes of apis mellifera feeding and apis cerana feeding;

FIG. 6 is a flow chart of bioinformatics analysis;

FIG. 7 is a diagram of sequencing randomness analysis of mRNA;

FIG. 8 is a graph showing the results of sequencing insert length tests;

FIG. 9 is a transcriptome data saturation simulation;

FIG. 10 is a SNP mutation type and density profile;

FIG. 11 is a SNP and InDel annotation classification chart;

FIG. 12 is a graph of the classification and quantity of alternative splicing;

FIG. 13 is a density profile of FPKM;

FIG. 14 is a box plot of FPKM distribution;

FIG. 15 is a difference gene volcano plot and cluster plot;

FIG. 16 is a differential expression gene GO classification map;

FIG. 17 is an enrichment analysis chart of the differentially expressed gene Pathway of EF1_ EF2_ EF3vsWF1_ WF2_ WF 3;

FIG. 18 is a fatty acid metabolic pathway map.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

Test materials:

the bee colony used in the test is Chinese bee and Italian bee raised in living frames in the teaching and scientific research park of Sichuan agricultural university (Yaan school district), and the test is completed in the bee-keeping base of Sichuan agricultural university in 2018 in 3-10 months.

The instrument comprises the following steps:

the morphological measurement adopts Microvision (VS-250DH) and a digital microscope (GL-0745 TI) to photograph the sample, and the sample is measured by using the matched software of the micro biomorphic measurement and data analysis system.

Example 1

A method for breeding Chinese bee queens comprises the following steps:

(1) preparing a Chinese bee queen:

mating: the mature queen cells are placed into a Chinese bee mating group, the quantity of the mating bee group is 1 splenic bee, when the Chinese bee queen feathers out of the house, mating is completed until spawning occurs, and a standby Chinese bee queen is obtained; the time of the whole mating process of the mature queen bee placed in the Chinese bee mating group to obtain the standby Chinese bee queen bee is as follows: 8-12 d.

(2) Preparation of a nursing group:

selecting a apis mellifera colony, rewarding for feeding, limiting a spawning space, enabling the apis mellifera colony to spawn on 1-2 empty spleens, putting the apis mellifera colony into an artificial climate box (with the temperature of 35.5 ℃, the relative humidity of 85 percent and no illumination) for centralized eclosion before the spleens are emerged out of the room, and enabling apis mellifera larvae emerging out of the room to form a feeding colony; the amount of the nursing swarms is not less than 3 splenic bees;

(3) preparation of spawning group:

placing a apis cerana empty honeycomb and an apis cerana honeycomb with honey and pollen in a new beehive, shaking a feeding group consisting of apis cerana brood in the step (2) into the new beehive, transferring the apis cerana queen for standby in the step (1) into the new beehive, feeding royal jelly of the apis cerana queen by using the apis cerana brood group in the step (2) as the feeding group, and replacing the apis cerana queen with a new apis cerana empty honeycomb after eggs are fully produced on the apis cerana empty honeycomb; the feeding group supplements 1 splenic apis mellifera baby bee every 7 days; the feeding group is in a dense group, and the relationship between the bees and the spleen is that more bees are kept than the spleen.

Effect example 1

Test groups: on the basis of example 1, 5 groups of apis mellifera larvae (apis mellifera queen-free group) with the same number of spleens and basically consistent bee quantity were constructed as feeding groups, which were respectively numbered as WF1, WF2, WF3, WF4 and WF5(Western honeybee workers fed), 5 healthy normal-laying apis mellifera queens obtained in the preparation process of apis mellifera in example 1 were selected and artificially introduced into the feeding groups (apis mellifera queen-free group) according to the preparation process of the oviposition group in example 1.

Control group:

the control group and the test group differ only in that: 5 groups of Chinese bee broods (Chinese bee queen-free groups) with the same number of spleens and basically consistent bee quantity are established as feeding groups, which are respectively numbered as EF1, EF2, EF3, EF4 and EF5(Eastern honeybee workers fed), 5 healthy and normal-spawning Chinese bee queens obtained in the preparation process of the Chinese bee queens in the embodiment 1 are selected and are respectively artificially induced into the feeding groups (Chinese bee queen-free groups) according to the preparation process of the spawning groups in the embodiment 1. The test group and the control group ensure that the honeycombs are the same, the bee quantity is sufficient, the feeding management modes of all bee colonies are consistent during the test period, feeding is rewarded in the whole process, and the worker bees freely enter and exit the honeycomb for collection.

In order to avoid the influence of individual genetic material difference, the test records the physiological change data of the weight change, the egg laying amount, the egg weight and the like of the queen bee of the same apis cerana in two different environments of the apis cerana worker bee colony and the apis cerana worker bee colony respectively (namely WF1 and EF1, WF2 and EF2, WF3 and EF3, WF4 and EF4, WF5 and EF5 are the same queen bee), so that the offspring queen bees and the worker bees cultured by the test group and the control group corresponding to the same serial number are the same female parent, and the genetic materials of the bees of the test group and the control group are basically consistent. In addition, the different development time after the queen bee mating, its physiological change also has very big difference, and the queen bee that chooses for use in this effect example is the queen bee that spawns comparatively stable after mating.

1. Method for measuring daily egg laying amount, egg weight and egg shape of queen bee

After the queen bees on the empty honeycombs of the Chinese bees lay eggs fully, a new empty honeycomb (honey powder and enough egg laying space) of the Chinese bees is used for replacing the queen bees, and the replacement frequency of the empty honeycombs of the Chinese bees is 24 h.

And (3) completely and softly taking out the bee eggs at the bottoms of 15 cells on a glass slide by using a larva transfer needle, photographing the sample by using an integrated digital microscope and the software of a micro biomorphic measurement and data analysis system, and measuring the length and the width of the bee eggs. As the weight of one bee egg is about 0.2mg, in order to reduce the weighing error, 10 bee eggs are taken in a centrifugal tube in each test, the bee eggs are weighed by using an electronic balance, and the weight of 10 bee eggs is obtained by subtracting the weight of an empty centrifugal tube from the total weight. The test records the egg laying amount of queen bee in 7 days and the egg weight and egg shape data.

2. Method for measuring weight of queen bees and worker bees

The primary body weights of the queen bees and the worker bees of each group were measured 10 individuals. The measuring method of the initial weight of the queen bee comprises the steps of weighing the weight of the queen bee cage by using an electronic balance, then placing the queen bee in the queen bee cage, weighing the weight, and subtracting the weight of the empty queen bee cage from the weight of the queen bee cage to obtain the weight of the queen bee.

Virgin queen of virgin weight should be weighed when the queen of bee just emerged from the room. The weighing method of the primary weight of the worker bees and the weight of the spawning queen bees is the same as the weighing method of the primary weight of the queen bees. When weighing the spawning queen bees, the actions should be gentle, so that the queen bees are prevented from being stressed too much, and the queen bees should be put back to the bee colony immediately after weighing.

3. Method for cultivating and measuring morphological measurement sample

3.1 morphological index cultivation method

After the control groups EF 1-EF 5 queen bees lay eggs and hatch, artificially transferring insects and breeding queen bees in each group respectively, and measuring the weight of each queen bee after the queen bee exits from the chamber; and the rest larvae are put back into the original bee colony for continuous cultivation, and the weight of the newly born bee is measured after the newly developed worker bee exits from the bee nest. The morphometric sample is killed by boiling water to make its rhynchopsis part extend out, and preserved in 75% alcohol to make morphometric specimen.

Because the pheromone of the Chinese bee larvae is different from the pheromone of the Italian bee larvae, the eggs of the Chinese bee are cleaned by worker bees of the Italian bee after hatching, the eggs of test groups WF 1-WF 5 queen bees are taken out and put into worker bee colonies of the Chinese bee for breeding, and the test method is the same as that of a control group.

3.2 morphological index measurement method

In this effect example, all bee colonies were subjected to the same breeding management method, and 19 morphological indexes having a closer correlation with productivity and environmental adaptability (such as a kiss length (a), a right front wing length (FL), a right front wing width (FB)) were selected for measurement with reference to 38 bee morphological measurement indexes summarized by Ruttner (1988), and 19 indexes such as a wing vein angle and a chromaticity index were not included in the measurement.

The determination method comprises the following steps: taking out the bee samples for the morphometry cultivated in the step 3 and the cultivation of the morphometric samples from the alcohol, and respectively selecting 15 worker bee samples with complete limbs and completely extended lips for each group to dissect. Coating vaseline on the glass slide, respectively taking down the rhynchophorus mollis, the right wing, the right hindfoot, the third back plate, the fourth back plate, the third web plate and the sixth web plate of the worker bee, cleaning the muscle and soft tissue of the worker bee, and paving the cleaned muscle and soft tissue on the glass slide to prepare a morphology determination sample, wherein the morphology determination sample is shown in figure 1; and measuring related morphological indexes by using a digital microscope and micro-biological morphological detection and data analysis software according to the bee morphological index measuring standard summarized by Ruttner. The morphological measurement has great influence with human factors, and all the morphological measurement work of the test is completed by the same person, so that the accuracy of data is ensured.

5. Data statistics and analysis

The statistical analysis work of the test data is completed by an SPSS (19.0 version) software tool, the data of the test group and the data of the comparison group are completely randomly compared, and the LSD method is used for carrying out difference significance analysis on various queen production data and the shape data of the later worker bees recorded in the test.

6 results and analysis

6.1 relationship between queen bee weight and daily egg laying amount

From the comparison of figure 2 between the body types of apis mellifera feeding and apis cerana feeding queen, the body type of apis mellifera queen feeding as feeding group is significantly larger than that of apis cerana queen feeding as feeding group.

As can be seen from figures 3 and 4, the weight of the apis cerana queen fluctuates in the apis cerana colony, and the weight of the apis cerana queen obviously increases after entering the apis cerana worker colony.

In five groups of experiments, the daily egg laying amount of the queen bees of the same queen bee is remarkably improved by about 56.92 percent in different food environments of the test group and the control group, wherein the difference of WF2 is remarkable (P is less than 0.05) compared with EF2 and WF4 compared with EF 4; compared with EF1 and WF3, the difference of WF1 in comparison with EF3 and WF5 in comparison with EF5 reaches an extremely significant level (P is less than 0.01), and the daily egg laying amount of WF3 in comparison with EF3 in queen bees is improved by 105.91%. As can be seen from the graph of the weight change of the queen bee and the daily egg laying amount change, the weight change of the queen bee and the daily egg laying amount are in positive correlation.

6.2 egg weight and egg shape variation

Due to the difference of genetic materials among different queen bee individuals, the difference of the weight of laid eggs, the length of laid eggs and the width of laid eggs is extremely large, so that the effect example only analyzes and compares the length and the width of eggs of the same queen bee egg, and the comparison of egg shape of queen bee fed by apis mellifera and egg shape of queen bee fed by apis cerana is shown in figure 5.

As can be seen from the test results (table 1), queen bee eggs in the test group were all heavier than the control group, but did not reach the significant level of the statistical analysis. From the oval changes, the egg length of WF1, WF4 queen bees was significantly greater than EF1, EF4, respectively (P <0.05), while the egg width difference was not significant; the difference of the egg length of WF2 compared with EF2 and WF5 compared with EF5 queen bees is not significant, while the egg width of WF2 queen bees is greatly larger than that of EF2 queen bees (P is less than 0.01), and the egg width of WF5 queen bees is greatly larger than that of EF5 queen bees (P is less than 0.05); the egg length and the egg width of WF3 queen bees are both obviously larger than those of EF3 queen bees, and the egg length difference reaches an extremely obvious level (P is less than 0.01).

From the data, it can be seen that by the queen bee fed by the apis mellifera, both queen egg length and egg width ratio are increased in the eggs laid in the apis cerana; the difference analysis proves that the apis cerana queen lays eggs after being fed by the apis mellifera larva swarm as a feeding swarm, the length or width of the eggs can be increased to different degrees (the difference reaches a very significant level), and the shape and the size of the eggs are influenced.

TABLE 1 Queen egg shape and egg weight differential analysis

Colony numbering	Egg length mm	Egg width mm	10 eggs weight mg
				EF1	1.7807±0.0415*	0.4301±0.0133	1.8657±0.2366
WF1	1.8183±0.0655*	0.4323±0.0144	1.9514±0.1753
				EF2	1.7986±0.0608	0.4374±0.0127**	1.9414±0.2310
WF2	1.8121±0.0203	0.4533±0.0115**	2.0029±0.1467
				EF3	1.7702±0.0611**	0.4439±0.0132*	1.9086±0.2107
WF3	1.8437±0.0411**	0.4518±0.0076*	1.9886±0.1281
				EF4	1.7802±0.0425*	0.4413±0.0124	1.9014±0.1099
WF4	1.8187±0.0659*	0.435±0.0132	1.9243±0.1451
				EF5	1.8163±0.0491	0.4174±0.0105*	1.7729±0.0795
WF5	1.8404±0.0392	0.4255±0.0131*	1.8029±0.1013

Note: the upper right-hand corner indicates that the difference reached a very significant level (P <0.01) and that the difference reached a significant level (P < 0.05).

6.3 the weight of the queen bee and worker bee

Through differential analysis (table 2), the weight average of queen bee mating generation living body of queen bee mating generation living body of queen bee mating group as feeding group is lighter than that of queen bee mating generation living body of queen bee mating group as feeding group, except that WF1 and EF1 show significant difference (P <0.05), the other two repeated tests show no significant difference in statistical analysis. On the contrary, the weight of the worker bee birth body of the queen bee eating Italian bee royal jelly descendant is heavier than that of the worker bee birth body of the queen bee eating Chinese royal jelly, but the difference between the weight of the worker bee birth body of the WF2 and that of the worker bee birth of the EF2 queen bee is not obvious, the weight of the worker bee birth body of the WF3 descendant is obviously higher than that of the EF3(P is less than 0.05), and the weight of the worker bee birth body of the WF1 descendant is greatly higher than that of the worker bee birth body of the EF1 (P is less than 0.01).

TABLE 2 Queen bee offspring weight differential analysis

Colony numbering	Weight g of offspring queen bee	Offspring worker bee weight g
			EF1	0.1695±0.0053*	0.0929±0.0042**
WF1	0.1630±0.0069*	0.0989±0.0016**
			EF2	0.1696±0.0072	0.0980±0.0046
WF2	0.1648±0.0085	0.1008±0.0041
			EF3	0.1706±0.0077	0.0873±0.0044*
WF3	0.1655±0.0073	0.0921±0.0041*

6.4 offspring worker bee morphological index analysis

From the results of morphometry of the offspring worker bees bred by the two processed queen bees (Table 3), among the 19 morphometric indexes, the right anterior winged breadth (FB), the postpodosomal tarsal ganglion length (ML), the postpodosomal tarsal ganglion width (MT), the third ventral plate length (S3),

The third web wax lens slant length (WT), the elbow pulse index (a/b), the fin hook number (Nh) and other 7 index values have no significant difference with the control group; 4 length indexes such as the hindfoot shin length (Fe), the fourth back plate smooth zone length (4b), the sixth web length (L6), the sixth web width (T6) and the like are all obviously larger than those of a control group; in addition, in the three replicates, 8 length indices, such as kiss length (a), right anterior Fin Length (FL), hindfoot femoral knot length (Fe), third back panel length (T3), fourth back panel length (T4), fourth back panel fuzz zone length (4a), third web wax mirror length (WL), and third web wax mirror spacing (WD), showed very significantly different changes.

TABLE 3 analysis table of morphological differences of descendants

Note: the upper right upper case of the numbers indicates that the difference reached a very significant level (P <0.01) and the lower case indicates that the difference reached a significant level (P < 0.05).

6.5 results summary

The body size of the apis mellifera larva swarm used as a feeding swarm for feeding the apis cerana queen is significantly larger than that of the apis cerana larva swarm used as a feeding swarm for feeding the apis cerana queen.

1. The reproductive performance comparison of the apis cerana queen fed by the apis mellifera and the apis cerana queen fed by the apis cerana shows that after the apis cerana queen enters the apis cerana group from the apis cerana group, the weight and the daily egg laying amount of the apis cerana queen are both improved, the daily egg laying amount of the apis cerana queen fed by the apis cerana is improved by 23.56-105.91% compared with that of the apis cerana fed by the apis cerana, the average egg laying amount is improved by about 56.92%, and the daily egg laying amount is obviously improved; egg length and egg width had different increases, but the egg weight variation was not significant. The breeding performance of the Chinese bee queen oviposition bee fed by worker bees of Italian bees is obviously improved.

2. The initial body weight of queen bee offspring in the feeding of apis mellifera is slightly lower than that in the feeding group of apis cerana but does not reach a remarkable level; and the initial reproduction of the bee is opposite to the initial reproduction of the bee, and has statistical significance.

3. The multiple indexes of the shapes of the worker bees of the Italian bee feeding queen and the offspring of the Chinese bee feeding queen have different increases, and the difference of partial indexes reaches a remarkable and extremely remarkable level.

7. Transcriptome analysis of copulation queen ovary based on RNA-seq technique

7.1 test Material

7.1.1 test specimens

Randomly selecting 3 queen bees from test group and control group, and using CO₂And (3) after anesthesia, dissecting, taking ovarian tissues, subpackaging the ovarian tissues into sterile RNA enzyme-free cryopreservation tubes, labeling, temporarily storing in liquid nitrogen, and after the collection of the samples is finished, transferring the samples into an ultralow temperature refrigerator at minus 80 ℃ until RNA and DNA are extracted, so as to facilitate the subsequent transcriptome sequencing and related analysis.

7.1.2 Main instrumentation

Gel imaging System, BIO-RAD, USA

Electronic balance, CP225D, Sartorius

Ultra-low temperature refrigerator, DW-86L828, Haier

High speed refrigerated centrifuge, Eppendorf 15804R

Ultraviolet spectrophotometer, Q5000, Quawell, USA

Autoclave, GR60DA, Shimao (Xiamen) instruments Co., Ltd

Ultrapure Water System, Milli-Q AdvantageA10, USA

Constant temperature water bath, HH, Changzhou national instruments manufacturing Co., Ltd

Electrophoresis apparatus, DYCP-31BN, Beijing Liuyi

PCR Instrument, Bio-Rad, USA

7.1.3 Main reagents and consumables

Trizol (Invitrogen), DNA Marker2000 (Beijing Tiangen), Taq DNA polymerase (Fermentas), M-MLV reverse transcriptase (Promega), reverse transcription kit (TOYOBO), 6 × Loading buffer, chloroform, isopropanol, agarose, absolute ethanol, centrifuge tube (Axygen), pipette tip, etc., and the reagents for molecular biology related tests were prepared in accordance with the standard.

7.2 test methods

7.2.1 RNA extraction of Queen bee ovary tissue

The test uses Trizol method to extract total RNA in queen bee ovary sample tissue, 0.05g of queen bee ovary tissue is taken out, ground in liquid nitrogen to be powder, then Trizol reagent is added and vortex mixing is carried out, insoluble substances are removed through high-speed freezing and centrifugation; adding chloroform into the supernatant to separate water phase, freezing and centrifuging, and repeating the steps to remove impurities such as nucleoprotein in the solution; adding isopropanol to precipitate RNA; pouring out the supernatant, drying, repeatedly washing RNA by using 75% ethanol solution, and removing the supernatant; finally, the total RNA is obtained by dissolving with DEPC water. And (3) ensuring that no RNA enzyme exists in the reagent and the consumable material in the extraction process, and detecting the purity of the obtained RNA by using a Q5000 ultraviolet spectrophotometer.

7.2.2 transcriptome library construction

After the RNA detection is qualified, the mRNA of the queen bee ovary tissue is purified and enriched by using Sera-mag Magnetic Oligo (dT) Beads. After purification was complete, fragmentation buffer was added to fragment the mRNA into 100-500bp fragments. The broken mRNA is used as a template by reverse transcriptase, DNA polymerase and random primers, and double-stranded cDNA is synthesized by reverse transcription. The synthesized cDNA was purified using AMPure XP beads. The purified double-stranded cDNA is end-filled and then subjected to phosphorylation modification, 3' end adenylation and ligation of sequencing adaptors. After the connection of the sequencing adaptor is completed, AMPure XP beads are used for preferentially selecting the length of a double-stranded cDNA fragment of 240bp, and finally, the double-stranded cDNA added with the adaptor is used as a template to carry out PCR amplification so as to enrich cDNA fragments with proper length and recover products, thereby constructing a cDNA library. The constructed library is qualified by quality inspection of the Aglient 2100 Bioanalyzer, and then is sequenced by a Tllumina HiSeqTM 4000 sequencer, wherein the sequencing read length is 150bp of double-ended sequencing (Paired-End). Qualified libraries were used for on-machine sequencing (done by bekkiso biotechnology limited, beijing).

7.2.3 bioinformatics analysis procedure summaries

Screening the original Data obtained by sequencing to obtain Clean Data, comparing the Clean Data with reference genes of bees to obtain Mapped Data, and evaluating the quality of the library through fragment randomness test and fragment length; analyzing the sequence structure by alternative splicing type; and analyzing the number of the differentially expressed genes according to the expression quantity of the genes among different samples, and performing analysis such as annotation, enrichment and the like on the differentially expressed genes in each gene bank. The information analysis flow is shown in fig. 6.

7.3 analysis of results

7.3.1 transcriptome library quality assessment

To test the quality of the 6 sample libraries, the invention was validated from three aspects (mRNA fragment randomization, insert length, and data saturation), respectively.

7.3.1.1 random test for mRNA fragmentation

By analyzing the distribution of Mappled reads over 6 sample transcripts, the probability that spliced reads will be extracted in each sequence measured will be more similar if the randomness of the mRNA disruption is better. As can be seen from FIG. 7 (the sequencing randomness analysis chart of mRNA, from left to right, and from top to bottom, respectively: EF1, EF4, EF5, WF1, WF3, WF4), the distribution of reads at each part of the gene is relatively balanced, which indicates that the randomness of fragment disruption is better during library construction.

7.3.1.2 insert Length test

Because of the presence of introns in eukaryotic genes, the distance between the start and stop points of mRNA obtained during transcriptome sequencing is less than the length of a reference gene, and the length can be calculated by comparing the length with the reference gene. The purification effect of the selected fragment during library construction can be reflected by the statistical analysis of the length of the inserted fragment. The results of the 6 sample sequencing insert length check (FIG. 8, the sequencing insert length check, from left to right, top to bottom, EF1, EF4, EF5, WF1, WF3, WF4, respectively) are shown, FIG. 8 illustrates: most of the insert was between 200 and 400bp in length. .

7.3.1.3 transcriptome sequencing data saturation test

Due to the limited number of genes in a species and the specificity of expression, sufficient sequencing data is required to accurately quantify genes with low expression levels. To determine whether the sequencing depth meets the requirements of the subsequent analysis, the saturation of the number of genes in the obtained transcript needs to be detected. By taking 10% of the obtained Mapped data amount as a gradient, sampling is performed randomly from sequencing data in sequence, saturation simulation graphs of genes with different expression levels of 6 sample transcriptomes are respectively drawn (FIG. 9, the saturation simulation graphs of the transcriptomes are from left to right and from top to bottom, namely EF1, EF4, EF5, WF1, WF3 and WF4), and FIG. 9 shows that the saturation of the number of the transcripts of 6 samples is better and can meet the requirement of further detection.

7.3.2 screening and analysis of transcriptome sequencing data

Through the transcriptome sequencing technology, raw reads of 143.317G are obtained from 6 test sample libraries (EF1, EF4, EF5, WF1, WF3 and WF4) in total, Clean Data of 141.376G is obtained after Data containing a joint is filtered out, low-quality reads are removed through sequencing quality screening, and finally 40.32Gb Clean Data are obtained, wherein the Data amount of the Clean Data of each sample is about 6.54 Gb. The GC specific ratio of 6 sequencing samples is between 35.22% and 36.35% on average, and the percentage of Q30 bases is not less than 93.03%. The effective reads obtained by sequencing is between 43,648,000-46,643,016, and the transcriptome coverage rate is higher, which shows that the sequencing result of the research has good quality and higher accuracy rate, and can be used for subsequent bioinformatics analysis. Data throughput statistics for each sample are shown in table 4.

TABLE 4 Gene coverage statistics

Sample numbering	Clean reads	Clean bases	GC content (%)	≥Q30(％)
					EF1	23,321,508	6,983,698,442	35.58％	93.42％
EF4	22,477,745	6,732,240,324	35.22％	93.50％
					EF5	22,312,315	6,683,212,282	35.54％	93.03％
WF1	22,798,102	6,817,750,388	35.72％	93.61％
					WF3	21,824,000	6,538,146,590	35.99％	93.24％
WF4	21,936,456	6,565,706,192	36.35％	93.63％

7.3.3 alignment of transcriptome data with reference genomic sequences

7.3.3.1 statistics of alignment efficiency

By using the HISAT2 to perform comparative analysis on the data after quality evaluation and the bee Amel _4.5 reference genome, 43,105,291 (92.42%), 41,444,616 (92.19%), 41,098,194 (92.10%), 42,113,653 (92.36%), 40,544,776 (92.89%), 40,377,907 (92.03%) of reads in 6 sequencing samples of the test group and the control group can be matched with the reference genes of the bee. The numbers of reads of the reference genes which are matched uniquely are 42,278,261, 40,606,156, 40,204,365, 41,179,039, 39,595,052 and 39,468,586, and respectively account for 90.64%, 90.33%, 90.09%, 90.31%, 90.71% and 89.96% of the total number of reads.

From the statistics of the alignment results, the alignment efficiency of clean reads of 6 samples to the reference genome is between 92.03% and 92.89%, and the unique alignment rate is between 89.96% and 90.71% (table 5).

TABLE 5 alignment of sequencing data and reference genome

7.3.4 SNP and InDel analysis

An SNP is a genetic marker formed by the conversion or transversion between single nucleotides on a gene, and the influence of the SNP on the gene expression can be analyzed by identifying potential SNP sites on the gene. We used the GATK software to identify potential SNP and InDel sites of 6 sequencing samples and count the number and type of the potential SNP and InDel sites respectively, based on the criterion that the number of consecutive single-base mismatches is less than 3 and the SNP quality value is greater than 2.0 within 35bp of the base sequence length (Table 6). The density values of SNP sites on 6 sample genes are calculated, and a density distribution diagram of SNP (FIG. 10, SNP mutation type and density distribution diagram) and an SNP and InDel annotation classification diagram (FIG. 11) are drawn. It can be seen that: the total number of SNPs in the WF group is higher than that in the EF group, and the difference of the other indexes is not obvious.

TABLE 6 statistical table of SNP loci

Sample numbering

Total number of SNPs

Gene region SNP

Intergenic region SNP

Conversion ratio

Ratio of transversion

Heterozygosity ratio

EF1

79,516

67,251

12,265

84.25％

15.75％

46.10％

EF4

103,180

85,820

17,360

83.83％

16.17％

44.70％

EF5

81,692

68,328

13,364

84.00％

16.00％

45.89％

WF1

77,243

65,378

11,865

84.01％

15.99％

45.81％

WF3

75,294

64,145

11,149

84.37％

15.63％

46.74％

WF4

85,557

72,400

13,157

84.04％

15.96％

45.20％

7.3.5 statistics of the number of alternative splicing events

Alternative Splicing (AS), also known AS alternative splicing, allows the selection of different exons for the precursor mRNA of eukaryotic genes, thus allowing the synthesis of multiple mrnas for the translation of different proteins, exerting different biological functions and adapting to changes in external conditions.

The alternative splicing patterns of 6 samples were analyzed using the ASProfile software. The five alternative splicing types were subdivided by the Asprofile software into 12 alternative splicing event types. The results of software analysis found that of the 12 different alternative splicing event types, TSS (first exon variable splicing) and TTS (last exon variable splicing) are the most abundant and the most predominant alternative splicing types in the queen transcriptome. The next are 3 types of AE (variable 5 'or 3' end splicing), SKIP (single exon skipping) and IR (single intron retention), with few other types of variable splicing events. Because queen bees are all from the same female parent and the genetic materials are basically consistent, the difference of the variable splicing types of each sample is basically consistent, and the statistical result of the number of the predicted variable splicing events in each sample is shown (figure 12).

7.3.6 analysis of Gene expression level

7.3.6.1 Gene expression Profile

The number of transcript fragments and the amount of data obtained by sequencing, the length of the transcripts and the level of expression of the transcripts are all relevant. Therefore, by counting the number of the Mapped Reads obtained from the sample and the length of the transcript, we can know the expression of the 6 sample transcripts. FPKM values were used to reflect gene expression levels of 6 transcripts, which were calculated as follows:

the curves in the density profile (fig. 13) of FPKM represent different samples, the abscissa of the point on the curve represents the log value of the FRKM for the corresponding sample, and the ordinate of the point represents the probability density.

As can be seen from the density distribution graph (fig. 13) of FPKM and the box plot (fig. 14) of FPKM distribution, the gene expression level distribution of 6 samples was similar and was not significantly different.

7.3.6.2 differential expression Gene screening

Gene expression was calculated by the RPKM method, and differential expression among sample groups was analyzed by using edgeR (F.gtoreq.2, P < 0.05). As sequencing sampling is random, in order to avoid misestimation of difference of sequence length on the calculated gene expression quantity, the p value is corrected again in the calculation process of differential expression analysis, so as to obtain the false discovery rate (FDR <0.01), and then the differentially expressed genes are screened for the genes with the differential expression of the queen ovary of the test group and the control group according to the FDR < 0.01. The screening results showed (fig. 15, differential gene volcano and cluster maps), 323 genes were differentially expressed compared with queen bee of apis cerana feeding group and apis cerana feeding group, of which 269 genes were up-regulated and 54 genes were down-regulated. A large number of genes which are possibly related to the reproductive performance of the queen bees are obtained through transcriptome sequencing, wherein the genes comprise FASN, EF1A, F-actin, HSPs, SF3b, PRMT1, HCRTR and other genes related to the growth and reproduction and nutrient metabolism of the queen bees.

7.3.6.3 functional annotation and enrichment analysis of differentially expressed genes

The differentially expressed genes were functionally annotated into databases, and the number of differentially expressed genes annotated into each database was counted (7). A total of 279 differentially expressed genes were annotated, representing 86.38% of the total. Wherein the results of the NR, eggNOG, Pfam, GO, KOG, Swiss-Prot, KEGG and COG database alignments are 278 (86.07%), 237 (73.38%), 195 (60.37%), 173 (53.56%), 159 (49.23%), 147 (45.51%), 96 (29.72%) and 79 (24.46%), respectively.

Table 7 annotated results of DEGs

Database with a plurality of databases	Number of annotated DEGs	Note the percentage of DEGs
			COG	79	24.46％
GO	173	53.56％
			KEGG	96	29.72％
KOG	159	49.23％
			NR	278	86.07％
Pfam	195	60.37％
			Swiss-Prot	147	45.51％
eggNOG	237	73.38％
			Total	279	86.38％

Note: total: number of differentially expressed genes noted.

7.3.6.4 differential expression Gene GO Classification

Expression difference genes between the ovary samples of the queen bee fed by worker bees and the ovary samples of the queen bee fed by worker bees of the apis cerana are analyzed through GO annotation, and the result shows that (figure 16, a difference expression gene GO classification chart) 174 difference genes are enriched in total in GO, and that 13 function categories related to biological processes are found, including stress, metabolic processes, signal transduction and the like; there are 16 functional classes of cellular components involved, including cytoplasm, organelles, extracellular domain parts, etc.; there are 9 functional classes involved in molecular function, including transport activity, signal transduction, molecular transduction, and the like.

7.3.6.5 DEGs pathway enrichment assay

In order to further understand the function of the genes differentially expressed by the ovary of the queen bee and the ovary of the common queen bee in the feeding of the apis mellifera, the marked enrichment analysis of the passage is carried out on the 174 screened genes differentially expressed (FC is more than or equal to 2, FDR is less than 0.05) through the analysis of a KEGG database, and the passage with the marked enrichment in the differentially expressed genes with the genome of the sample is found out.

As a result, 49 significantly enriched pathways (P <0.05) were found in two comparisons (EF1_ EF4_ EF5_ vs _ WF1_ WF3_ WF4), wherein the first 20 pathways with the most significant enrichment were found (see fig. 17, EF1_ EF2_ EF3vsWF1_ WF2_ WF3 enrichment analysis chart of differentially expressed gene Pathway). Most of these pathways are involved in the synthesis of amino acids, carbohydrates and fats, such as valine, leucine and isoleucine degradation, glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, glycosaminoglycan biosynthesis chondroitin sulfate/dermatan sulfate, pyruvate metabolism, etc.

According to the test result of the physiological condition of queen bee of the test group, the fatty acid metabolic pathway is shown in fig. 18.

7.4 Small knot

1. The effect example analyzes the transcriptome change of ovary tissues of a queen bee and a common queen bee in feeding of apis mellifera through an RNA-seq technology, and explores a control mechanism of apis mellifera royal jelly on the reproductive performance of the queen bee. Differential expression genes between two queen bees were obtained.

2. By performing alternative splicing analysis on the sequencing data, TSS and TTS were found to be the most prominent types of alternative splicing events in Chinese bees.

3. The RNA-seq sequencing result shows that 323 genes are differentially expressed between queen bee fed by Italian bee and a control group, wherein 269 genes are up-regulated in the queen bee fed by Italian bee group, and only 54 genes are up-regulated in the control group, and genes such as FASN, HSPs, EF1A, F-actin, PRMT1, HCRTR and the like which are possibly related to the reproductive performance of queen bee are obtained through differential gene screening.

4. The differential expression genes are enriched and analyzed by the GO and KEGG databases, and the result shows that the two genes differentially expressed by the queen bee ovary are obviously enriched on the molecular functions and signal paths related to the metabolism and synthesis of amino acid, sugar and amino acid.

In this effect example, 323 differentially expressed genes were obtained between the queen bee fed by the Apis mellifera and the queen bee fed by the Apis in the control group by gene differential expression analysis, 54 genes were up-regulated in the control group, and 269 genes were up-regulated in the ovarian tissue of the queen bee fed by the Apis mellifera. Among the differentially expressed genes, genes involved in the growth and development of queen bees, nucleotide synthesis and nutritional metabolism may be related to the reproductive performance of queen bees, and genes such as FASN (fatty acid synthase), HSPs (heat shock proteins), EF1A (splicing factor), F-actin (actin fibers), PRMT1 (arginine methylation), HCRTR (orexin receptor) and the like are obtained by screening.

The bee eggs contain nucleotide, protein and other nutrient substances required by bee embryonic cells to develop into larvae, and the nutrient substances are synthesized in queen bees, so that the increase of the egg laying amount of the queen bees necessarily needs more nutrition and energy metabolism, and in the transcriptome of the queen bee nursing and the queen bee nursing, the gene expression amount of FASN and HSPs of the queen bee in the queen bee nursing group is obviously lower than that of the queen bee in the queen bee nursing group.

From the above analysis, it is known that the egg laying amount and weight gain of queen bee in the apis mellifera feeding group are significantly higher than those in the apis cerana feeding group, and thus, it is presumed that the FASN is related to the growth and reproduction process of queen bee by HSPs. In this test, the expression level of HSPs in queen bees in queen bee nursing group is higher than that in queen bee nursing group compared with queen bees in queen bee nursing group, and it is presumed that the result may be correlated with the environmental stress of queen bees.

Furthermore, biological functions related to amino acid, carbohydrate and fat metabolic synthesis as well as pathways were most significant by GO functional annotation and pathway analysis. Phenotypic differences in queen bee appearance between the apis mellifera and apis cerana feeding groups may be associated with these pathways. Among the KEGG pathway enrichments, the first 3 pathways that are most significantly enriched are glycosaminoglycan biosynthesis, glycolysis/gluconeogenesis, and amino sugar and nucleotide sugar metabolism, respectively. It is proved that chondroitin sulfate has the function of promoting cell nucleic acid synthesis so as to promote cell propagation. Glycolysis is a metabolically important pathway for the breakdown of glucose to produce energy in organisms. Gluconeogenesis is a process in which organisms convert non-carbohydrate nutrients such as pyruvic acid and amino acids into carbohydrates. The improvement of the reproductive performance of the queen bee of the apis mellifera feeding group can be that certain substances in the apis mellifera royal jelly induce and regulate the differential expression of the pathway genes, thereby meeting the requirement of a great amount of nutrition for the queen bee to breed and lay eggs.

Control group 2

The only difference from example 1 is that: the feeding group supplements 1 splenic apis mellifera baby bee every 10 days

Control group 3

The only difference from example 1 is that: the feeding group supplements 1 spleen Italian bee baby bee every 5 days

Control group 4

The only difference from example 1 is that: the feeding group supplements 2 spleen Italian bee baby bees every 7 days

Through detection, 1 spleen apis mellifera young bee is supplemented every 7 days, sufficient royal jelly can be obtained from the apis cerana queen in the feeding group, the feeding capacity of the bee group is vigorous, and the apis cerana queen can continuously lay eggs and reproduce.

Egg shape and egg weight of queen bee, morphological index condition:

and (4) conclusion: the control example 1 and the control groups 1-3 supplement the amount and frequency of apis mellifera brood in different feeding groups, and the result shows that the feeding groups supplement one spleen brood every 7 days to maintain the vigorous feeding enthusiasm, so that the weight of queen bees and the daily egg laying amount of apis cerana are remarkably improved, and the egg weight, the egg length and the birth weight of offspring worker bees are increased to different degrees.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims

1. A method for breeding Chinese bee queens is characterized by comprising the following steps:

(1) preparing a Chinese bee queen:

mating: putting the mature queen bee platforms into a Chinese bee mating group, finishing mating until the Chinese bee queen bees emerge from the hive after the feathering, and laying eggs to obtain a spare Chinese bee queen bee;

(2) preparation of a nursing group:

selecting Italian bee colonies, rewarding for feeding, limiting oviposition space, after emerging from the room by the spleen, putting the Italian bee colonies into an artificial climate box for centralized emerging, and forming feeding colonies by the Italian bee broods emerging from the room;

(3) preparation of spawning group:

the new beehive is internally provided with a apis cerana empty honeycomb, an apis cerana honeycomb with honey and pollen storage, a feeding group consisting of apis cerana larva bees is shaken into the new beehive, a standby apis cerana queen is transferred into the new beehive, the feeding group is used for feeding the standby apis cerana queen royal jelly, and after the standby apis cerana queen on the apis cerana empty honeycomb is full of eggs, the new apis cerana empty honeycomb is used for replacing.

2. The method for breeding the queen bee of the Chinese bees according to claim 1, wherein in the step (1), the bee quantity of the Chinese bee mating group is 1 splenic bee quantity.

3. The method for breeding Chinese bee queens according to claim 1, wherein in the step (1), the whole mating process time of the mature queens from the placement of the mature queens in the Chinese bee mating group to the obtaining of the standby Chinese bee queens is as follows: 8-12 d.

4. The method of claim 1, wherein in step (2), the bee quantity of the nursing group is not less than 3 splenic bee quantities.

5. The method for breeding queen bees of Chinese bees according to claim 1, wherein in the step (2), the oviposition limiting space is implemented by allowing the apis mellifera colony to lay eggs on 1-2 empty spleens.

6. The method of claim 1, wherein in step (3), the feeding group supplements 1 splenic apis mellifera larvae every 7 days.

7. The method of claim 1, wherein in step (3), the relationship between the combs of the nursing group is to keep more bees than spleens.